RNA Activation of CEBPA in PBMCs Enhances α-L-Iduronidase Expression A Translational Adjuvant Therapy for MPS I After Bone Marrow Transplantation

RNA Activation of CEBPA in PBMCs Enhances α-L-Iduronidase Expression A Translational Adjuvant Therapy for MPS I After Bone Marrow Transplantation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article RNA Activation of CEBPA in PBMCs Enhances α-L-Iduronidase Expression A Translational Adjuvant Therapy for MPS I After Bone Marrow Transplantation Vikash Reebye, Konstantina S Stathaki, Konstantinos Vanezis, Minsun Song, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7346641/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Hurler syndrome, the most severe form of mucopolysaccharidosis type I (MPS I), is a rare genetic disorder caused by mutations in the IDUA gene, leading to a deficiency of the α-L-iduronidase enzyme. While current treatments offer some benefits, there remains a significant unmet medical need. We have identified a potential new therapeutic approach using MTL-CEBPA, a drug that upregulates the transcription factor CEBPA , which in turn regulates IDUA expression. Results In vitro studies demonstrated significant upregulation of IDUA in various cell lines following MTL-CEBPA treatment. In vivo experiments in both wild-type and MPS I mouse models showed a two-fold increased IDUA expression and enzyme activity for up to four weeks after a single dose. Analysis of archival samples from cancer patients treated with MTL-CEBPA revealed a correlation between increased CEBPA expression and IDUA expression, with approximately half of the patients showing elevated plasma IDUA enzyme activity post-treatment. Conclusions These findings provide proof-of-concept evidence supporting the potential use of MTL-CEBPA as a treatment for MPS I patients. We propose that this therapeutic oligonucleotide approach offers a favourable safety profile, allowing for multiple dosing to maintain elevated IDUA enzyme activity in bone marrow transplanted patients over extended periods, potentially addressing the limitations of current treatments and improving patient outcomes. Therapeutic oligonucleotides. RNA activation Small activating RNA Hurler Syndrome MPS-I α-L-iduronidase Enzyme deficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Mucopolysaccharidosis type I (MPS I) is a rare, autosomal recessive lysosomal storage disorder caused by deficiency of the enzyme α-L-iduronidase (IDUA). This leads to accumulation of the glycosaminoglycans (GAGs), heparan sulfate and dermatan sulfate in lysosomes, resulting in multisystem organ dysfunction. Clinical features include cardiac valve thickening, cardiomyopathy, respiratory disease, cognitive decline, hepatosplenomegaly, skeletal dysplasia, and vision impairment. MPS I is classified by severity into severe (Hurler syndrome), intermediate (Hurler-Scheie), and attenuated (Scheie) forms, based on residual enzyme activity, age of onset, and clinical phenotype. Untreated, severe MPS I (Hurler syndrome) leads to death within the first decade, often earlier. Attenuated forms present later and progress more slowly, with variable life expectancy ( 1 ). Currently, two main treatments are available for MPS I: enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT) ( 6 ). ERT involves weekly infusions of recombinant enzyme, which is costly but effective in improving somatic symptoms affecting organs such as the heart, lungs, liver, spleen, and kidneys ( 2 , 3 ). However, ERT cannot cross the blood-brain barrier, so it does not address neurological symptoms. Its efficacy is also limited in poorly vascularised tissues such as heart valves and cartilage, making it less effective for cardiac and skeletal manifestations. Long-term ERT can lead to the development of anti-IDUA antibodies, potentially causing immune reactions ( 4 , 5 ). HSCT is recommended for patients diagnosed before 2.5 years of age, ideally before irreversible symptoms develop, and is often combined with ERT ( 6 ). Donor stem cells provide a permanent source of enzyme, enabling cross-correction of deficient cells via the mannose-6-phosphate receptor. Although the blood-brain barrier is not fully permeable, HSCT can facilitate the delivery of enzyme-secreting cells to the brain, supporting ongoing neurodevelopment ( 7 ). Both therapies have limitations, but HSCT remains the only option with potential to address both somatic and neurological aspects of MPS I. Hematopoietic stem cell transplantation (HSCT) often fails to resolve glycosaminoglycan (GAG) accumulation in avascular tissues (such as bone and cartilage) and non-dividing structures (such as the cornea) in patients with severe mucopolysaccharidosis type I (MPS I). This persistent substrate accumulation results in ongoing skeletal deformities, cardiac complications, and a continued need for surgical interventions—representing a significant unmet medical challenge ( 8 ). Post-transplant outcomes are closely linked to leukocyte α-L-iduronidase (IDUA) activity: engraftment with wild-type donor cells leads to minimal tissue substrate deposition, whereas transplants from carrier donors are associated with higher levels of residual GAG accumulation ( 9 ). Raised serum IDUA levels following HSCT are associated with reduced disease burden and a lower incidence of surgical procedures for carpal tunnel syndrome and spinal abnormalities ( 10 ). Ex vivo lentiviral IDUA gene transduction in autologous HSCT has shown promise in achieving supra-physiological enzyme levels, potentially addressing residual disease following transplantation ( 11 ). However, this irreversible procedure carries significant risks, particularly for patients who have previously undergone transplantation and have a limited pool of stem cells. This report presents a novel strategy to enhance the effectiveness of HSCT: RNA activation to naturally increase IDUA production from existing transplanted cells in patients with MPS I. This approach aims to boost enzymatic activity without genetic modification, potentially offering a safer and more effective solution for managing residual disease post-transplantation. Methods Cell culture and transfection A549 cells, IMR90 cells, and MSCs (ATCC) were grown in ATCC-formulated RPMI medium supplemented with 10% fetal bovine serum following manufacturer’s protocols. Cells were maintained in a 5% CO2 incubator. Unless otherwise specified, for transfections, the cells were seeded at 1.5 x 10 5 cells per well in a 24-well plate and reverse transfected immediately after seeding with the indicated oligonucleotide concentration using 1 uL per well of Lipofectamine 2000 (Life Technologies). In silico PROMO analysis PROMO was used to screen the 5’ regulatory DNA sequence of IDUA gene http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 ). RNA isolation and qPCR from cell lines RNA was isolated from cultured cells using the RNeasy Mini Kit (QIAGEN). RNA was quantitated using a QiaXPERT spectrophotometer (Qiagen), and 500 ng was reverse transcribed using the Quantitect Reverse Transcription Kit (QIAGEN). Relative expression levels were determined by qPCR using Quantifast SYBR Green Master Mix (QIAGEN) on an QuantStudio thermal cycler (LifeTechnologies). Quantitect Primer Assays (QIAGEN) were used to probe for transcript levels of IDUA and Actin B as the house keeping gene. Relative expression was determined using the DDCt method normalized to GAPDH expression. Assessment of MTL-CEBPA in wild-type mice All work was carried out under UK Home Office legislation. Male C57Bl/6J (6 weeks old) were purchased from Charles River UK. Animals were each allocated a unique tag number. A random number generator was used to randomise the animals by tag numbers into each treatment groups. Animals were dosed intravenously via tail vein injection with 2 single doses, 24 hour apart of 2mg/kg of MTL-CEBPA or NOV340-FLUC (formulated CEBPA-51 (saCEBPA) and siFLUC previously described ( 12 , 13 , 17 ). The Animals were weighed three times weekly. Bone marrow samples were taken at termination. The left femur was excised and the femoral cells flushed out using 1mL PBS. An equal volume of 70% ethanol was added and the solution was mixed by pipetting. The mixture was then transferred to RNeasy columns following the manufacturer’s instructions. After the wash steps, 30ul of water was used for eluting the RNA from the column. RNA Samples were quantified using the QIAGEN QIAxpert spectrophotometer according to the manufacturer’s protocol. QC of the samples was performed using the ScreenTape Assay for Tapestation (Agilent) according to the manufacturer’s recommended protocol. Samples with RIN greater than 7.0 were considered as meeting the threshold quality for qPCR amplification. Samples were subjected to reverse transcription using the Qiagen Quantitect RT Kit with 300–500 ng RNA input which was standardised across the samples. Reverse transcription was performed according to manufacturer’s protocol. Oligo-dT primer (QIAGEN) was substituted for the default random hexamer/oligo-dT primer mix in the kit. In brief, for the RT reaction, 2µL of the gDNA wipeout was added to 12ul diluted RNA for 2 minutes at 42ÆC followed by 1µL RT enzyme, 1µL oligodT primer, and 4µL 5xRT buffer for a total of 20µL each sample. Following the RT step, 140µL of water was added to each cDNA sample for an 8-fold dilution prior to qPCR amplification. qPCR SYBR qPCR was performed for the analysis of CEBPA gene expression. Relative gene expression was measured in cDNA samples by qPCR using the ThermoFisher PowerUp SYBR Green Master Mix on a QuantStudio 5 qPCR machine using QIAGEN QuantiTect murine primer assays for CEBPA and 4 housekeeping genes: GAPDH, HPRT, B2M, ACTB. Reactions were run in triplicate with each 10µL reaction volume composed of the following: 1µL primer assay, 5µL 2x SYBR Green mastermix, 4µL diluted cDNA from above. The following cycling conditions were used: initial heat activation step 2 minutes @95 o C then 40 cycles comprising 1 second @95 o C, 30 seconds @60 o C followed by melt curve analysis. geNORM analysis revealed that the most consistent housekeeping genes for these samples were HPRT and B2M so a mean of these 2 was used for normalisation. Taqman qPCR was performed for the analysis of IDUA gene expression. Relative gene expression of IDUA was measured in cDNA samples by qPCR using the Thermo Fisher TaqMan Fast Advanced Master Mix according to the manufacturer’s protocol on a QuantStudio 5 qPCR machine with a Life Tech Taqman assay for murine IDUA. Reactions were run in triplicate with each 10µL reaction volume composed of the following: 0.5µL primer assay, 5µL 2x SYBR Green mastermix, 4µL diluted cDNA from above, 0.5µL water. The following cycling conditions were used: initial heat activation step 20 seconds @95 o C then 40 cycles comprising 1 second @95 o C, 20 seconds @60 o C. The maximum amount of blood (600µL -800µL) was collected by cardiac puncture and transferred to K2 EDTA treated tubes. Tubes were spun down at 300g for 5’ at 4 º C and the plasma aliquoted and stored at -80 º C until analysis. IDUA enzyme activity assay Preparation of serum from blood samples After blood was collected and allowed to clot, serum was separated following standard centrifugation guidelines (5000rpm for 15’). In duplicate, 25µL of serum was mixed with 25µL of 360µM 4-methylumbelliferyl alpha-L-iduronide (Glycosynthe) in 0.4M Formate buffer at pH3.5 in a microplate. Sample blanks were prepared by mixing 25µL 0.2% BSA in PBS with 25µL of 360µM 4-methylumbelliferyl alpha-L-iduronide in 0.4M Formate buffer at pH 3.5. A 10-point standard curve of 4-methylumbelliferone in 0.4M Formate buffer at pH 3.5 ranging from 0.04ug/mL to 0.4ug/mL was prepared at the same time. Plates were then protected from light and incubated for 30’ at 37 º C. The reaction was stopped by the addition of 200µL of glycine carbonate buffer. Samples were then read in a Biotek plate reader at 365nm excitation and 450nm emission. Enzyme activity was calculated by dividing activity in ng/h by mL of serum using the formula: Activity in nmol/h/ml where specific activity was summarised as ng/h/ml divided by FW of 4MU (176.17). In vivo assessment of MTL-CEBPA in humanised MPS-1 mice Generation of the MPS 1 Disease Mouse Model To generate the MPS1 mouse model, breeding challenges were encountered due to reproductive limitations. Females homozygous for all three mutations (Idua W392X , scid, and Il2Rγ null ) exhibited poor maternal behaviour, while males homozygous for the Idua W392X mutation, homozygous for scid, and hemizygous for the X-linked Il2Rγ null mutation demonstrated subfertility, leading to a higher incidence of non-productive breeding pairs. To maintain a viable colony, breeding strategies involved crossing females heterozygous for the Idua W392X mutation and homozygous for the scid and Il2Rγ null mutations with males heterozygous for the Idua W392X allele, homozygous for the scid mutation, and hemizygous for the X-linked Il2Rγ null mutation. NOD.Cg-Idua tm1Clk Prkdc scid Il2rg tm1Wjl /J (NSG-MPS1) heterozygous mice were obtained from Jackson Laboratories and incorporated into the breeding strategy. Genotyping by rhAmp SNP Assays Total genomic DNA was extracted from mouse tail samples (5 mm of tail placed into a 1.5 mL tube) using DirectPCR Lysis Reagent (VivaGen Biotech), following the manufacturer’s protocol. For extraction, 200 µL of DNA lysis buffer and 2 µL of 20 mg/mL Proteinase K solution were added to each sample. The samples were incubated overnight at 55°C and then heated at 85°C for 45 minutes to inactivate Proteinase K. DNA was purified via phenol/chloroform extraction and ethanol precipitation, and the concentration was determined. Purified DNA was stored at − 20°C until use. Genotyping was performed using rhAmp technology, which employs dual-enzyme chemistry based on RNase H2-dependent PCR (rhPCR) and universal reporters. Each assay utilized two allele-specific primers along with a locus-specific primer. Synthetic gBlocks™ gene fragments representing known genotypes served as controls for each SNP assay: one for the wild-type allele (WT), one for the mutant allele (MA), and a 1:1 mixture to represent the heterozygous genotype. Primers were designed using the rhAmp® Genotyping Design Tool (IDT; https://eu.idtdna.com/site/order/designtool/index/GENOTYPING_PREDESIGN ) as detailed in Table 1 . The rhAmp genotyping assay was performed according to the manufacturer’s instructions. Briefly, 2 µL (10 ng) of DNA sample was mixed with 5.3 µL of rhAmp Genotyping Mix [1 mL of rhAmp Genotyping Master Mix 2× (Cat. no. 1076017; IDT) and 50 µL of rhAmp Reporter Mix (Cat. no. 1076028; IDT)] and 0.5 µL of custom rhAmp SNP assays (IDT), achieving a final volume of 10 µL. Standard samples and negative template controls used in the HRM analysis were also included. The SNP genotyping assays were prepared with 0.25 µL of rhAmp SNP Assays (20X), 2.65 µL of combined rhAmp Genotyping Master Mix (2X) and rhAmp Reporter Mix (40X), 0.10 µL of nuclease-free water, 2 µL of sample DNA, and 2 µL of control template (gBlocks fragment controls) or nuclease-free water for no-template controls. Reactions were conducted on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), and data were analyzed using CFX Maestro Software version 2.3 (Bio-Rad). Thermal cycling conditions followed the standard protocol: 95°C for 10 minutes, followed by 40 cycles at 95°C for 10 seconds, 60°C for 30 seconds, and 68°C for 20 seconds ( www.idtdna.com/rhAmp-SNP-protocol ; accessed on 29 February 2024). Bi-allelic specificity was achieved using two probes, one labeled with Amidite-fluorescein (FAM™) dye and the other with Hexachloro-fluorescein (HEX™) dye (Table 2 ). These dyes were detected independently with appropriate excitation sources and emission filters at their respective wavelengths. All genomic DNA samples were quantified, and each specimen was classified as resistant (RR), susceptible (SS), or heterozygous (RS) based on the melting temperature obtained. The results were displayed as puncta on an XY scatter plot based on relative fluorescent units (RFU), facilitating allelic discrimination through CFX Maestro software (Bio-Rad Laboratories). Table 1 Primer Type Sequence 5’ → 3’ Quencher Common CCT GGG GAT TCC TTC CAC Wild type Reverse GGC ACA GTG ACC CAG GAG Mutant Reverse GGC TCT ATG GCT TCT GAG G WT probe CTG CCA AGG TCA CCA ATG T FAM MUT probe CCT GCC AAG GTC ACG AG HEX Table 2 Different excitation and emission spectra for fluorophores. Quencher Excitation nm Emission nm FAM 450 533 HEX 483 568 Ethics statement All animal care and experimental procedures were conducted in accordance with protocols reviewed and approved by the City of Hope Institutional Animal Care and Use Committee (IACUC), under the principal investigator for this study, John Rossi (IACUC 23017). Human fetal liver tissue was obtained from Cercle Allocation Services, Inc., a nonprofit organization, in compliance with all applicable federal and state regulations. The vendor maintains its own Institutional Review Board (IRB) and adheres to human subject protection requirements. Humanised Mouse Generation To generate humanised NSG-MPS1 mice, two preparative methods were employed. First, 5–8-week-old NSG-MPS1 mice were irradiated with 230 cGy and subsequently transplanted via intravenous (i.v.) injection with 2 × 10 5 to 1 × 10 6 CD34 + hematopoietic stem cells (HSCs) isolated from human fetal liver tissue. Alternatively, a second approach involved administering intraperitoneal (IP) injections of busulfan (35 mg/kg, adjusted by weight) for four consecutive days. On the fifth day, 130,000 CD34 + human fetal liver stem cells were injected through the tail vein to establish humanization. Tail vein injections were performed using sterile strainers, with proper disposal of needles in biohazard containers without recapping. Engraftment was monitored through retro-orbital blood collection, and mice were observed for 10–20 weeks post-transplantation. At 12 weeks, engraftment was evaluated by flow cytometry analysis of peripheral blood samples. Blood Collection and Analysis Baseline blood samples were collected for IDUA analysis. Subsequent blood samples were collected weekly for a duration of 7 weeks. The blood plasma IDUA enzyme activity was assessed using the 4-methylumbelliferyl-α-L-iduronide (4Mu) enzyme colorimetric assay. Statistical Methods GraphPad Prism (version 8.1.1, GraphPad Software, Inc) was used to graph and analyse all data. In vitro transfection data was analysed for saCEBPA vs Fluc comparison using unpaired t-test with Welch’s correction. In vivo mouse and white blood cell patient data were analysed by two-way ANOVA. Results CEBPA Activation Enhances IDUA Expression Across Multiple Cell Types To gain a comprehensive understanding of IDUA biology across different cellular states within a lung tissue context, we utilised both transformed (A549) and non-transformed (IMR90) cell lines. We previously developed an RNAactivation (CEBPA51) designed to upregulate CEBPA expression, known as saCEBPA ( 12 ). To determine whether CEBPA is a bona fide activator of IDUA expression, we transfected A549 cells with saCEBPA. This resulted in a 3-fold increase in CEBPA mRNA levels (Fig. 1 A) and a corresponding 4-fold increase in IDUA mRNA expression (Fig. 1 B) relative to control (FLUC). We then investigated the transcriptional effect of saCEBPA on IDUA expression in non-transformed IMR90 cells. Cells were transfected with increasing doses of saCEBPA (1nM, 5nM, 10nM, and 20nM). FLUC control was transfected at 20nM. Gene expression was assessed by RT-PCR at 72 hours post-transfection for both CEBPA and IDUA. Results showed CEBPA expression was significantly upregulated at 10nM and 20nM saCEBPA. Values were normalised to untreated cells (Fig. 1 C). IDUA gene expression also increased within the same dose range of 10nM to 20nM of saCEBPA (Fig. 1 D). To further explore the transcriptional relationship, we assessed the effect in mesenchymal stem cells. We observed increased expression of both IDUA and CEBPA, which persisted for seven days (Fig. 1 E and 1 F). These findings demonstrated that CEBPA activation consistently lead to increased IDUA expression across multiple cell types, including transformed lung cells, non-transformed lung fibroblasts, and mesenchymal stem cells. In vivo evidence that MTL-CEBPA increases IDUA expression and activity To evaluate the in vivo pharmacodynamic effect of saCEBPA on upregulation of IDUA enzyme activity, wild-type C57/BL6 female mice were administered 2x bolus injection of 2mg/kg of MTL-CEBPA via tail vein IV infusion over a 24-hour period so that the animals would have received a total of 4mg/kg of the RNAa (n = 5 for each group). The effects of MTL-CEBPA was measured over a period of 10 weeks. The pharmacodynamic activity of MTL-CEBPA was determined by measuring CEBPA and IDUA mRNA in the bone marrow and IDUA activity in plasma with a fluorometric assay using 4-methylumbelliferyl alpha-L-iduronide (4-MU iduronide) as substrate. As shown in Fig. 2 A, there was a significant increase in CEBPA mRNA expression at 4-, 6-, and 8-weeks post-administration relative to control. A significant increase in IDUA mRNA in bone marrow was observed at week 4 (Fig. 2 B). Interestingly, we observed that the increase in IDUA expression was correlated with an increase in IDUA activity in plasma at 6 weeks post treatment (Fig. 2 C). Collectively, these data support our in vitro evidence that saCEBPA increases IDUA expression. Further, these data indicate that the increase in IDUA by MTL-CEBPA (the NOV340 in vivo carrier formulated RNAa) is detected in the circulation where it can facilitate cross-correction. Collectively, these data provide in vivo POC that IV administration of MTL-CEBPA can increase the plasma levels of IDUA activity after treatment at baseline. In vivo effects of MTL-CEBPA in humanised bone marrow transplanted MPS 1 mice To explore the potential for MTL-CEBPA to be used as a treatment to enhance the efficacy of bone marrow transplant in MPS I mice, we assessed the benefits of MTL-CEBPA treatment in NSG-MPS I mice (Jackson Lab). 8-week old NSG-MPS 1 mice were irradiated with 230cGy and subsequently underwent i.v transplantation with healthy CD34 + hematopoeitc stem cells (HSCs) isolated from human fetal liver tissue. Following successful engraftment confirmation (> 10% hCD45 + cells in blood at 18 weeks post-transplantation, Supplementary Fig. 2), we initiated a twice-weekly MTL-CEBPA treatment regimen to assess its long-term effects over a three-month period. Compared to the control groups (saline and NOV340-FLUC), animals in the MTL-CEBPA treated groups exhibited elevated IDUA activity levels throughout the study. While the increase was modest in weeks 1 and 2, IDUA activity peaked at week 3, reaching levels at least twofold higher than the control groups (158 nmol/h/mL in the MTL-CEBPA arm versus 88 nmol/h/mL in the NOV340-FLUC arm), Fig. 3 A. The impact of MTL-CEBPA treatment varied among different genotype groups by the end of the study period, with the most pronounced effect observed in the homozygous cohort, which relied entirely on donor cells for active iduronidase production. In contrast, the heterozygous and wildtype groups showed a comparatively less dramatic response to the treatment (Fig. 3 B). This differential response pattern suggests that MTL-CEBPA's efficacy may be particularly significant in cases where endogenous iduronidase production is absent or severely limited, highlighting its potential as a targeted therapeutic approach for specific genetic profiles. Relationship Between CEBPA and IDUA in Clinical Trial Samples To show clinical proof of concept that MTL-CEBPA administered IV to a human can lead to increased IDUA gene expression and IDUA enzyme activity in the circulation, human biospecimens from legacy clinical studies [NCT02716012] ( 16 , 17 ) that investigated the effects of MTLCEBPA in cancer patients, were leveraged and examined to determine the relationship between CEBPA and IDUA. Levels of IDUA and CEBPA protein were measured in monocytes samples collected from the TIMEPOINT clinical trial ( 17 ), where a positive correlation was observed (R 2 = 0.571) (Fig. 4 A). Further, legacy plasma samples from patients enrolled in the OUTREACH and TIMEPOINT clinical trial were tested for IDUA activity pre- and post-MTL-CEBPA administration (Fig. 4 B; note: due to biospecimen availability it was not possible to measure every timepoint in each patient). Approximately half of the patients had an increase in IDUA activity post MTL-CEBPA. Finally, we observed a trend that potentially indicates that patients with the lowest baseline plasma IDUA activity had the greatest increase (Fig. 4 C). Collectively, these data support the application of MTL-CEBPA in the clinic for the purposes of increasing IDUA activity in MPS I patients who have received HSCT. Discussion Clinical evidence demonstrates that even moderate boosts in IDUA enzyme activity can meaningfully improve outcomes in MPS I subtypes like Scheie and Hurler-Scheie syndromes. While current therapies focus on exogenous enzyme supply strategies (hematopoietic stem cell transplantation or recombinant IDUA), these approaches face limitations including suboptimal enzyme levels post-transplant and immunogenicity challenges with repeated ERT administration. Our research identified CEBPA as a novel transcriptional regulator of IDUA. RNA activation experiments confirmed this relationship across cellular models, showing CEBPA upregulation enhances IDUA activity in human fibroblasts and mesenchymal stem cells. Building on prior development of MTL-CEBPA (CEBPA-51 RNAa encapsulated in NOV340 liposomes) for HCC, we tested its potential in MPS I models. In humanised bone marrow-transplanted MPS I mice, MTL-CEBPA induced sustained IDUA elevation compared to baseline therapy. Retrospective analysis of clinical trial samples revealed on-target effects: increased CEBPA protein correlated with higher IDUA levels in monocytes, and > 50% of patients showed elevated plasma IDUA activity. Conclusions The collective evidence presented demonstrates that MTL-CEBPA represents a promising therapeutic strategy for addressing residual disease in hematopoietic stem cell transplant (HSCT)-treated MPS I patients. While donor-derived HSCT provides wild-type IDUA enzyme expression, substantial residual pathology persists in most recipients due to incomplete correction of glycosaminoglycan (GAG) accumulation in avascular tissues such as bone and cartilage, as well as in post-mitotic structures like the cornea ( 9 ). RNA activation (RNAa) therapeutics offer several distinct advantages in this context. Their favourable safety profile permits repeated dosing, which enables sustained therapeutic IDUA levels to address chronic GAG deposition. Furthermore, systemically administered RNAa may achieve better tissue penetration in anatomical sites that are typically refractory to HSCT-derived enzyme distribution. Compared to viral vector-based gene therapies, RNAa also benefits from streamlined manufacturing, reduced production costs, simplified intravenous administration, and a lower risk of insertional mutagenesis. Collectively, these characteristics position RNAa as a clinically adoptable solution for enhancing IDUA activity in MPS I, potentially bridging the therapeutic gap left by HSCT alone. Abbreviations CEBPA CCAAT enhancer binding protein alpha IDUA α-L-iduronidase MPSI mucopolysaccharidosis type GAGs Glycosaminoglycans RNAa RNA activation saCEBPA RNA activation to CEBPA HSCT Hematopoietic stem cell transplantation ERT Enzyme Replacement Therapy ChIP Chromatin Immunoprecipitation 4-MU 4-methylumbelliferyl alpha-L-iduronide ZGBT zygomatic bone thickness DEXA Dual-energy X-ray absorptiometry NOV340 Novosome 340 lipid nanoparticle Declarations Ethics statement All animal care and experimental procedures were conducted in accordance with protocols reviewed and approved by the City of Hope Institutional Animal Care and Use Committee (IACUC), under the principal investigator for this study, John Rossi (IACUC 23017). Human fetal liver tissue was obtained from Cercle Allocation Services, Inc., a nonprofit organization, in compliance with all applicable federal and state regulations. The vendor maintains its own Institutional Review Board (IRB) and adheres to human subject protection requirements. Consent for publication This manuscript does not contain data from any individual person. Data availability All data are contained within the manuscript Conflict of interests NH, K-WH, JR, RH, JN, VR, PS have equity in MiNA Therapeutics Ltd. K.V, K.SS, R.H, K.P, G.P, J.T, NR, P.A, JV, B.R and R.H are employees of MiNA Therapeutics Ltd. SAJ: received consulting fees for MiNA therapeutics, Orchard therapeutics and Sanofi Funding and acknowledgment: This study was funded by MiNA Therapeutics Ltd Authors' contributions V.R, M.P, KV, KS. S, R. H contributed to writing the manuscript. V.R, K.V, K.S.S, K.P, R.Hodsgon, K.P, G.P, J.T, S.J, N.F, P.A J.V, M-S.S, H.Li contributed to the invitro and in vivo data for the figures. 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Long-term efficacy and safety of laronidase in the treatment of mucopolysaccharidosis I. N Engl J Med. (2009); 361(6), 583-593. Setten RL, Lightfoot HL, Habib N, Rossi JJ. Development of MTL-CEBPA: Small Activating RNA Drug for Hepatocellular Carcinoma. Current Pharmaceutical Biotechnology (2018); 19 (8) 611-621. Sarker D, Plummer R, Meyer T, Sodergren MH, Basu B, Chee CE, Huang KW, Palmer DH, Ma YT, Evans TRJ, Spalding DC, Pai M, Sharma R, Pinato D, Spicer J, Hunter S, Kwatra V, Nicholls JP, Collin D, Nutbrown R, Glenny H, Fairbairn S, Reebye V, Voutila J, Dorman S, Andrikakou P, Lloyd P, Felstead S, Vasara J, Habib R, Wood C, Saetrom P, Huber HE, Blakey DC, Rossi JJ, Habib N. MTL-CEBPA, a Small Activating RNA Therapeutic Upregulating C/EBP-α, in Patients with Advanced Liver Cancer: A First-in-Human, Multicenter, Open-Label, Phase I Trial Clin Can Res (2020); 26(15): 3936-3946. Hashimoto A, Sarker D, Reebye V, Javis S, Sodergren MH, Kossenkov A, Sansviero E, Raulf N, Vasara J, Andrikakou P, Meyer T, Huang KW, Plummer R, Chee CE, Spalding D, Pai M, Khan S, Pinato DJ, Sharma R, Basu B, Palmer D, Ma YT, Evans J, Habib R, Martirosyan A, Elasri N, Reynaud A, Rossi JJ, Cobbold M, Habib, NA, Gabrilovich DI. Upregulation of C/EBPα Inhibits Suppressive Activity of Myeloid Cells and Potentiates Antitumor Response in Mice and Patients with Cancer. Clin Cancer Res (2021); 27(21): 5961-5978. Supplementary Files SupplementalInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7346641","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":500022350,"identity":"8d96d826-5d13-4b07-82f7-dbc5c78f2309","order_by":0,"name":"Vikash Reebye","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYLACxgYGBn5mKEeCaC2SzSRrMThArBaDA7wPH3zccVjO+DjzA4YfNQyJMxsIamE3Npx55rCx2WE2A8aeYwyJswnZItnAxibN23Y4cdthHgYG3gaGxHlEaGH//bftcP3mZh4Gxr/EaOFnYGNjZmw7nGDAzMPADLKFoMP4mdmYJXvPpBvOAPrlsMwxCWOC3mdjb2P88HOHtTx//+GHD9/U2MjOOEDIGkikQ+LxALERCQJ1RKscBaNgFIyCEQgA1fI4VxmG+oIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0811-5323","institution":"Imperial College London","correspondingAuthor":true,"prefix":"","firstName":"Vikash","middleName":"","lastName":"Reebye","suffix":""},{"id":500022351,"identity":"67413b0e-1a6d-4a7a-8ab2-3c693d5b5125","order_by":1,"name":"Konstantina S Stathaki","email":"","orcid":"","institution":"MiNA Therapeutics Ltd","correspondingAuthor":false,"prefix":"","firstName":"Konstantina","middleName":"S","lastName":"Stathaki","suffix":""},{"id":500022352,"identity":"ad364b31-dbb6-4434-b8ed-27f5310e43e9","order_by":2,"name":"Konstantinos Vanezis","email":"","orcid":"","institution":"MINA Therapeutics LTd","correspondingAuthor":false,"prefix":"","firstName":"Konstantinos","middleName":"","lastName":"Vanezis","suffix":""},{"id":500022353,"identity":"d84cd185-3663-43b6-a0d0-b357624b2583","order_by":3,"name":"Minsun Song","email":"","orcid":"","institution":"City of Hope Beckman Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Minsun","middleName":"","lastName":"Song","suffix":""},{"id":500022354,"identity":"7092b498-a97c-468b-954a-c4672b79f9d2","order_by":4,"name":"Michael J. 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Rossi","email":"","orcid":"","institution":"City of Hope Beckman Research Institute","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"J.","lastName":"Rossi","suffix":""},{"id":500022373,"identity":"f850d7d0-379d-4b4a-904a-45815c09bf3f","order_by":23,"name":"Shunji Tomatsu","email":"","orcid":"","institution":"Nemours Children's Hospital Delaware","correspondingAuthor":false,"prefix":"","firstName":"Shunji","middleName":"","lastName":"Tomatsu","suffix":""},{"id":500022374,"identity":"9f4e51ad-0bda-4ec0-96a8-8e9d4956a656","order_by":24,"name":"Shaukat Khan","email":"","orcid":"","institution":"Nemours Children's Hospital Delaware","correspondingAuthor":false,"prefix":"","firstName":"Shaukat","middleName":"","lastName":"Khan","suffix":""},{"id":500022375,"identity":"851f0dd3-4afa-4089-8d2d-17ab04f6a37f","order_by":25,"name":"Laura Furness","email":"","orcid":"","institution":"Manchester University NHS Foundation Trust","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Furness","suffix":""},{"id":500022376,"identity":"4b4efc56-3afc-4f9d-82c8-2642e2211c69","order_by":26,"name":"Robert Wynn","email":"","orcid":"","institution":"Manchester University","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Wynn","suffix":""},{"id":500022377,"identity":"94cd5c39-1d3d-449a-b04b-6c6cc1d5d4e0","order_by":27,"name":"Chester B. Whitley","email":"","orcid":"","institution":"University of Minnesota Medical Center Fairview: M Health Fairview University of Minnesota Medical Center East Bank","correspondingAuthor":false,"prefix":"","firstName":"Chester","middleName":"B.","lastName":"Whitley","suffix":""},{"id":500022378,"identity":"d0d891d5-dee1-4e33-8723-6793111c826f","order_by":28,"name":"Simon A. Jones","email":"","orcid":"","institution":"Manchester University NHS Foundation Trust","correspondingAuthor":false,"prefix":"","firstName":"Simon","middleName":"A.","lastName":"Jones","suffix":""},{"id":500022379,"identity":"491fbe1b-a518-47f8-9f52-ce969bb5d9b4","order_by":29,"name":"Nagy A. Habib","email":"","orcid":"","institution":"Imperial College of Science Technology and Medicine: Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Nagy","middleName":"A.","lastName":"Habib","suffix":""}],"badges":[],"createdAt":"2025-08-11 13:07:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7346641/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7346641/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89563442,"identity":"332a928a-7829-423f-8a35-58a7417699d4","added_by":"auto","created_at":"2025-08-21 10:30:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":403393,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003esaCEBPA transcriptionally activates IDUA.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eqRT-PCR for RNA from A549 cells showing CEBPA mRNA increases following treatment with saCEBPA relative to control (FLUC) and untreated cells. \u003cstrong\u003e(B)\u003c/strong\u003eqRT-PCR for RNA from A549 cells showing IDUA mRNA increases following treatment with saCEBPA relative to control (FLUC) and untreated cells. \u003cstrong\u003e(C)\u003c/strong\u003e. qRT-PCR for RNA from IMR90 fibroblast cells showing CEBPA mRNA increases following treatment with saCEBPA relative to control (FLUC) and untreated cells at a range of doses. \u003cstrong\u003e(D)\u003c/strong\u003e qRT-PCR for RNA from IMR90 cells showing IDUA mRNA increases following treatment with saCEBPA relative to control (FLUC) and untreated cells. \u003cstrong\u003e(E) \u003c/strong\u003emRNA analysis of CEBPA in MSC cells at 196 hours relative to control (FLUC) and untreated cells. \u003cstrong\u003e(F)\u003c/strong\u003e mRNA analysis of IDUA in MSC cells at 196 hours relative to control (FLUC) and untreated cells gene in same conditions as above. N=3 biological replicates. Error bars are -/+ SD and statistics are performed using unpaired \u003cem\u003et\u003c/em\u003e-tests (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001). In all figure panels saCEBPA denotes CEBPA-51.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7346641/v1/2cb207af58af6e9bfa1b9fd8.png"},{"id":89564679,"identity":"3f282477-6ad2-4741-9301-fde083d88196","added_by":"auto","created_at":"2025-08-21 10:38:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":501636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDurable increase of IDUA expression and activity in wild-type mice treated with MTL-CEBPA. (A) \u003c/strong\u003eCEBPA mRNA expression in bone marrow of mice treated with MTL-CEBPA at baseline and then followed bi-weekly for 10 weeks. \u003cstrong\u003e(B)\u003c/strong\u003eIDUA mRNA expression in the bone marrow and \u003cstrong\u003e(C)\u003c/strong\u003e IDUA enzyme activity in the plasma of MTL-CEBPA treated mice for a period of 10 weeks. Animals were treated with 2x2 mg/kg infusions at baseline, 24 hours apart. N=5 mice per group. Error bars are -/+ SD and statistics are performed using unpaired \u003cem\u003et\u003c/em\u003e-tests (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7346641/v1/d17fed61707cc11750ecc05b.png"},{"id":89563446,"identity":"8eebe048-38f4-48cc-8d6a-83d9ac057564","added_by":"auto","created_at":"2025-08-21 10:30:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":628376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDurable increase of IDUA enzymatic activity in MPS-1 following human CD45 engraftment. \u003c/strong\u003eIDUA enzyme activity in the serum of humanised wild-type, heterozygous, and homozygous mice MPS I mice were measured following baseline infusion of 4 mg/kg MTL-CEBPA \u003cstrong\u003e(A).\u003c/strong\u003e Summary at Week 3 of IDUA enzyme activity \u003cstrong\u003e(B).\u003c/strong\u003e Data are relative to saline treated and MTL-FLUC treated animals. Data are expressed as mean ± SD of the animals in each group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7346641/v1/14a029f0363812c05b6a432b.png"},{"id":89564681,"identity":"5442bb87-2033-49f7-bf7a-9d613906d233","added_by":"auto","created_at":"2025-08-21 10:38:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":588112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreases in IDUA enzymatic activity in cancer patients following treatment with MTL-CEBPA. \u003c/strong\u003eCorrelation between IDUA protein and CEBPA protein measured in WBCs from patients 24 hours post treatment with MTL-CBEPA \u003cstrong\u003e(A).\u003c/strong\u003e Comparison of IDUA activity in patients enrolled in the TIMEPOINT and OUTREACH clinical trials. Treatment infusions are on Day 1 and Day 8 \u003cstrong\u003e(B).\u003c/strong\u003e Representation of changes in IDUA activity in patients categories based on baseline IDUA activity \u003cstrong\u003e(C).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7346641/v1/1b36886b3ff5d320b3ec40bd.png"},{"id":91592102,"identity":"c283f05d-1b65-43e7-9be2-fd566c8e1a72","added_by":"auto","created_at":"2025-09-18 06:52:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3209915,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7346641/v1/810c7538-2798-42b3-ad90-3338e221e4b0.pdf"},{"id":89564691,"identity":"0390cf15-46b4-44ce-ac77-a846b6b200a5","added_by":"auto","created_at":"2025-08-21 10:38:22","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2653502,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7346641/v1/8ee43111e3b7a6cdb7648b0d.docx"}],"financialInterests":"","formattedTitle":"RNA Activation of CEBPA in PBMCs Enhances α-L-Iduronidase Expression A Translational Adjuvant Therapy for MPS I After Bone Marrow Transplantation","fulltext":[{"header":"Background","content":"\u003cp\u003eMucopolysaccharidosis type I (MPS I) is a rare, autosomal recessive lysosomal storage disorder caused by deficiency of the enzyme α-L-iduronidase (IDUA). This leads to accumulation of the glycosaminoglycans (GAGs), heparan sulfate and dermatan sulfate in lysosomes, resulting in multisystem organ dysfunction. Clinical features include cardiac valve thickening, cardiomyopathy, respiratory disease, cognitive decline, hepatosplenomegaly, skeletal dysplasia, and vision impairment. MPS I is classified by severity into severe (Hurler syndrome), intermediate (Hurler-Scheie), and attenuated (Scheie) forms, based on residual enzyme activity, age of onset, and clinical phenotype. Untreated, severe MPS I (Hurler syndrome) leads to death within the first decade, often earlier. Attenuated forms present later and progress more slowly, with variable life expectancy (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCurrently, two main treatments are available for MPS I: enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eERT involves weekly infusions of recombinant enzyme, which is costly but effective in improving somatic symptoms affecting organs such as the heart, lungs, liver, spleen, and kidneys (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, ERT cannot cross the blood-brain barrier, so it does not address neurological symptoms. Its efficacy is also limited in poorly vascularised tissues such as heart valves and cartilage, making it less effective for cardiac and skeletal manifestations. Long-term ERT can lead to the development of anti-IDUA antibodies, potentially causing immune reactions (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHSCT is recommended for patients diagnosed before 2.5 years of age, ideally before irreversible symptoms develop, and is often combined with ERT (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Donor stem cells provide a permanent source of enzyme, enabling cross-correction of deficient cells via the mannose-6-phosphate receptor. Although the blood-brain barrier is not fully permeable, HSCT can facilitate the delivery of enzyme-secreting cells to the brain, supporting ongoing neurodevelopment (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBoth therapies have limitations, but HSCT remains the only option with potential to address both somatic and neurological aspects of MPS I. Hematopoietic stem cell transplantation (HSCT) often fails to resolve glycosaminoglycan (GAG) accumulation in avascular tissues (such as bone and cartilage) and non-dividing structures (such as the cornea) in patients with severe mucopolysaccharidosis type I (MPS I). This persistent substrate accumulation results in ongoing skeletal deformities, cardiac complications, and a continued need for surgical interventions\u0026mdash;representing a significant unmet medical challenge (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePost-transplant outcomes are closely linked to leukocyte α-L-iduronidase (IDUA) activity: engraftment with wild-type donor cells leads to minimal tissue substrate deposition, whereas transplants from carrier donors are associated with higher levels of residual GAG accumulation (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Raised serum IDUA levels following HSCT are associated with reduced disease burden and a lower incidence of surgical procedures for carpal tunnel syndrome and spinal abnormalities (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Ex vivo lentiviral IDUA gene transduction in autologous HSCT has shown promise in achieving supra-physiological enzyme levels, potentially addressing residual disease following transplantation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, this irreversible procedure carries significant risks, particularly for patients who have previously undergone transplantation and have a limited pool of stem cells.\u003c/p\u003e\u003cp\u003eThis report presents a novel strategy to enhance the effectiveness of HSCT: RNA activation to naturally increase IDUA production from existing transplanted cells in patients with MPS I. This approach aims to boost enzymatic activity without genetic modification, potentially offering a safer and more effective solution for managing residual disease post-transplantation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture and transfection\u003c/h2\u003e\u003cp\u003eA549 cells, IMR90 cells, and MSCs (ATCC) were grown in ATCC-formulated RPMI medium supplemented with 10% fetal bovine serum following manufacturer\u0026rsquo;s protocols. Cells were maintained in a 5% CO2 incubator. Unless otherwise specified, for transfections, the cells were seeded at 1.5 x 10\u003csup\u003e5\u003c/sup\u003e cells per well in a 24-well plate and reverse transfected immediately after seeding with the indicated oligonucleotide concentration using 1 uL per well of Lipofectamine 2000 (Life Technologies).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIn silico PROMO analysis\u003c/h3\u003e\n\u003cp\u003ePROMO was used to screen the 5\u0026rsquo; regulatory DNA sequence of \u003cem\u003eIDUA\u003c/em\u003e gene \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3\u003c/span\u003e\u003cspan address=\"http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eRNA isolation and qPCR from cell lines\u003c/h3\u003e\n\u003cp\u003eRNA was isolated from cultured cells using the RNeasy Mini Kit (QIAGEN). RNA was quantitated using a QiaXPERT spectrophotometer (Qiagen), and 500 ng was reverse transcribed using the Quantitect Reverse Transcription Kit (QIAGEN). Relative expression levels were determined by qPCR using Quantifast SYBR Green Master Mix (QIAGEN) on an QuantStudio thermal cycler (LifeTechnologies). Quantitect Primer Assays (QIAGEN) were used to probe for transcript levels of IDUA and Actin B as the house keeping gene. Relative expression was determined using the DDCt method normalized to GAPDH expression.\u003c/p\u003e\n\u003ch3\u003eAssessment of MTL-CEBPA in wild-type mice\u003c/h3\u003e\n\u003cp\u003eAll work was carried out under UK Home Office legislation. Male C57Bl/6J (6 weeks old) were purchased from Charles River UK. Animals were each allocated a unique tag number. A random number generator was used to randomise the animals by tag numbers into each treatment groups. Animals were dosed intravenously via tail vein injection with 2 single doses, 24 hour apart of 2mg/kg of MTL-CEBPA or NOV340-FLUC (formulated CEBPA-51 (saCEBPA) and siFLUC previously described (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The Animals were weighed three times weekly. Bone marrow samples were taken at termination. The left femur was excised and the femoral cells flushed out using 1mL PBS. An equal volume of 70% ethanol was added and the solution was mixed by pipetting. The mixture was then transferred to RNeasy columns following the manufacturer\u0026rsquo;s instructions. After the wash steps, 30ul of water was used for eluting the RNA from the column. RNA Samples were quantified using the QIAGEN QIAxpert spectrophotometer according to the manufacturer\u0026rsquo;s protocol. QC of the samples was performed using the ScreenTape Assay for Tapestation (Agilent) according to the manufacturer\u0026rsquo;s recommended protocol. Samples with RIN greater than 7.0 were considered as meeting the threshold quality for qPCR amplification. Samples were subjected to reverse transcription using the Qiagen Quantitect RT Kit with 300\u0026ndash;500 ng RNA input which was standardised across the samples. Reverse transcription was performed according to manufacturer\u0026rsquo;s protocol. Oligo-dT primer (QIAGEN) was substituted for the default random hexamer/oligo-dT primer mix in the kit. In brief, for the RT reaction, 2\u0026micro;L of the gDNA wipeout was added to 12ul diluted RNA for 2 minutes at 42\u0026AElig;C followed by 1\u0026micro;L RT enzyme, 1\u0026micro;L oligodT primer, and 4\u0026micro;L 5xRT buffer for a total of 20\u0026micro;L each sample. Following the RT step, 140\u0026micro;L of water was added to each cDNA sample for an 8-fold dilution prior to qPCR amplification.\u003c/p\u003e\n\u003ch3\u003eqPCR\u003c/h3\u003e\n\u003cp\u003eSYBR qPCR was performed for the analysis of CEBPA gene expression. Relative gene expression was measured in cDNA samples by qPCR using the ThermoFisher PowerUp SYBR Green Master Mix on a QuantStudio 5 qPCR machine using QIAGEN QuantiTect murine primer assays for CEBPA and 4 housekeeping genes: GAPDH, HPRT, B2M, ACTB. Reactions were run in triplicate with each 10\u0026micro;L reaction volume composed of the following: 1\u0026micro;L primer assay, 5\u0026micro;L 2x SYBR Green mastermix, 4\u0026micro;L diluted cDNA from above. The following cycling conditions were used: initial heat activation step 2 minutes @95\u003csup\u003eo\u003c/sup\u003eC then 40 cycles comprising 1 second @95\u003csup\u003eo\u003c/sup\u003eC, 30 seconds @60\u003csup\u003eo\u003c/sup\u003eC followed by melt curve analysis. geNORM analysis revealed that the most consistent housekeeping genes for these samples were HPRT and B2M so a mean of these 2 was used for normalisation. Taqman qPCR was performed for the analysis of IDUA gene expression. Relative gene expression of IDUA was measured in cDNA samples by qPCR using the Thermo Fisher TaqMan Fast Advanced Master Mix according to the manufacturer\u0026rsquo;s protocol on a QuantStudio 5 qPCR machine with a Life Tech Taqman assay for murine IDUA. Reactions were run in triplicate with each 10\u0026micro;L reaction volume composed of the following: 0.5\u0026micro;L primer assay, 5\u0026micro;L 2x SYBR Green mastermix, 4\u0026micro;L diluted cDNA from above, 0.5\u0026micro;L water. The following cycling conditions were used: initial heat activation step 20 seconds @95\u003csup\u003eo\u003c/sup\u003eC then 40 cycles comprising 1 second @95 \u003csup\u003eo\u003c/sup\u003eC, 20 seconds @60 \u003csup\u003eo\u003c/sup\u003eC. The maximum amount of blood (600\u0026micro;L -800\u0026micro;L) was collected by cardiac puncture and transferred to K2 EDTA treated tubes. Tubes were spun down at 300g for 5\u0026rsquo; at 4 \u003csup\u003e\u0026ordm;\u003c/sup\u003eC and the plasma aliquoted and stored at -80 \u003csup\u003e\u0026ordm;\u003c/sup\u003eC until analysis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eIDUA enzyme activity assay\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003ePreparation of serum from blood samples\u003c/h2\u003e\u003cp\u003eAfter blood was collected and allowed to clot, serum was separated following standard centrifugation guidelines (5000rpm for 15\u0026rsquo;). In duplicate, 25\u0026micro;L of serum was mixed with 25\u0026micro;L of 360\u0026micro;M 4-methylumbelliferyl alpha-L-iduronide (Glycosynthe) in 0.4M Formate buffer at pH3.5 in a microplate. Sample blanks were prepared by mixing 25\u0026micro;L 0.2% BSA in PBS with 25\u0026micro;L of 360\u0026micro;M 4-methylumbelliferyl alpha-L-iduronide in 0.4M Formate buffer at pH 3.5. A 10-point standard curve of 4-methylumbelliferone in 0.4M Formate buffer at pH 3.5 ranging from 0.04ug/mL to 0.4ug/mL was prepared at the same time. Plates were then protected from light and incubated for 30\u0026rsquo; at 37 \u003csup\u003e\u0026ordm;\u003c/sup\u003eC. The reaction was stopped by the addition of 200\u0026micro;L of glycine carbonate buffer. Samples were then read in a Biotek plate reader at 365nm excitation and 450nm emission. Enzyme activity was calculated by dividing activity in ng/h by mL of serum using the formula: Activity in nmol/h/ml where specific activity was summarised as ng/h/ml divided by FW of 4MU (176.17).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eassessment of MTL-CEBPA in humanised MPS-1 mice\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eGeneration of the MPS 1 Disease Mouse Model\u003c/h3\u003e\n\u003cp\u003eTo generate the MPS1 mouse model, breeding challenges were encountered due to reproductive limitations. Females homozygous for all three mutations (Idua\u003csup\u003eW392X\u003c/sup\u003e, scid, and Il2Rγ\u003csup\u003enull\u003c/sup\u003e) exhibited poor maternal behaviour, while males homozygous for the Idua\u003csup\u003eW392X\u003c/sup\u003e mutation, homozygous for scid, and hemizygous for the X-linked Il2Rγ\u003csup\u003enull\u003c/sup\u003e mutation demonstrated subfertility, leading to a higher incidence of non-productive breeding pairs. To maintain a viable colony, breeding strategies involved crossing females heterozygous for the Idua\u003csup\u003eW392X\u003c/sup\u003e mutation and homozygous for the scid and Il2Rγ\u003csup\u003enull\u003c/sup\u003e mutations with males heterozygous for the Idua\u003csup\u003eW392X\u003c/sup\u003e allele, homozygous for the scid mutation, and hemizygous for the X-linked Il2Rγ\u003csup\u003enull\u003c/sup\u003e mutation. NOD.Cg-Idua\u003csup\u003etm1Clk\u003c/sup\u003e Prkdc\u003csup\u003escid\u003c/sup\u003e Il2rg\u003csup\u003etm1Wjl\u003c/sup\u003e/J (NSG-MPS1) heterozygous mice were obtained from Jackson Laboratories and incorporated into the breeding strategy.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eGenotyping by rhAmp SNP Assays\u003c/h2\u003e\u003cp\u003eTotal genomic DNA was extracted from mouse tail samples (5 mm of tail placed into a 1.5 mL tube) using DirectPCR Lysis Reagent (VivaGen Biotech), following the manufacturer\u0026rsquo;s protocol. For extraction, 200 \u0026micro;L of DNA lysis buffer and 2 \u0026micro;L of 20 mg/mL Proteinase K solution were added to each sample. The samples were incubated overnight at 55\u0026deg;C and then heated at 85\u0026deg;C for 45 minutes to inactivate Proteinase K. DNA was purified via phenol/chloroform extraction and ethanol precipitation, and the concentration was determined. Purified DNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use. Genotyping was performed using rhAmp technology, which employs dual-enzyme chemistry based on RNase H2-dependent PCR (rhPCR) and universal reporters. Each assay utilized two allele-specific primers along with a locus-specific primer. Synthetic gBlocks\u0026trade; gene fragments representing known genotypes served as controls for each SNP assay: one for the wild-type allele (WT), one for the mutant allele (MA), and a 1:1 mixture to represent the heterozygous genotype. Primers were designed using the rhAmp\u0026reg; Genotyping Design Tool (IDT; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://eu.idtdna.com/site/order/designtool/index/GENOTYPING_PREDESIGN\u003c/span\u003e\u003cspan address=\"https://eu.idtdna.com/site/order/designtool/index/GENOTYPING_PREDESIGN\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The rhAmp genotyping assay was performed according to the manufacturer\u0026rsquo;s instructions. Briefly, 2 \u0026micro;L (10 ng) of DNA sample was mixed with 5.3 \u0026micro;L of rhAmp Genotyping Mix [1 mL of rhAmp Genotyping Master Mix 2\u0026times; (Cat. no. 1076017; IDT) and 50 \u0026micro;L of rhAmp Reporter Mix (Cat. no. 1076028; IDT)] and 0.5 \u0026micro;L of custom rhAmp SNP assays (IDT), achieving a final volume of 10 \u0026micro;L. Standard samples and negative template controls used in the HRM analysis were also included.\u003c/p\u003e\u003cp\u003eThe SNP genotyping assays were prepared with 0.25 \u0026micro;L of rhAmp SNP Assays (20X), 2.65 \u0026micro;L of combined rhAmp Genotyping Master Mix (2X) and rhAmp Reporter Mix (40X), 0.10 \u0026micro;L of nuclease-free water, 2 \u0026micro;L of sample DNA, and 2 \u0026micro;L of control template (gBlocks fragment controls) or nuclease-free water for no-template controls. Reactions were conducted on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), and data were analyzed using CFX Maestro Software version 2.3 (Bio-Rad). Thermal cycling conditions followed the standard protocol: 95\u0026deg;C for 10 minutes, followed by 40 cycles at 95\u0026deg;C for 10 seconds, 60\u0026deg;C for 30 seconds, and 68\u0026deg;C for 20 seconds (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.idtdna.com/rhAmp-SNP-protocol\u003c/span\u003e\u003cspan address=\"http://www.idtdna.com/rhAmp-SNP-protocol\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed on 29 February 2024).\u003c/p\u003e\u003cp\u003eBi-allelic specificity was achieved using two probes, one labeled with Amidite-fluorescein (FAM\u0026trade;) dye and the other with Hexachloro-fluorescein (HEX\u0026trade;) dye (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These dyes were detected independently with appropriate excitation sources and emission filters at their respective wavelengths. All genomic DNA samples were quantified, and each specimen was classified as resistant (RR), susceptible (SS), or heterozygous (RS) based on the melting temperature obtained. The results were displayed as puncta on an XY scatter plot based on relative fluorescent units (RFU), facilitating allelic discrimination through CFX Maestro software (Bio-Rad Laboratories).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence 5\u0026rsquo; \u0026rarr; 3\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eQuencher\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCommon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCT GGG GAT TCC TTC CAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWild type Reverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGGC ACA GTG ACC CAG GAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMutant Reverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGGC TCT ATG GCT TCT GAG G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWT probe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTG CCA AGG TCA CCA ATG T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFAM\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMUT probe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCT GCC AAG GTC ACG AG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHEX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferent excitation and emission spectra for fluorophores.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQuencher\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExcitation nm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEmission nm\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFAM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e450\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e533\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHEX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e483\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e568\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003e All animal care and experimental procedures were conducted in accordance with protocols reviewed and approved by the City of Hope Institutional Animal Care and Use Committee (IACUC), under the principal investigator for this study, John Rossi (IACUC 23017). Human fetal liver tissue was obtained from Cercle Allocation Services, Inc., a nonprofit organization, in compliance with all applicable federal and state regulations. The vendor maintains its own Institutional Review Board (IRB) and adheres to human subject protection requirements.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eHumanised Mouse Generation\u003c/h2\u003e\u003cp\u003eTo generate humanised NSG-MPS1 mice, two preparative methods were employed. First, 5\u0026ndash;8-week-old NSG-MPS1 mice were irradiated with 230 cGy and subsequently transplanted via intravenous (i.v.) injection with 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CD34\u0026thinsp;+\u0026thinsp;hematopoietic stem cells (HSCs) isolated from human fetal liver tissue. Alternatively, a second approach involved administering intraperitoneal (IP) injections of busulfan (35 mg/kg, adjusted by weight) for four consecutive days. On the fifth day, 130,000 CD34\u0026thinsp;+\u0026thinsp;human fetal liver stem cells were injected through the tail vein to establish humanization. Tail vein injections were performed using sterile strainers, with proper disposal of needles in biohazard containers without recapping. Engraftment was monitored through retro-orbital blood collection, and mice were observed for 10\u0026ndash;20 weeks post-transplantation. At 12 weeks, engraftment was evaluated by flow cytometry analysis of peripheral blood samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eBlood Collection and Analysis\u003c/h2\u003e\u003cp\u003eBaseline blood samples were collected for IDUA analysis. Subsequent blood samples were collected weekly for a duration of 7 weeks. The blood plasma IDUA enzyme activity was assessed using the 4-methylumbelliferyl-α-L-iduronide (4Mu) enzyme colorimetric assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Methods\u003c/h2\u003e\u003cp\u003eGraphPad Prism (version 8.1.1, GraphPad Software, Inc) was used to graph and analyse all data. \u003cem\u003eIn vitro\u003c/em\u003e transfection data was analysed for saCEBPA vs Fluc comparison using unpaired t-test with Welch\u0026rsquo;s correction. \u003cem\u003eIn vivo\u003c/em\u003e mouse and white blood cell patient data were analysed by two-way ANOVA.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCEBPA Activation Enhances IDUA Expression Across Multiple Cell Types\u003c/h2\u003e\u003cp\u003eTo gain a comprehensive understanding of IDUA biology across different cellular states within a lung tissue context, we utilised both transformed (A549) and non-transformed (IMR90) cell lines. We previously developed an RNAactivation (CEBPA51) designed to upregulate CEBPA expression, known as saCEBPA (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). To determine whether CEBPA is a bona fide activator of IDUA expression, we transfected A549 cells with saCEBPA. This resulted in a 3-fold increase in CEBPA mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and a corresponding 4-fold increase in IDUA mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) relative to control (FLUC). We then investigated the transcriptional effect of saCEBPA on IDUA expression in non-transformed IMR90 cells. Cells were transfected with increasing doses of saCEBPA (1nM, 5nM, 10nM, and 20nM). FLUC control was transfected at 20nM. Gene expression was assessed by RT-PCR at 72 hours post-transfection for both CEBPA and IDUA. Results showed CEBPA expression was significantly upregulated at 10nM and 20nM saCEBPA. Values were normalised to untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). IDUA gene expression also increased within the same dose range of 10nM to 20nM of saCEBPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To further explore the transcriptional relationship, we assessed the effect in mesenchymal stem cells. We observed increased expression of both IDUA and CEBPA, which persisted for seven days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These findings demonstrated that \u003cem\u003eCEBPA\u003c/em\u003e activation consistently lead to increased \u003cem\u003eIDUA\u003c/em\u003e expression across multiple cell types, including transformed lung cells, non-transformed lung fibroblasts, and mesenchymal stem cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eevidence that MTL-CEBPA increases IDUA expression and activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the \u003cem\u003ein vivo\u003c/em\u003e pharmacodynamic effect of saCEBPA on upregulation of IDUA enzyme activity, wild-type C57/BL6 female mice were administered 2x bolus injection of 2mg/kg of MTL-CEBPA via tail vein IV infusion over a 24-hour period so that the animals would have received a total of 4mg/kg of the RNAa (n\u0026thinsp;=\u0026thinsp;5 for each group). The effects of MTL-CEBPA was measured over a period of 10 weeks. The pharmacodynamic activity of MTL-CEBPA was determined by measuring CEBPA and IDUA mRNA in the bone marrow and IDUA activity in plasma with a fluorometric assay using 4-methylumbelliferyl alpha-L-iduronide (4-MU iduronide) as substrate. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, there was a significant increase in CEBPA mRNA expression at 4-, 6-, and 8-weeks post-administration relative to control. A significant increase in IDUA mRNA in bone marrow was observed at week 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Interestingly, we observed that the increase in IDUA expression was correlated with an increase in IDUA activity in plasma at 6 weeks post treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Collectively, these data support our \u003cem\u003ein vitro\u003c/em\u003e evidence that saCEBPA increases IDUA expression. Further, these data indicate that the increase in IDUA by MTL-CEBPA (the NOV340 in vivo carrier formulated RNAa) is detected in the circulation where it can facilitate cross-correction. Collectively, these data provide \u003cem\u003ein vivo\u003c/em\u003e POC that IV administration of MTL-CEBPA can increase the plasma levels of IDUA activity after treatment at baseline.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eeffects of MTL-CEBPA in humanised bone marrow transplanted MPS 1 mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the potential for MTL-CEBPA to be used as a treatment to enhance the efficacy of bone marrow transplant in MPS I mice, we assessed the benefits of MTL-CEBPA treatment in NSG-MPS I mice (Jackson Lab). 8-week old NSG-MPS 1 mice were irradiated with 230cGy and subsequently underwent i.v transplantation with healthy CD34\u003csup\u003e+\u003c/sup\u003e hematopoeitc stem cells (HSCs) isolated from human fetal liver tissue. Following successful engraftment confirmation (\u0026gt;\u0026thinsp;10% hCD45\u0026thinsp;+\u0026thinsp;cells in blood at 18 weeks post-transplantation, Supplementary Fig.\u0026nbsp;2), we initiated a twice-weekly MTL-CEBPA treatment regimen to assess its long-term effects over a three-month period. Compared to the control groups (saline and NOV340-FLUC), animals in the MTL-CEBPA treated groups exhibited elevated IDUA activity levels throughout the study. While the increase was modest in weeks 1 and 2, IDUA activity peaked at week 3, reaching levels at least twofold higher than the control groups (158 nmol/h/mL in the MTL-CEBPA arm versus 88 nmol/h/mL in the NOV340-FLUC arm), Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe impact of MTL-CEBPA treatment varied among different genotype groups by the end of the study period, with the most pronounced effect observed in the homozygous cohort, which relied entirely on donor cells for active iduronidase production. In contrast, the heterozygous and wildtype groups showed a comparatively less dramatic response to the treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThis differential response pattern suggests that MTL-CEBPA's efficacy may be particularly significant in cases where endogenous iduronidase production is absent or severely limited, highlighting its potential as a targeted therapeutic approach for specific genetic profiles.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRelationship Between\u003c/b\u003e \u003cb\u003eCEBPA\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eIDUA\u003c/b\u003e \u003cb\u003ein Clinical Trial Samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo show clinical proof of concept that MTL-CEBPA administered IV to a human can lead to increased \u003cem\u003eIDUA\u003c/em\u003e gene expression and IDUA enzyme activity in the circulation, human biospecimens from legacy clinical studies [NCT02716012] (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) that investigated the effects of MTLCEBPA in cancer patients, were leveraged and examined to determine the relationship between CEBPA and IDUA.\u003c/p\u003e\u003cp\u003eLevels of IDUA and CEBPA protein were measured in monocytes samples collected from the TIMEPOINT clinical trial (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), where a positive correlation was observed (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.571) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Further, legacy plasma samples from patients enrolled in the OUTREACH and TIMEPOINT clinical trial were tested for IDUA activity pre- and post-MTL-CEBPA administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; note: due to biospecimen availability it was not possible to measure every timepoint in each patient). Approximately half of the patients had an increase in IDUA activity post MTL-CEBPA. Finally, we observed a trend that potentially indicates that patients with the lowest baseline plasma IDUA activity had the greatest increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Collectively, these data support the application of MTL-CEBPA in the clinic for the purposes of increasing IDUA activity in MPS I patients who have received HSCT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eClinical evidence demonstrates that even moderate boosts in IDUA enzyme activity can meaningfully improve outcomes in MPS I subtypes like Scheie and Hurler-Scheie syndromes. While current therapies focus on exogenous enzyme supply strategies (hematopoietic stem cell transplantation or recombinant IDUA), these approaches face limitations including suboptimal enzyme levels post-transplant and immunogenicity challenges with repeated ERT administration.\u003c/p\u003e\u003cp\u003eOur research identified CEBPA as a novel transcriptional regulator of IDUA. RNA activation experiments confirmed this relationship across cellular models, showing CEBPA upregulation enhances IDUA activity in human fibroblasts and mesenchymal stem cells.\u003c/p\u003e\u003cp\u003eBuilding on prior development of MTL-CEBPA (CEBPA-51 RNAa encapsulated in NOV340 liposomes) for HCC, we tested its potential in MPS I models. In humanised bone marrow-transplanted MPS I mice, MTL-CEBPA induced sustained IDUA elevation compared to baseline therapy. Retrospective analysis of clinical trial samples revealed on-target effects: increased CEBPA protein correlated with higher IDUA levels in monocytes, and \u0026gt;\u0026thinsp;50% of patients showed elevated plasma IDUA activity.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe collective evidence presented demonstrates that MTL-CEBPA represents a promising therapeutic strategy for addressing residual disease in hematopoietic stem cell transplant (HSCT)-treated MPS I patients. While donor-derived HSCT provides wild-type IDUA enzyme expression, substantial residual pathology persists in most recipients due to incomplete correction of glycosaminoglycan (GAG) accumulation in avascular tissues such as bone and cartilage, as well as in post-mitotic structures like the cornea (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRNA activation (RNAa) therapeutics offer several distinct advantages in this context. Their favourable safety profile permits repeated dosing, which enables sustained therapeutic IDUA levels to address chronic GAG deposition. Furthermore, systemically administered RNAa may achieve better tissue penetration in anatomical sites that are typically refractory to HSCT-derived enzyme distribution. Compared to viral vector-based gene therapies, RNAa also benefits from streamlined manufacturing, reduced production costs, simplified intravenous administration, and a lower risk of insertional mutagenesis. Collectively, these characteristics position RNAa as a clinically adoptable solution for enhancing IDUA activity in MPS I, potentially bridging the therapeutic gap left by HSCT alone.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCEBPA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCCAAT enhancer binding protein alpha\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIDUA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eα-L-iduronidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMPSI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emucopolysaccharidosis type\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGAGs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlycosaminoglycans\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRNAa\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRNA activation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003esaCEBPA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRNA activation to CEBPA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHSCT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHematopoietic stem cell transplantation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eERT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEnzyme Replacement Therapy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eChIP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChromatin Immunoprecipitation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e4-MU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e4-methylumbelliferyl alpha-L-iduronide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eZGBT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ezygomatic bone thickness\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDEXA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDual-energy X-ray absorptiometry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNOV340\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNovosome 340 lipid nanoparticle\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal care and experimental procedures were conducted in accordance with protocols reviewed and approved by the City of Hope Institutional Animal Care and Use Committee (IACUC), under the principal investigator for this study, John Rossi (IACUC 23017). Human fetal liver tissue was obtained from Cercle Allocation Services, Inc., a nonprofit organization, in compliance with all applicable federal and state regulations. The vendor maintains its own Institutional Review Board (IRB) and adheres to human subject protection requirements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript does not contain data from any individual person.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are contained within the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNH, K-WH, JR, RH, JN, VR, PS have equity in MiNA Therapeutics Ltd.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK.V, K.SS, R.H, K.P, G.P, J.T, NR, P.A, JV, B.R and R.H are employees of MiNA Therapeutics Ltd. SAJ: received consulting fees for MiNA therapeutics, Orchard therapeutics and Sanofi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding and acknowledgment:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by MiNA Therapeutics Ltd\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eV.R, M.P, KV, KS. S, R. H contributed to writing the manuscript. V.R, K.V, K.S.S, K.P, R.Hodsgon, K.P, G.P, J.T, S.J, N.F, P.A J.V, M-S.S, H.Li contributed to the invitro and in vivo data for the figures. J.N, S.H, P.S, B.R, R.H, K-W.H, F.F, J.R, S.T, L.O, P.O, C.R.W, C.B.W, L.F, S.A.J and N.H contributed to project management, manuscript revisions and key project concepts. All authors approved the final manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArn P, Bruce IA, Wraith JE, Travers H, Fallet S. Airway-related symptoms and surgeries in patients with mucopolysaccharidosis I. Ann Otol Rhinol Laryngo (2014); 124:198-205. \u003c/li\u003e\n\u003cli\u003eWraith JE, Clark LA, Beck M, Kolodny EH, Pastores GM, Muenzer J et al., Enzyme replacement therapy for mucopolysaccharidosis 1: aransomized, double-blinded, placebo-controlled, multinational study of recombinant human alpha-L-iduronidase (laronidase). Journal of Pediatrics (2004); 144: 581-588. \u003c/li\u003e\n\u003cli\u003ePastores GM. Laronidase (Aldurazyme\u0026reg;): enzyme replacement therapy for mucopolysaccharidosis type I. Expert Opinion on Biological Therapy (2008); 8 (7): 1003-1009.\u003c/li\u003e\n\u003cli\u003ede Ru MH , Boelens JJ, Das AM, Jones SA, van der Lee JH, Mahlaoui N et al., Enzyme replacement therapy and/or hematopoietic stem cell transplantation at diagnosis in patients with mucopolysaccharidosis type I: results of a European consensus procedure. Orphanet J Rare Dis (2011); 10 (6):55. \u003c/li\u003e\n\u003cli\u003eLangereis EJ, van Vlies N, Church HJ, Geskus RB, Hollak CE, Jones SA et al., Biomarker responses correlate with antibody status in mucopolysaccharidosis type I patients on long-term enzyme replacement therapy. Mol Genet Metab (2015); 114(2):129-37. \u003c/li\u003e\n\u003cli\u003eGomez-Ospina N, Scharenberg SG, Mostrel N, Bak RO, Mantri S, Quadros RM et al., Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type 1. Nature Communications (2019); 10: 4045.\u003c/li\u003e\n\u003cli\u003eWynn RF, Wraith JE, Mercer J, O\u0026rsquo;Meara A, Tylee K, Thornley M et al., Improved metabolic correction in patients with liposomal storage disease treated with hematopoietic stem cell transplant compared with enzyme replacement therapy. J Pediatr (2009); 154:609-611.\u003c/li\u003e\n\u003cli\u003eM, Wynn RF, Orchard PJ, O\u0026rsquo;Meara A, Veys P, Fischer A et al., Long-term outcome of Hurler syndrome patients after hematopoietic cell transplantation: an international multicentre study. Blood (2015); 125:2164-2172. \u003c/li\u003e\n\u003cli\u003eAldenhoven M, van den Broek BTA, Wynn RF, O\u0026rsquo;Meara A, Veys P, Rovelli A et al., Quality of life of Hurler syndrome patients after successful hematopoietic stem cell transplantation. Blood Adv (2017); 1(24): 2236-2242. \u003c/li\u003e\n\u003cli\u003eLum S.H, Stepien K.M, Ghosh. A, Broomfield A, Church H, Mercer J, Jones S, Wynn RF. Long term survival and cardiopulmonary outcome in children with Hurler syndrome after haematopoietic stem cell transplantation J Inherit Metab Dis. (2017); 40(3):455-460.\u003c/li\u003e\n\u003cli\u003eGentner B, Tucci F, Galimberti S, Fumagalli F, Pellegrin MD, Silvani P et al., Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome. N Engl J Med (2021) ;385:1929-1940. \u003c/li\u003e\n\u003cli\u003eVoutila J, Reebye V, Roberts T.C, Protopapa P, Andrikakou P, Blakey D.C, Habib R, Huber H, Saetrom P, Rossi J.J, Habib N.A. Development and Mechanism of Small Activating RNA Targeting CEBPA, a Novel Therapeutic in Clinical Trials for Liver Cancer. Mol Ther (2017); 25(12):2705-2714.\u003c/li\u003e\n\u003cli\u003eReebye V, Huang K-W, Lin V, Jarvis S, Cutilas P, Dorman S, Ciriello S, Andrikakou P, Voutila J, Saetrom P, Mintz P.J, Reccia I, Rossi J.J, Huber H, Habib R, Kostomitsopoulos N, Blakey D.C, Habib N.A. Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer. Oncogene (2018); 37(24):3216-3228. \u003c/li\u003e\n\u003cli\u003eClarke, L. A., Wraith, J. E., Beck, M., Kolodny, E. H., Pastores, G. M., Muenzer, J., ... \u0026amp; Tylki-Szymańska, A. Long-term efficacy and safety of laronidase in the treatment of mucopolysaccharidosis I. N Engl J Med. (2009); 361(6), 583-593. \u003c/li\u003e\n\u003cli\u003eSetten RL, Lightfoot HL, Habib N, Rossi JJ. Development of MTL-CEBPA: Small Activating RNA Drug for Hepatocellular Carcinoma. Current Pharmaceutical Biotechnology (2018); 19 (8) 611-621.\u003c/li\u003e\n\u003cli\u003eSarker D, Plummer R, Meyer T, Sodergren MH, Basu B, Chee CE, Huang KW, Palmer DH, Ma YT, Evans TRJ, Spalding DC, Pai M, Sharma R, Pinato D, Spicer J, Hunter S, Kwatra V, Nicholls JP, Collin D, Nutbrown R, Glenny H, Fairbairn S, Reebye V, Voutila J, Dorman S, Andrikakou P, Lloyd P, Felstead S, Vasara J, Habib R, Wood C, Saetrom P, Huber HE, Blakey DC, Rossi JJ, Habib N. MTL-CEBPA, a Small Activating RNA Therapeutic Upregulating C/EBP-\u0026alpha;, in Patients with Advanced Liver Cancer: A First-in-Human, Multicenter, Open-Label, Phase I Trial Clin Can Res (2020); 26(15): 3936-3946.\u003c/li\u003e\n\u003cli\u003eHashimoto A, Sarker D, Reebye V, Javis S, Sodergren MH, Kossenkov A, Sansviero E, Raulf N, Vasara J, Andrikakou P, Meyer T, Huang KW, Plummer R, Chee CE, Spalding D, Pai M, Khan S, Pinato DJ, Sharma R, Basu B, Palmer D, Ma YT, Evans J, Habib R, Martirosyan A, Elasri N, Reynaud A, Rossi JJ, Cobbold M, Habib, NA, Gabrilovich DI. Upregulation of C/EBP\u0026alpha; Inhibits Suppressive Activity of Myeloid Cells and Potentiates Antitumor Response in Mice and Patients with Cancer. Clin Cancer Res (2021); 27(21): 5961-5978.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Therapeutic oligonucleotides. RNA activation, Small activating RNA, Hurler Syndrome, MPS-I, α-L-iduronidase, Enzyme deficiency","lastPublishedDoi":"10.21203/rs.3.rs-7346641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7346641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eHurler syndrome, the most severe form of mucopolysaccharidosis type I (MPS I), is a rare genetic disorder caused by mutations in the IDUA gene, leading to a deficiency of the α-L-iduronidase enzyme. While current treatments offer some benefits, there remains a significant unmet medical need. We have identified a potential new therapeutic approach using MTL-CEBPA, a drug that upregulates the transcription factor \u003cem\u003eCEBPA\u003c/em\u003e, which in turn regulates \u003cem\u003eIDUA\u003c/em\u003e expression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e studies demonstrated significant upregulation of \u003cem\u003eIDUA\u003c/em\u003e in various cell lines following MTL-CEBPA treatment. \u003cem\u003eIn vivo\u003c/em\u003e experiments in both wild-type and MPS I mouse models showed a two-fold increased \u003cem\u003eIDUA\u003c/em\u003e expression and enzyme activity for up to four weeks after a single dose. Analysis of archival samples from cancer patients treated with MTL-CEBPA revealed a correlation between increased \u003cem\u003eCEBPA\u003c/em\u003e expression and \u003cem\u003eIDUA\u003c/em\u003e expression, with approximately half of the patients showing elevated plasma IDUA enzyme activity post-treatment.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThese findings provide proof-of-concept evidence supporting the potential use of MTL-CEBPA as a treatment for MPS I patients. We propose that this therapeutic oligonucleotide approach offers a favourable safety profile, allowing for multiple dosing to maintain elevated IDUA enzyme activity in bone marrow transplanted patients over extended periods, potentially addressing the limitations of current treatments and improving patient outcomes.\u003c/p\u003e","manuscriptTitle":"RNA Activation of CEBPA in PBMCs Enhances α-L-Iduronidase Expression A Translational Adjuvant Therapy for MPS I After Bone Marrow Transplantation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 10:30:17","doi":"10.21203/rs.3.rs-7346641/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ac3ace62-b1e2-4eb6-9ce9-f8a31dec0dd4","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-18T06:44:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-21 10:30:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7346641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7346641","identity":"rs-7346641","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}