Improved therapeutic efficacy in two mouse models of methylmalonic acidemia (MMA) using a second-generation mRNA therapy.

Isolated methylmalonic acidemia/aciduria (MMA) due to MMUT enzyme deficiency is an ultra-rare pediatric disease with high morbidity and mortality, with no approved disease-altering therapies. Previous publications showed that systemic treatment with a codon-optimized mRNA encoding wild-type human MMUT (MMUT) is a promising strategy for treatment of MMA. We developed a second-generation drug product, mRNA-3705, comprised of an mRNA encoding the MMUT enzyme formulated in a lipid nanoparticle (LNP) with incorporation of enhancements over the previous clinical candidate mRNA-3704. Both drug products produced functional MMUT in rat livers when dosed IV, and showed long-term safety and efficacy in two mouse models of MMA. mRNA-3705 produced 2.1-3.4-fold higher levels of hepatic MMUT protein expression than the first-generation drug product mRNA-3704 when given at an identical dose level, which resulted in greater and more sustained reductions in plasma methylmalonic acid. The data presented herein provide comprehensive preclinical pharmacology to support the clinical development of mRNA-3705.

Clinical outcomes and survival rates of a uncemented modular revision stem system in hip arthroplasty: a 10-year single-institution study on a frail population.

INTRODUCTION: The increasing prevalence of primary hip arthroplasty has led to a parallel rise in revision cases. Femoral revision often entails compromised bone integrity, requiring consideration of various solutions for optimal reconstructive options. Despite technological advancements, there is limited evidence on the clinical outcomes of the latest modular revision stems. This study aimed to evaluate the clinical outcomes and survival rates of next generation uncemented modular revision stem in patients undergoing hip revision surgery. MATERIALS AND METHODS: This retrospective single-center study assessed the survival and failure causes of a specific uncemented modular stem in 48 patients undergoing hip revision surgery between 2012 and 2022. Data included preoperative parameters, surgical details, and postoperative outcomes measured through clinical and radiographic assessments. Forty-eight patients (25 males, 23 females; mean age 72 years) were included, with a mean Charlson Comorbidity Index of 5. Preoperative diagnoses varied, with periprosthetic joint infection (PJI) being the most common (45.8%), followed by periprosthetic fractures (27.1%). Partial revisions occurred in 60.4%, total revisions in 39.6%. According to Paprosky classification of femoral bone loss, type II and III were the most represented, respectively 35.4% and 50%. RESULTS: At a mean follow-up of 4.6 years, stem survival was 92.5%. Complications (20%) included dislocation, PJI, fracture, and loosening; the overall reoperation rate was 12.5%. The SF-12 physical score was 43.6, while the mental score was 51.1. The HOOS score was 71.8, and the HHS score was 71.4. Radiographic analysis identified nonprogressive osteolysis in 15.1% of patients. CONCLUSIONS: This study on this uncemented modular revision stem demonstrated favorable outcomes in an elder fragile population with moderate to severe femoral bone loss. The implant's modularity provides versatility in addressing various defects, without any implant breakage observed during the study period. Literature comparison highlighted similar outcomes despite sample size differences. The promising results warrant continued investigation into the long-term survivorship of this modular stem system.

Lipid Nanoparticle-Mediated Delivery of mRNA Into the Mouse and Human Retina and Other Ocular Tissues.

Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in LNPs with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA-carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and RPE. Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mouse eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge, we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time. Translational Relevance: Ocular gene-replacement therapies using non-viral vector methods are a promising alternative to adeno-associated virus (AAV) vectors. Our studies show that mRNA LNP delivery can be used to transfect retinal cells in both mouse and human tissues without inducing significant inflammation. This methodology could be used to transfect retinal cell lines, tissue explants, mice, or potentially as gene-replacement therapy in a clinical setting in the future.

Unraveling epigenomic signatures and effectiveness of electroconvulsive therapy in treatment-resistant depression patients: a prospective longitudinal study.

BACKGROUND: Electroconvulsive therapy (ECT) benefits patients with treatment-resistant depression (TRD), but the underlying biological processes are unclear. We conducted an epigenome-wide association study in 32 TRD patients undergoing ECT to depict ECT-associated methylation changes. Illness severity and ECT outcomes were assessed with the Montgomery-Åsberg Depression Rating Scale at baseline (T0) and 1 month after its end (T1). Methylation was profiled at T0 and T1 with the Illumina Infinium Methylation EPIC BeadChip array. RESULTS: Longitudinal T0-T1 analyses showed 3 differentially methylated probes (DMPs) with nominal p values ≤ 10-5, with 2 annotated in the genes CYB5B and PVRL4. Including covariates, we found 4 DMPs for symptoms variation, annotated in FAM20C, EPB41, OTUB1 and ADARB1, and 3 DMPs for response status, with 2 annotated in IQCE and FAM20C. Regional analysis revealed 54 differentially methylated regions (DMRs) with nominal p value area ≤ 0.05, with 9 presenting adjusted p-value area ≤ 0.10, annotated in MCF2L, SLC25A24, RUNX3, MIR637, FOXK2, FAM180B, POU6F1, ALS2CL and CCRL2. Considering covariates, we found 21 DMRs for symptoms variation and 26 DMRs for response (nominal p value area ≤ 0.05), with 4 presenting adjusted p-value area ≤ 0.10 for response, annotated in SNORD34, NLRP6, GALNT2 and SFT2D3. None remained significant after false discovery rate correction. Notably, ADARB1 variants are associated with suicide attempt in patients with psychiatric disorders, and SLC25A24 relates to conduct disorder. Several DMPs and DMRs are annotated in genes associated with inflammatory/immune processes. Longitudinal analyses on females (n = 22) revealed statistically significant DMRs (adjusted p value area ≤ 0.05) and trend-significant DMRs (adjusted p value area ≤ 0.07) for symptoms variation and response status, annotated in genes related to psychiatric disorders (ZFP57, POLD4, TRIM10, GAS7, ADORA2A, TOLLIP), trauma exposure (RIPOR2) and inflammatory/immune responses (LAT, DLX4, POLD4, FAM30A, H19). Pathway analysis on females revealed enrichment for transcriptional activity, growth factors, DNA maintenance, and immune pathways including IRF7 and IRF2. CONCLUSION: Although no significant results were found for the whole cohort, the study provides insights into ECT-associated methylation changes, highlighting DMPs and DMRs related to ECT outcomes. Analyses on females revealed significant DMRs and pathways related to psychiatric disorders and inflammatory/immune processes.

South Tyrol Healthcare Company: A Look at a Peculiar Model of Claims Management in Italy and Analysis of Its Last 11 Years.

BACKGROUND: Alternative Disputes Resolution (ADR) systems are becoming increasingly important tools in recent years for the management and resolution of health responsibility cases, but their dissemination and efficiency are still poorly described. The purpose of this paper is to present an ADR system in the autonomous province of Bolzano: the Conciliation Commission. METHODS: systematic collection of all claims of the South Tyrol Sanitary Service that were dealt with in the Conciliation Commission from 1 January 2012 to 31 December 2022. RESULTS: closing times of the applications received turn out to be less than a year in 63.8% of the cases, with a number of cases managed rather stably in the time, even if minimal if compared to the number of complaints received to the South Tirol Health Service. Only 5.3% of the application continued the legal process before a civil court. CONCLUSIONS: the Conciliation Commission of South Tirol appears to be an excellent instrument for the resolution of disputes in the healthcare field, with rapid resolution times and little to zero costs for the healthcare company, a public health institution. Despite its effectiveness, it seems to be a tool that is still little-known in South Tyrol.

Mid-Term Outcomes of a Short Modular Neck-Preserving Cementless Hip Stem: A Retrospective Study With a 6-Year Minimum Follow-Up.

Background: The neck-preserving cementless short stem represents a valid therapeutic option for total hip replacement in high-functional-demand patients, but few studies are available about the use of modularity in the last-generation short stem. The aim of the study was to evaluate the mid-term survival of a specific implant design that combines partial collum short hip stem with neck modularity; assessing the functional status was the second endpoint. Methods: A retrospective single-center cohort study was conducted on 75 patients aged 35 to 80 years, with a minimum 6-year follow-up. Patients with neurological/rheumatic pathologies and previous hip surgeries were excluded. All the patients underwent total hip replacement with a short modular neck-preserving cementless hip stem. Clinical outcomes, complications, revisions, and the Western Ontario and McMaster Universities Osteoarthritis Index, Harris hip score, and Short Form 12-Item Health Survey (SF-12) questionnaires were evaluated. The results were compared with healthy population's data extracted from the literature, stratified by age. Results: The Kaplan-Meier analysis revealed a 10-year implant survival rate of 96.7%, coupled with a revision rate of 1.3%. Results showed a Harris hip score and physical SF-12 significantly lower and a mental SF-12 higher when compared to healthy population. No statistically significant differences emerged when comparing groups based on neck modularity. Conclusions: The short modular neck-preserving cementless hip stem emerged as a reasonable choice for patients with elevated functional demands, ensuring good clinical outcomes while preserving bone integrity. The use of a modular neck in short stems didn't show any mechanical problems in the mid-term.

Characterizing the mechanism of action for mRNA therapeutics for the treatment of propionic acidemia, methylmalonic acidemia, and phenylketonuria.

Messenger RNA (mRNA) therapeutics delivered via lipid nanoparticles hold the potential to treat metabolic diseases caused by protein deficiency, including propionic acidemia (PA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Herein we report results from multiple independent preclinical studies of mRNA-3927 (an investigational treatment for PA), mRNA-3705 (an investigational treatment for MMA), and mRNA-3210 (an investigational treatment for PKU) in murine models of each disease. All 3 mRNA therapeutics exhibited pharmacokinetic/pharmacodynamic (PK/PD) responses in their respective murine model by driving mRNA, protein, and/or protein activity responses, as well as by decreasing levels of the relevant biomarker(s) when compared to control-treated animals. These preclinical data were then used to develop translational PK/PD models, which were scaled allometrically to humans to predict starting doses for first-in-human clinical studies for each disease. The predicted first-in-human doses for mRNA-3927, mRNA-3705, and mRNA-3210 were determined to be 0.3, 0.1, and 0.4 mg/kg, respectively.

A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate.

Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.

Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia.

Despite the implementation of lifesaving newborn screening programs and a galactose-restricted diet, many patients with classic galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that the administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which decreased not only galactose-1 phosphate (gal-1P) in the liver but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, noninvasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of galactosemia. Although our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that targets the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety.

DNA methylation changes in association with trauma-focused psychotherapy efficacy in treatment-resistant depression patients: a prospective longitudinal study.

Background: Stressful events increase the risk for treatment-resistant depression (TRD), and trauma-focused psychotherapy can be useful for TRD patients exposed to early life stress (ELS). Epigenetic processes are known to be related to depression and ELS, but there is no evidence of the effects of trauma-focused psychotherapy on methylation alterations.Objective: We performed the first epigenome-wide association study to investigate methylation changes related to trauma-focused psychotherapies effects in TRD patients.Method: Thirty TRD patients assessed for ELS underwent trauma-focused psychotherapy, of those, 12 received trauma-focused cognitive behavioural therapy, and 18 Eye Movement Desensitization and Reprocessing (EMDR). DNA methylation was profiled with Illumina Infinium EPIC array at T0 (baseline), after 8 weeks (T8, end of psychotherapy) and after 12 weeks (T12 - follow-up). We examined differentially methylated CpG sites and regions, as well as pathways analysis in association with the treatment.Results: Main results obtained have shown 110 differentially methylated regions (DMRs) with a significant adjusted p-value area associated with the effects of trauma-focused psychotherapies in the entire cohort. Several annotated genes are related to inflammatory processes and psychiatric disorders, such as LTA, GFI1, ARID5B, TNFSF13, and LST1. Gene enrichment analyses revealed statistically significant processes related to tumour necrosis factor (TNF) receptor and TNF signalling pathway. Stratified analyses by type of trauma-focused psychotherapy showed statistically significant adjusted p-value area in 141 DMRs only for the group of patients receiving EMDR, with annotated genes related to inflammation and psychiatric disorders, including LTA, GFI1, and S100A8. Gene set enrichment analyses in the EMDR group indicated biological processes related to inflammatory response, particularly the TNF signalling pathway.Conclusion: We provide preliminary valuable insights into global DNA methylation changes associated with trauma-focused psychotherapies effects, in particular with EMDR treatment.

Stressful events increase treatment-resistant depression, and trauma-focused psychotherapy can be useful for these patients.Epigenome-wide data shows changes associated with trauma-focused psychotherapies, especially eye movement desensitization and reprocessing therapy, in treatment-resistant depression patients.Genes and biological pathways related to inflammatory and immune systems are among the most statistically significant results.

CSF-1R in Cancer: More than a Myeloid Cell Receptor.

Colony-stimulating factor 1 receptor (CFS-1R) is a myeloid receptor with a crucial role in monocyte survival and differentiation. Its overexpression is associated with aggressive tumors characterized by an immunosuppressive microenvironment and poor prognosis. CSF-1R ligands, IL-34 and M-CSF, are produced by many cells in the tumor microenvironment (TME), suggesting a key role for the receptor in the crosstalk between tumor, immune and stromal cells in the TME. Recently, CSF-1R expression was reported in the cell membrane of the cancer cells of different solid tumors, capturing the interest of various research groups interested in investigating the role of this receptor in non-myeloid cells. This review summarizes the current data available on the expression and activity of CSF-1R in different tumor types. Notably, CSF-1R+ cancer cells have been shown to produce CSF-1R ligands, indicating that CSF-1R signaling is positively regulated in an autocrine manner in cancer cells. Recent research demonstrated that CSF-1R signaling enhances cell transformation by supporting tumor cell proliferation, invasion, stemness and drug resistance. In addition, this review covers recent therapeutic strategies, including monoclonal antibodies and small-molecule inhibitors, targeting the CSF-1R and designed to block the pro-oncogenic role of CSF-1R in cancer cells.

mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria.

The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [18F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.

Pancreatojejunostomy: standing on the shoulders of giants. A single centre retrospective analysis.

Gaining experience in pancreatic surgery could be demanding especially when minimally invasive approach is used. Pancreatojejunostomy (PJ) is one of the most critical steps during pancreatoduodenectomy (PD). Our aim was to investigate the impact of a surgeon's experience in performing PJ, especially in a subgroup of patients undergoing laparoscopic PD (LPD). Data of consecutive patients undergoing PD from 2017 to 2022 were prospectively collected and retrospectively analyzed. Patients were divided into two groups: M group included patients in which PJ was performed by an experienced surgeon, D group included those receiving PJ by a less experienced one. The groups were compared in terms of postoperative outcomes. 187 patients were selected (157 in group M and 30 in group D). The cohorts differed in terms of median age (68 vs 74 years, p = 0.016), and previous abdominal surgery (41.4% vs 66.7%, p = 0.011), while no difference was found regarding risk of postoperative pancreatic fistula (POPF). The groups did not differ in terms of surgical outcomes. POPF rate was 15.9% and 10% in the M and D group (p = 0.578), respectively. Among patients undergoing laparoscopic PJ POPF rate was 16.0% and 17.7% in the M and D group (p = 0.867), respectively, without difference. No difference was found in terms of POPF in patients undergoing PD independently from the surgeon who performed the PJ, even during LPD. Moderate/high FRS, BMI > 30 kg/m2 and male sex, but not the surgeon who performed the PJ anastomosis, were independent predictors of POPF.

The incidence of movement disorder increases with age and contrasts with subtle and limited neuroimaging abnormalities in argininosuccinic aciduria.

Argininosuccinate lyase (ASL) is integral to the urea cycle detoxifying neurotoxic ammonia and the nitric oxide (NO) biosynthesis cycle. Inherited ASL deficiency causes argininosuccinic aciduria (ASA), a rare disease with hyperammonemia and NO deficiency. Patients present with developmental delay, epilepsy and movement disorder, associated with NO-mediated downregulation of central catecholamine biosynthesis. A neurodegenerative phenotype has been proposed in ASA. To better characterise this neurodegenerative phenotype in ASA, we conducted a retrospective study in six paediatric and adult metabolic centres in the UK in 2022. We identified 60 patients and specifically looked for neurodegeneration-related symptoms: movement disorder such as ataxia, tremor and dystonia, hypotonia/fatigue and abnormal behaviour. We analysed neuroimaging with diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) in an individual with ASA with movement disorders. We assessed conventional and DTI MRI alongside single photon emission computer tomography (SPECT) with dopamine analogue radionuclide 123 I-ioflupane, in Asl-deficient mice treated by hASL mRNA with normalised ureagenesis. Movement disorders in ASA appear in the second and third decades of life, becoming more prevalent with ageing and independent from the age of onset of hyperammonemia. Neuroimaging can show abnormal DTI features affecting both grey and white matter, preferentially basal ganglia. ASA mouse model with normalised ureagenesis did not recapitulate these DTI findings and showed normal 123 I-ioflupane SPECT and cerebral dopamine metabolomics. Altogether these findings support the pathophysiology of a late-onset movement disorder with cell-autonomous functional central catecholamine dysregulation but without or limited neurodegeneration of dopaminergic neurons, making these symptoms amenable to targeted therapy.

Induction of long-term tolerance to a specific antigen using anti-CD3 lipid nanoparticles following gene therapy.

Development of factor VIII (FVIII) inhibitors is a serious complication in the treatment of hemophilia A (HemA) patients. In clinical trials, anti-CD3 antibody therapy effectively modulates the immune response of allograft rejection or autoimmune diseases without eliciting major adverse effects. In this study, we delivered mRNA-encapsulated lipid nanoparticles (LNPs) encoding therapeutic anti-CD3 antibody (αCD3 LNPs) to overcome the anti-FVIII immune responses in HemA mice. It was found that αCD3 LNPs encoding the single-chain antibodies (Fc-scFv) can efficiently deplete CD3+ and CD4+ effector T cells, whereas αCD3 LNPs encoding double-chain antibodies cannot. Concomitantly, mice treated with αCD3 (Fc-scFv) LNPs showed an increase in the CD4+CD25+Foxp3+ regulatory T cell percentages, which modulated the anti-FVIII immune responses. All T cells returned to normal levels within 2 months. HemA mice treated with αCD3 LNPs prior to hydrodynamic injection of liver-specific FVIII plasmids achieved persistent FVIII gene expression without formation of FVIII inhibitors. Furthermore, transgene expression was increased and persistent following secondary plasmid challenge, indicating induction of long-term tolerance to FVIII. Moreover, the treated mice maintained their immune competence against other antigens. In conclusion, our study established a potential new strategy to induce long-term antigen-specific tolerance using an αCD3 LNP formulation.

Protocol for generation of a time-resolved cellular interactome during tissue remodeling in adult mice.

Efficient skeletal muscle regeneration necessitates fine-tuned coordination among multiple cell types through an intricate network of intercellular communication. We present a protocol for generation of a time-resolved cellular interactome during tissue remodeling. We describe steps for isolating distinct cell populations from skeletal muscle of adult mice after acute damage and extracting RNA from purified cells prior to the generation of RNA sequencing data. We then detail procedures for generating and deciphering a time- and lineage-resolved model of intercellular crosstalk. For complete details on the use and execution of this protocol, please refer to Groppa et al. (2023).1.

Hydrogel Coating versus Calcium Sulphate Beads as a Local Antibiotic Carrier for Debridement Procedures in Acute Periprosthetic Joint Infection: A Preliminary Study.

Periprosthetic joint infections (PJI) are among the most difficult complications to treat in orthopaedic surgery. Debridement, antibiotics, and implant retention (DAIR) represent an efficient strategy for acute PJI, especially when resorbable local antibiotic carriers and coatings are used. The aim of this pilot study was to evaluate the difference between using antibiotic-loaded hydrogel (ALH) and calcium sulphate (CS) beads in the DAIR procedure. We analysed 16 patients who had been treated since 2018 for acute PJI, namely eight patients with knee PJI (50%), seven with hip PJI (43.7%), and one with shoulder PJI (6.2%). Nine patients were treated with the Debridement, Antibiotic Coating and Retention of the Implant (DACRI) method, while seven were treated with the Debridement, Antibiotic Pearls, Retention of the Implant (DAPRI) method. We found no significant differences between the two groups in terms of age, sex, the American Society of Anesthesiologists risk score, Charlson Comorbidity Index, localisation, days from onset to diagnosis and pathogenesis. Furthermore, no differences were found between the DACRI and DAPRI groups in terms of infection control (15 patients, 93.75% with p = 0.36) and last C-Reactive Protein values (p = 0.26), with a mean follow-up of 26.1 ± 7.7 months. Treatment for one patient affected by knee Candida albicans PJI in the DACRI group was not successful. In conclusion, DAPRI and DACRI appear to be safe and effective treatments for PJIs. This evidence will encourage the development of new clinical research into local carriers and coatings for use in acute implant-associated infections.

GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals.

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.

Lipid nanoparticle-mediated delivery of mRNA into the mouse and human retina and other ocular tissues.

Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in lipid nanoparticles (LNPs) with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and retinal pigment epithelium (RPE). Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mice eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time.