Molecular Study of the Fukutin-Related Protein (FKRP) Gene in Patients from Southern Italy with Duchenne/Becker-like Phenotype.

Pathogenic variants localized in the gene coding for the Fukutin-Related Protein (FKRP) are responsible for Limb-Girdle Muscular Dystrophy type 9 (LGMDR9), Congenital Muscular Dystrophies type 1C (MDC1C), Walker-Warburg Syndrome (WWS), and Muscle-Eye-Brain diseases (MEBs). LGMDR9 is the fourth most common hereditary Limb Girdle Muscular Dystrophy in Italy. LGMDR9 patients with severe disease show an overlapping Duchenne/Becker phenotype and may have secondary dystrophin reduction on muscle biopsy. We conducted a molecular analysis of the FKRP gene by direct sequencing in 153 patients from Southern Italy (Calabria) with Duchenne/Becker-like phenotypes without confirmed genetic diagnosis. Mutational screening of the patients (112 men and 41 women, aged between 5 and 84 years), revealed pathogenic variants in 16 subjects. The most frequent variants identified were c.427C > A, p.R143S, and c.826C > A, p.L276I (NM_024301.5). The results obtained show that the Duchenne/Becker-like phenotype is frequently determined by mutations in the FKRP gene in our cohort and highlight the importance of considering LGMDR9 in the differential diagnosis of dystrophinopathies in Calabria. Finally, this study, which, to our knowledge, is the first conducted on Calabrian subjects, will contribute to the rapid identification and management of LGMDR9 patients.

Biosynthesis of Arabinoside from Sucrose and Nucleobase via a Novel Multi-Enzymatic Cascade.

Arabinoside and derived nucleoside analogs, a family of nucleoside analogs, exhibit diverse typically biological activities and are widely used as antibacterial, antiviral, anti-inflammatory, antitumor, and other drugs in clinical and preclinical trials. Although with a long and rich history in the field of medicinal chemistry, the biosynthesis of arabinoside has only been sporadically designed and studied, and it remains a challenge. Here, we constructed an in vitro multi-enzymatic cascade for the biosynthesis of arabinosides. This artificial biosystem was systematically optimized, involving an exquisite pathway design, NADP+ regeneration, meticulous enzyme selection, optimization of the key enzyme dosage, and the concentration of inorganic phosphate. Under the optimized conditions, we achieved 0.37 mM of vidarabine from 5 mM of sucrose and 2 mM of adenine, representing 18.7% of the theoretical yield. Furthermore, this biosystem also has the capability to produce other arabinosides, such as spongouridine, arabinofuranosylguanine, hypoxanthine arabinofuranoside, fludarabine, and 2-methoxyadenine arabinofuranoside, from sucrose, and corresponding nucleobase by introducing different nucleoside phosphorylases. Overall, our biosynthesis approach provides a pathway for the biosynthesis of arabinose-derived nucleoside analogs, offering potential applications in the pharmaceutical industry.

Uracil phosphoribosyltransferase is required to establish a functional cytochrome b6f complex.

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme and plants lacking this enzyme are strongly compromised in chloroplast function. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial to establish a fully functional photosynthesis as the RIESKE iron sulfur protein (PetC) is almost absent, leading to a block in photosynthetic electron transport. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis showed that protein homeostasis but not gene expression is most likely responsible for this observation and high light provoked an upregulation of protease levels, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery in UPP amiRNA lines. Strongly reduced PetC amounts were not only detected by immunoblot from mature plants but in addition in a de-etiolation experiment with young seedlings and are causing reduced high light-induced non-photochemical quenching Φ(NPQ) but increased unregulated energy dissipation Φ(NO). This impaired photosynthesis results in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline, and fumarate were found to be reduced. In sum, our work suggests that UPP assists in stabilization PetC during import, processing or targeting to the thylakoid membrane, or protects it against proteolytic degradation.

Characterization of a Xylosyltransferase from Panax notoginseng Catalyzing Ginsenoside 2'-O Glycosylation in the Biosynthesis of Notoginsenosides.

Notoginsenosides are important bioactive compounds from Panax notoginseng (Burk.) F. H. Chen, most of which have xylose in their sugar chains. However, the xylosyltransferases involved in the generation of notoginsenosides remain poorly understood, posing a bottleneck for further study of the biosynthesis of notoginsenosides. In this work, a new xylosyltransferase gene, PnUGT57 (named UGT94BW1), was identified from P. notoginseng, which has a distinct sequence and could catalyze the 2'-O glycosylation of ginsenosides Rh1 and Rg1 to produce notoginsenosides R2 and R1, respectively. We first characterized the optimal conditions for the PnUGT57 activity and its enzymatic kinetic parameters, and then, molecular docking and site-directed mutagenesis were performed to elucidate the catalytic mechanism of PnUGT57. Combined with the results of site-directed mutagenesis, Glu26, Ser266, Glu267, Trp347, Ser348, and Glu352 in PnUGT57 were identified as the key residues involved in 2'-O glycosylation of C-6 O-Glc, and PnUGT57R175A and PnUGT57G237A could significantly improve the catalytic activity of PnUGT57. These findings not only provide a new xylosyltransferase gene for augmenting the plant xylosyltransferase database but also identify the pivotal sites and catalytic mechanism of the enzyme, which would provide reference for the modification and application of xylosyltransferases in the future.

Periplasmic binding proteins Bug69 and Bug27 from Bordetella pertussis are in vitro high-affinity quinolinate binders with a potential role in NAD biosynthesis.

Bordetella's genome contains a large family of periplasmic binding proteins (PBPs) known as Bugs, whose functions are mainly unassigned. Two members, Bug27 and Bug69, have previously been considered potential candidates for the uptake of small pyridine precursors, possibly linked to NAD biosynthesis. Here, we show an in vitro affinity of Bug27 and Bug69 for quinolinate in the submicromolar range, with a marked preference over other NAD precursors. A combined sequence similarity network and genome context analysis identifies a cluster of Bug69/27 homologs that are genomically associated with the NAD transcriptional regulator NadQ and the enzyme quinolinate phosphoribosyltransferase (QaPRT, gene nadC), suggesting a functional linkage to NAD metabolism. Integrating molecular docking and structure-based multiple alignments confirms that quinolinate is the preferred ligand for Bug27 and Bug69.

Cryo-EM structure of 1-deoxy-D-xylulose 5-phosphate synthase DXPS from Plasmodium falciparum reveals a distinct N-terminal domain.

Plasmodium falciparum is the main causative agent of malaria, a deadly disease that mainly affects children under five years old. Artemisinin-based combination therapies have been pivotal in controlling the disease, but resistance has arisen in various regions, increasing the risk of treatment failure. The non-mevalonate pathway is essential for the isoprenoid synthesis in Plasmodium and provides several under-explored targets to be used in the discovery of new antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of the pathway. Despite its importance, there are no structures available for any Plasmodium spp., due to the complex sequence which contains large regions of high disorder, making crystallisation a difficult task. In this manuscript, we use cryo-electron microscopy to solve the P. falciparum DXPS structure at a final resolution of 2.42 Å. Overall, the structure resembles other DXPS enzymes but includes a distinct N-terminal domain exclusive to the Plasmodium genus. Mutational studies show that destabilization of the cap domain interface negatively impacts protein stability and activity. Additionally, a density for the co-factor thiamine diphosphate is found in the active site. Our work highlights the potential of cryo-EM to obtain structures of P. falciparum proteins that are unfeasible by means of crystallography.

NAPRT Silencing in FH-Deficient Renal Cell Carcinoma Confers Therapeutic Vulnerabilities via NAD+ Depletion.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by loss of function mutations in fumarate hydratase (FH) and results in an aggressive subtype of renal cell carcinoma with limited treatment options. Loss of FH leads to accumulation of fumarate, an oncometabolite that disrupts multiple cellular processes and drives tumor progression. High levels of fumarate inhibit alpha ketoglutarate-dependent dioxygenases, including the ten-eleven translocation (TET) enzymes, and can lead to global DNA hypermethylation. Here, we report patterns of hypermethylation in FH-mutant cell lines and tumor samples are associated with the silencing of nicotinate phosphoribosyl transferase (NAPRT), a rate-limiting enzyme in the Preiss-Handler pathway of NAD+ biosynthesis, in a subset of HLRCC cases. NAPRT is hypermethylated at a CpG island in the promoter in cell line models and patient samples, resulting in loss of NAPRT expression. We find that FH-deficient RCC models with loss of NAPRT expression, as well as other oncometabolite-producing cancer models that silence NAPRT, are extremely sensitive to nicotinamide phosphoribosyl transferase inhibitors (NAMPTi). NAPRT silencing was also associated with synergistic tumor cell killing with PARP inhibitors and NAMPTis, which was associated with effects on PAR-mediated DNA repair. Overall, our findings indicate that NAPRT silencing can be targeted in oncometabolite-producing cancers and elucidates how oncometabolite-associated hypermethylation can impact diverse cellular processes and lead to therapeutically relevant vulnerabilities in cancer cells. Implications: NAPRT is a novel biomarker for targeting NAD+ metabolism in FH-deficient HLRCCs with NAMPTis alone and targeting DNA repair processes with the combination of NAMPTis and PARP inhibitors.

Improved taxadiene production by optimizing DXS expression and fusing short-chain prenyltransferases.

This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD600). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis.

Dual FKRP/FST gene therapy normalizes ambulation, increases strength, decreases pathology, and amplifies gene expression in LGMDR9 mice.

Recent clinical studies of single gene replacement therapy for neuromuscular disorders have shown they can slow or stop disease progression, but such therapies have had little impact on reversing muscle disease that was already present. To reverse disease in patients with muscular dystrophy, new muscle mass and strength must be rebuilt at the same time that gene replacement prevents subsequent disease. Here, we show that treatment of FKRPP448L mice with a dual FKRP/FST gene therapy packaged into a single adeno-associated virus (AAV) vector can build muscle strength and mass that exceed levels found in wild-type mice and can induce normal ambulation endurance in a 1-h walk test. Dual FKRP/FST therapy also showed more even increases in muscle mass and amplified muscle expression of both genes relative to either single gene therapy alone. These data suggest that treatment with single AAV-bearing dual FKRP/FST gene therapies can overcome loss of ambulation by improving muscle strength at the same time it prevents subsequent muscle damage. This design platform could be used to create therapies for other forms of muscular dystrophy that may improve patient outcomes.

Spondyloocular Syndrome: First Case of Rare Osseous and Ocular Syndrome from India with Novel Mutation and Expanded Phenotypic Spectrum.

Spondyloocular syndrome (SOS) is a rare autosomal recessive skeletal and ocular disorder with variable phenotypes. It is caused by pathogenic mutation in the XYLT2 gene, which encodes the enzyme xylo-transferase, necessary for the synthesis of proteoglycan. It is characterized by generalized osteoporosis, short stature, hearing impairment, eye abnormalities, and cardiac defects. Till date only 24 cases have been reported worldwide with no cases documented from India. We subjected the patient to relevant biochemical investigations and Dual Energy X-ray Absorptiometry (DEXA) scan along with Next Generation Clinical Exome Sequencing (NGCES). We report a case of 23-year-old male who presented with recurrent long bone fractures, congenital heart defects, eye abnormalities (bilateral corneal opacities and atrophic bulbi), and short stature. In addition, our patient also had genu valgum and right-sided hydrocele which have never been reported in SOS till date. On genetic analysis, NGCES revealed a novel pathogenic frameshift variant c.191_192 delCA, p.(Thr64fs*22) in the XYLT2 gene. The patient is doing well on six monthly zoledronic acid infusions.

Enhanced anti-tumor efficacy with multi-transgene armed mesenchymal stem cells for treating peritoneal carcinomatosis.

BACKGROUND: Mesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime. METHODS: In this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking. RESULTS: Firstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively. CONCLUSIONS: Taken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.

Biochemical Characterization of Rice Xylan Biosynthetic Enzymes in Determining Xylan Chain Elongation and Substitutions.

Grass xylan consists of a linear chain of ß-1,4-linked xylosyl residues that often form domains substituted only with either arabinofuranose (Araf) or glucuronic acid (GlcA)/methylglucuronic acid (MeGlcA) residues, and it lacks the unique reducing end tetrasaccharide sequence found in dicot xylan. The mechanism of how grass xylan backbone elongation is initiated and how its distinctive substitution pattern is determined remains elusive. Here, we performed biochemical characterization of rice xylan biosynthetic enzymes, including xylan synthases, glucuronyltransferases and methyltransferases. Activity assays of rice xylan synthases demonstrated that they required short xylooligomers as acceptors for their activities. While rice xylan glucuronyltransferases effectively glucuronidated unsubstituted xylohexaose acceptors, they transferred little GlcA residues onto (Araf)-substituted xylohexaoses and rice xylan 3-O-arabinosyltransferase could not arabinosylate GlcA-substituted xylohexaoses, indicating that their intrinsic biochemical properties may contribute to the distinctive substitution patterns of rice xylan. In addition, we found that rice xylan methyltransferase exhibited a low substrate binding affinity, which may explain the partial GlcA methylation in rice xylan. Furthermore, immunolocalization of xylan in xylem cells of both rice and Arabidopsis showed that it was deposited together with cellulose in secondary walls without forming xylan-rich nanodomains. Together, our findings provide new insights into the biochemical mechanisms underlying xylan backbone elongation and substitutions in grass species.

Defining clinical endpoints in limb girdle muscular dystrophy: a GRASP-LGMD study.

BACKGROUND: The Limb Girdle Muscular Dystrophies (LGMDs) are characterized by progressive weakness of the shoulder and hip girdle muscles as a result of over 30 different genetic mutations. This study is designed to develop clinical outcome assessments across the group of disorders. METHODS/DESIGN: The primary goal of this study is to evaluate the utility of a set of outcome measures on a wide range of LGMD phenotypes and ability levels to determine if it would be possible to use similar outcomes between individuals with different phenotypes. We will perform a multi-center, 12-month study of 188 LGMD patients within the established Genetic Resolution and Assessments Solving Phenotypes in LGMD (GRASP-LGMD) Research Consortium, which is comprised of 11 sites in the United States and 2 sites in Europe. Enrolled patients will be clinically affected and have mutations in CAPN3 (LGMDR1), ANO5 (LGMDR12), DYSF (LGMDR2), DNAJB6 (LGMDD1), SGCA (LGMDR3), SGCB (LGMDR4), SGCD (LGMDR6), or SGCG (LGMDR5, or FKRP-related (LGMDR9). DISCUSSION: To the best of our knowledge, this will be the largest consortium organized to prospectively validate clinical outcome assessments (COAs) in LGMD at its completion. These assessments will help clinical trial readiness by identifying reliable, valid, and responsive outcome measures as well as providing data driven clinical trial decision making for future clinical trials on therapeutic agents for LGMD. The results of this study will permit more efficient clinical trial design. All relevant data will be made available for investigators or companies involved in LGMD therapeutic development upon conclusion of this study as applicable. TRIAL REGISTRATION: Clinicaltrials.gov NCT03981289; Date of registration: 6/10/2019.

Specific protein interactions between rice members of the GT43 and GT47 families form various central cores of putative xylan synthase complexes.

Members of the glycosyltransferase (GT)43 and GT47 families have been associated with heteroxylan synthesis in both dicots and monocots and are thought to assemble into central cores of putative xylan synthase complexes (XSCs). Currently, it is unknown whether protein-protein interactions within these central cores are specific, how many such complexes exist, and whether these complexes are functionally redundant. Here, we used gene association network and co-expression approaches in rice to identify four OsGT43s and four OsGT47s that assemble into different GT43/GT47 complexes. Using two independent methods, we showed that (i) these GTs assemble into at least six unique complexes through specific protein-protein interactions and (ii) the proteins interact directly in vitro. Confocal microscopy showed that, when alone, all OsGT43s were retained in the endoplasmic reticulum (ER), while all OsGT47s were localized in the Golgi. co-expression of OsGT43s and OsGT47s displayed complexes that form in the ER but accumulate in Golgi. ER-to-Golgi trafficking appears to require interactions between OsGT43s and OsGT47s. Comparison of the central cores of the three putative rice OsXSCs to wheat, asparagus, and Arabidopsis XSCs, showed great variation in GT43/GT47 combinations, which makes the identification of orthologous central cores between grasses and dicots challenging. However, the emerging picture is that all central cores from these species seem to have at least one member of the IRX10/IRX10-L clade in the GT47 family in common, suggesting greater functional importance for this family in xylan synthesis. Our findings provide a new framework for future investigation of heteroxylan biosynthesis and function in monocots.

Genetic blueprint of congenital muscular dystrophies with brain malformations in Egypt: A report of 11 families.

Congenital muscular dystrophies (CMDs) are a group of rare muscle disorders characterized by early onset hypotonia and motor developmental delay associated with brain malformations with or without eye anomalies in the most severe cases. In this study, we aimed to uncover the genetic basis of severe CMD in Egypt and to determine the efficacy of whole exome sequencing (WES)-based genetic diagnosis in this population. We recruited twelve individuals from eleven families with a clinical diagnosis of CMD with brain malformations that fell into two groups: seven patients with suspected dystroglycanopathy and five patients with suspected merosin-deficient CMD. WES was analyzed by variant filtering using multiple approaches including splicing and copy number variant (CNV) analysis. We identified likely pathogenic variants in FKRP in two cases and variants in POMT1, POMK, and B3GALNT2 in three individuals. All individuals with merosin-deficient CMD had truncating variants in LAMA2. Further analysis in one of the two unsolved cases showed a homozygous protein-truncating variant in Feline Leukemia Virus subgroup C Receptor 1 (FLVCR1). FLVCR1 loss of function has never been previously reported. Yet, loss of function of its paralog, FLVCR2, causes lethal hydranencephaly-hydrocephaly syndrome (Fowler Syndrome) which should be considered in the differential diagnosis for dystroglycanopathy. Overall, we reached a diagnostic rate of 86% (6/7) for dystroglycanopathies and 100% (5/5) for merosinopathy. In conclusion, our results provide further evidence that WES is an important diagnostic method in CMD in developing countries to improve the diagnostic rate, management plan, and genetic counseling for these disorders.

Break Down of the Complexity and Inconsistency Between Levels of Matriglycan and Disease Phenotype in FKRP-Related Dystroglycanopathies: A Review and Model of Interpretation.

Dystroglycanopathies are a group of muscle degenerative diseases characterized with significant reduction in matriglycan expression critical in disease pathogenesis. Missense point mutations in the Fukutin-related protein (FKRP) gene cause variable reduction in the synthesis of matriglycan on alpha-dystroglycan (α-DG) and a wide range of disease severity. Data analyses of muscle biopsies from patients fail to show consistent correlation between the levels of matriglycan and clinical phenotypes. By reviewing clinical reports in conjunction with analysis of clinically relevant mouse models, we identify likely causes for the confusion. Nearly all missense FKRP mutations retain variable, but sufficient function for the synthesis of matriglycan during the later stage of muscle development and periods of muscle regeneration. These factors lead to a highly heterogenous pattern of matriglycan expression in diseased muscles, depending on age and stages of muscle regeneration. The limited size in clinical biopsy samples from different parts of even a single muscle tissue at different time points of disease progression may well mis-represent the residual function (base-levels) of the mutated FKRPs and phenotypes. We propose to use a simple Multi Point tool from ImageJ to more accurately measure the signal intensity of matriglycan expression on fiber membrane for assessing mutant FKRP function and therapeutic efficacy. A robust and sensitive immunohistochemical protocol would further improve reliability and comparability for the detection of matriglycan.

RETINAL MANIFESTATIONS OF WALKER-WARBURG SYNDROME IN TWO SIBLINGS WITH RXYLT1 MUTATIONS.

PURPOSE: We report two siblings with genetically confirmed Walker-Warburg syndrome (WWS), studied with multimodal imaging, who presented with different retinal manifestations. METHODS: This is a retrospective report of two WWS cases with ultra-widefield fundus photography, fluorescein angiography, and ultrasound. Molecular diagnosis was achieved using panel testing and targeted variant testing. RESULTS: Two siblings, one male and one female, born 17 months apart with a diagnosis of WWS underwent retinal examination with imaging. The 3-month-old female infant exhibited microphthalmia, persistent hyaloidal arteries, and retrolental membranes with total tractional retinal detachments on ultrasound in both eyes. The 22-day-old male newborn exhibited persistent hyaloidal arteries and extensive peripheral avascular retina on angiography in both eyes. Both were found to be positive for the same two pathogenic variants in the RXYLT1/TMEM5 gene, which accounts for approximately 9% of cases of genetically confirmed WWS. CONCLUSION: Siblings with genetically confirmed WWS can have variable presentations despite identical genotype. This highlights the phenotypic disease spectrum of WWS, which may be similar to that seen in familial exudative vitreoretinopathy.

Improved efficacy of FKRP AAV gene therapy by combination with ribitol treatment for LGMD2I.

Mutations in the fukutin-related protein (FKRP) gene cause dystroglycanopathy, with disease severity ranging from mild LGMD2I to severe congenital muscular dystrophy. Recently, considerable progress has been made in developing experimental therapies, with adeno-associated virus (AAV) gene therapy and ribitol treatment demonstrating significant therapeutic effect. However, each treatment has its strengths and weaknesses. AAV gene therapy can achieve normal levels of transgene expression, but it requires high doses, with toxicity concerns and variable distribution. Ribitol relies on residual FKRP function and restores limited levels of matriglycan. We hypothesized that these two treatments can work synergistically to offer an optimized therapy with efficacy and safety unmatched by each treatment alone. The most effective treatment is the combination of high-dose (5e-13 vg/kg) AAV-FKRP with ribitol, whereas low dose (1e-13 vg/kg) AAV-FKRP combined with ribitol showed a 22.6% increase in positive matriglycan fibers and the greater improvement in pathology when compared to low-dose AAV-FKRP alone. Together, our results support the potential benefits of combining ribitol with AAV gene therapy for treating FKRP-related muscular dystrophy. The fact that ribitol is a metabolite in nature and has already been tested in animal models and clinical trials in humans without severe side effects provides a safety profile for it to be trialed in combination with AAV gene therapy.

Single-centre experience with autosomal recessive limb-girdle muscular dystrophy: case series and literature review.

Limb-girdle muscular dystrophy (LGMD) is a group of myopathies that lead to progressive muscle weakness, predominantly involving the shoulder and pelvic girdles; it has a heterogeneous genetic etiology, with variation in the prevalence of subtypes according to the ethnic backgrounds and geographic origins of the populations. The aim of the present study was to analyze a series of patients with autosomal recessive LGMD (LGMD-R) to contribute to a better characterization of the disease and to find the relative proportion of the different subtypes in a Southern Brazil cohort. The sample population consisted of 36 patients with LGMD-R. A 9-gene targeted next-generation sequencing panel revealed variants in 23 patients with LGMD (64%), and it identified calpainopathy (LGMD-R1) in 26%, dysferlinopathy (LGMD-R2) in 26%, sarcoglycanopathies (LGMD-R3-R5) in 13%, telethoninopathy (LGMD-R7) in 18%, dystroglicanopathy (LGMD-R9) in 13%, and anoctaminopathy (LGMD-R12) in 4% of the patients. In these 23 patients with LGMD, there were 27 different disease-related variants in the ANO5, CAPN3, DYSF, FKRP, SGCA, SGCB, SGCG, and TCAP genes. There were different causal variants in different exons of these genes, except for the TCAP gene, for which all patients carried the p.Gln53* variant, and the FKRP gene, which showed recurrence of the p.Leu276Ile variant. We analyzed the phenotypic, genotypic and muscle immunohistochemical features of this Southern Brazilian cohort.

A distrofia muscular de cinturas (DMC) é um grupo de miopatias que leva à fraqueza muscular progressiva, e envolvendo predominante as cinturas escapular e pélvica. A DMCtem uma etiologia genética heterogênea, com variação na prevalência de subtipos de acordo com as origens étnicas e geográficas das populações. O objetivo deste estudo foi analisar uma série de pacientes com DMC do tipo autossômico recessivo (DMC-R) para contribuir para uma melhor caracterização da doença e encontrar a proporção relativa dos diferentes subtipos em uma coorte do Sul do Brasil. A população amostral foi composta por 36 pacientes com DMC-R. O painel de sequenciamento de nova geração com 9 genes revelou variantes em 23 pacientes com DMC (64%), e identificou calpainopatia (DMC-R1) em 26%, disferlinopatia (DMC-R2) em 26%, sarcoglicanopatias (DMC-R3­R5) em 13%, teletoninopatia (D-MCR7) em 18%, distroglicanopatia (D-MCR9) em 13%, e anoctaminopatia (DMC-R12) em 4% dos pacientes. Nesses 23 pacientes com DMC, havia 27 variantes diferentes nos genes ANO5, CAPN3, DYSF, FKRP, SGCA, SGCB, SGCG e TCAP. Foram encontradas diferentes variantes em diferentes éxons desses genes, com exceção do gene TCAP, para o qual todos os pacientes eram portadores da variante p.Gln53*, e do gene FKRP, que apresentou recorrência da variante p.Leu276Ile. As características fenotípicas, genotípicas e imuno-histoquímicas musculares desta coorte do Sul do Brasil foram analisadas.

CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy.

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of α-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of α-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC50) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products.