Mutant RNA Research Articles

Naringin Alleviates Glucose-Induced Aging by Reducing Fat Accumulation and Promoting Autophagy in Caenorhabditis elegans.

Naringin (Nar) is a dihydroflavonoid compound, widely found in citrus fruit and used in Chinese herbal medicine. As a phytochemical, it acts as a dietary supplement that can delay aging and prevent aging-related disease, such as obesity and diabetes. However, its exact mechanism remains unclear. In this study, the high-glucose-induced (HGI) Caenorhabditis elegans model was used to evaluate the anti-aging and anti-obesity effects of Nar. The mean lifespan and fast movement span of HGI worms were extended roughly 24% and 11%, respectively, by Nar treatment. Oil red O staining revealed a significant reduction in fat accumulation and dFP::LGG-labeled worms showed the promotion of autophagy. Additionally, whole transcriptome sequencing and gene set variation analysis suggested that Nar upregulated the lipid biosynthesis and metabolism pathways, as well as the TGF-β, Wnt and longevity signaling pathways. Protein-protein interaction (PPI) network analysis identified hub genes in these pathways for further analysis. Mutant worms and RNA interference were used to study mechanisms; the suppression of hlh-30, lgg-1, unc-51, pha-4, skn-1 and yap-1 disabled the fat-lowering, lifespan-prolonging, and health-promoting properties of Nar. Collectively, our findings indicate that Nar plays an important role in alleviating HGI-aging and anti-obesity effects by reducing fat accumulation and promoting autophagy.

Molecular basis of RNA recognition by TBP of HIV-1 from multiple molecular dynamics simulations and energy predictions.

The frequent outbreaks of the AIDS (Acquired Immune Deficiency Syndrome) pandemic and the limited availability of anti-Human Immunodeficiency Virus (HIV) drugs highlight the urgent need to develop new antiviral drugs. A detailed understanding of the interactions between TAR-Binding Proteins (TBP) and RNA will facilitate the discovery of new anti-AIDS drugs. In order to characterize and explore the key interactions between RNA and TBP, we focused on the wild type (WT) and three mutant TBPs (TBP6.9, TBP6.7, and TBP6.3) with RNA, multiple molecular dynamics simulation and energy computation were performed. The results showed that 12 key residues played a major role in the interaction between TBP and RNA. The mutated residues of TBP changed the interaction between their surrounding residues and RNA, thus affecting the binding of TBP to RNA. In addition, structural and energy analyses showed that in contrast with WT TBP-RNA complex, the mutated residues had little effect on the backbone structure of TBP, but changes in the van der Waals interactions and electrostatic interaction associated with the side chains are responsible for the altered the binding between three mutant TBPs and RNA complexes. The discovery of TBP-RNA recognition mechanism in our work provides some useful insights and new opportunities for the development of anti-aids drugs.

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5'-3' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

Antiviral Pills against SARS-CoV-2 Virus to Combat Future Epidemic Threats in Pakistan

Antiviral pills are oral medications that treat infections through the inhibition of the viral growth and replication cycle. Paxlovid by Pfizer and Molnupiravir by Merck are the two pills effective for high-risk SARS-CoV-2 patients. Paxlovid works by inhibiting the replication cycle of SARS-CoV-2 using cysteine residues in-vitro. These cysteine residues inhibit the main protease of the virus by functioning as reversible covalent inhibitors. Molnupiravir works by introducing a high rate of mutations in the viral RNA causing the virus to become biologically unstable and non-functional. Both antiviral drugs can bridge the gap in the preparedness for viral outbreaks in low-income countries like Pakistan by mitigating the chances of fatality and inpatient treatment in high-risk, unvaccinated individuals. Pakistan has been plagued by various epidemics over the years however SARS-CoV-2 outbreak caused many deaths along with an economic crisis. The country lacks the resources to endure high inpatient treatment rates in case of SARS-CoV-2 infections, which is why the need for antiviral pills like Paxlovid and Molnupiravir is empirical to overcome epidemics and viral outbreaks. This work outlines the antiviral pills and their efficacy against SARS-CoV-2 with a focus on how these drugs can overcome significant gaps in epidemic preparedness and response in Pakistan. We aim to highlight how antiviral pills against SARS-CoV-2 can ensure resilience to future epidemic threats in Pakistan.

Insulin Resistance in Mitochondrial Diabetes

Mitochondrial diabetes (MD) is generally classified as a genetic defect of β-cells. The main pathophysiology is insulin secretion failure in pancreatic β-cells due to impaired mitochondrial ATP production. However, several reports have mentioned the presence of insulin resistance (IR) as a clinical feature of MD. As mitochondrial dysfunction is one of the important factors causing IR, we need to focus on IR as another pathophysiology of MD. In this special issue, we first briefly summarized the insulin signaling and molecular mechanisms of IR. Second, we overviewed currently confirmed pathogenic mitochondrial DNA (mtDNA) mutations from the MITOMAP database. The variants causing diabetes were mostly point mutations in the transfer RNA (tRNA) of the mitochondrial genome. Third, we focused on these variants leading to the recently described "tRNA modopathies" and reviewed the clinical features of patients with diabetes. Finally, we discussed the pathophysiology of MD caused by mtDNA mutations and explored the possible mechanism underlying the development of IR. This review should be beneficial to all clinicians involved in diagnostics and therapeutics related to diabetes and mitochondrial diseases.

Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome.

The number of genetic variations in the SARS-CoV-2 genome has been increasing primarily due to continuous viral mutations. Here, we report that the human APOBEC3A (A3A) cytidine deaminase plays a critical role in the induction of C-to-U substitutions in the SARS-CoV-2 genome. Bioinformatic analysis of the chronological genetic changes in a sequence database indicated that the largest UC-to-UU mutation signature, consistent with APOBEC-recognized nucleotide motifs, was predominant in single-stranded RNA regions of the viral genome. In SARS-CoV-2-infected cells, exogenous expression of A3A but not expression of other APOBEC proteins induced UC-to-UU mutations in viral RNA (vRNA). Additionally, the mutated C bases were often located at the tips in bulge or loop regions in the vRNA secondary structure. Interestingly, A3A mRNA expression was drastically increased by interferons (IFNs) and tumour necrosis factor-α (TNF-α) in epithelial cells derived from the respiratory system, a site of efficient SARS-CoV-2 replication. Moreover, the UC-to-UU mutation rate was increased in SARS-CoV-2 produced from lung epithelial cells treated with IFN-ß and TNF-α, but not from CRISPR/Cas9-based A3A knockout cells. Collectively, these findings demonstrate that A3A is a primary host factor that drives mutations in the SARS-CoV-2 RNA genome via RNA editing.

Mutation density analyses on long noncoding RNA reveal comparable patterns to protein-coding RNA and prognostic value

Mutations and gene expression are the two most studied genomic features in cancer research. In the last decade, the combined advances in genomic technology and computational algorithms have broadened mutation research with the concept of mutation density and expanded the traditional scope of protein-coding RNA to noncoding RNAs. However, mutation density analysis had yet to be integrated with non-coding RNAs. In this study, we examined long non-coding RNA (lncRNA) mutation density patterns of 57 unique cancer types using 80 cancer cohorts. Our analysis revealed that lncRNAs exhibit mutation density patterns reminiscent to those of protein-coding mRNAs. These patterns include mutation peak and dip around transcription start sites of lncRNA. In many cohorts, these patterns justified statistically significant transcription strand bias, and the transcription strand bias was shared between lncRNAs and mRNAs. We further quantified transcription strand biases with a Log Odds Ratio metric and showed that some of these biases are associated with patient prognosis. The prognostic effect may be exerted due to strong Transcription-coupled repair mechanisms associated with the individual patient. For the first time, our study combined mutational density patterns with lncRNA mutations, and the results demonstrated remarkably comparable patterns between protein-coding mRNA and lncRNA, further illustrating lncRNA’s potential roles in cancer research.

Nano-biosensor for SARS-CoV-2/COVID-19 detection: methods, mechanism and interface design.

The epidemic of coronavirus disease 2019 (COVID-19) was a huge disaster to human society. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which led to COVID-19, has resulted in a large number of deaths. Even though the reverse transcription-polymerase chain reaction (RT-PCR) is the most efficient method for the detection of SARS-CoV-2, the disadvantages (such as long detection time, professional operators, expensive instruments, and laboratory equipment) limit its application. In this review, the different kinds of nano-biosensors based on surface-enhanced Raman scattering (SERS), surface plasmon resonance (SPR), field-effect transistor (FET), fluorescence methods, and electrochemical methods are summarized, starting with a concise description of their sensing mechanism. The different bioprobes (such as ACE2, S protein-antibody, IgG antibody, IgM antibody, and SARS-CoV-2 DNA probes) with different bio-principles are introduced. The key structural components of the biosensors are briefly introduced to give readers an understanding of the principles behind the testing methods. In particular, SARS-CoV-2-related RNA mutation detection and its challenges are also briefly described. We hope that this review will encourage readers with different research backgrounds to design SARS-CoV-2 nano-biosensors with high selectivity and sensitivity.

Current Technical Approaches to Study RNA–Protein Interactions in mRNAs and Long Non-Coding RNAs

It is commonly understood that RNA-binding proteins crucially determine the fate of their target RNAs. Vice versa, RNAs are becoming increasingly recognized for their functions in protein regulation and the dynamics of RNA-protein complexes. Long non-coding RNAs are emerging as potent regulators of proteins that exert unknown RNA-binding properties and moonlighting functions. A vast array of RNA- and protein-centric techniques have been developed for the identification of protein and RNA targets, respectively, including unbiased protein mass spectrometry and next-generation RNA sequencing as readout. Determining true physiological RNA and protein targets is challenging as RNA–protein interaction is highly dynamic, tissue- and cell-type-specific, and changes with the environment. Here I review current techniques for the analysis of RNA–protein interactions in living cells and in vitro. RNA-centric techniques are presented on the basis of cross-linking or the use of alternative approaches. Protein-centric approaches are discussed in combination with high-throughput sequencing. Finally, the impact of mutations in RNA–protein complexes on human disease is highlighted.

Tobacco mosaic virus movement protein complements a Potato spindle tuber viroid RNA mutant impaired for mesophyll entry but not mutants unable to enter the phloem.

Tobacco mosaic virus movement protein (TMV MP) is essential for virus spread between cells. To accomplish its task, TMV MP binds viral RNA, interacts with components of the cytoskeleton, and increases the size exclusion limit (SEL) of plasmodesmata. Plasmodesmata are gated intercellular channels that allow passage of small molecules and macromolecules, including RNA and protein, between plant cells. Moreover, plasmodesmata are diverse and those connecting different cell types appear to have unique mechanisms to regulate macromolecular trafficking, which likely contributes to the establishment of distinct cell boundaries. Consequently, TMV MP might be competent to mediate RNA transport through some but not all plasmodesmal gates. Due to a lack of viral mutants defective for movement between specific cell types, the ability of TMV MP in this regard is incompletely understood. In contrast, a number of trafficking impaired Potato spindle tuber viroid (PSTVd) mutants have been identified. PSTVd is a systemically infectious non-coding RNA that nevertheless can perform all functions required for replication as well as cell-to-cell and systemic spread. Previous studies have shown that PSTVd employs different structure and sequence elements to move between diverse cell types in host plants, and mutants defective for transport between specific cell types have been identified. Therefore, PSTVd may serve as a tool to analyze the functions of MPs of viral and cellular origin. To probe the RNA transport activity of TMV MP, transgenic plants expressing the protein were inoculated with PSTVd mutants. Remarkably, TMV MP complemented a PSTVd mutant defective for mesophyll entry but could not support two mutants impaired for phloem entry, suggesting it fails to productively interface with plasmodesmata at the phloem boundary and that additional viral and host factors may be required. Consistent with this idea, TMV co-infection, but not the combination of MP and coat protein (CP) expression, was able to complement one of the phloem entry mutants. These observations suggest that phloem loading is a critical impediment to establishing systemic infection that could involve the entire ensemble of TMV proteins. They also demonstrate a novel strategy for analysis of MPs.

RNA Foci Formation in a Retinal Glial Model for Spinocerebellar Ataxia Type 7.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder characterized by cerebellar ataxia and retinopathy. SCA7 is caused by a CAG expansion in the ATXN7 gene, which results in an extended polyglutamine (polyQ) tract in the encoded protein, the ataxin-7. PolyQ expanded ataxin-7 elicits neurodegeneration in cerebellar Purkinje cells, however, its impact on the SCA7-associated retinopathy remains to be addressed. Since Müller glial cells play an essential role in retinal homeostasis, we generate an inducible model for SCA7, based on the glial Müller MIO-M1 cell line. The SCA7 pathogenesis has been explained by a protein gain-of-function mechanism, however, the contribution of the mutant RNA to the disease cannot be excluded. In this direction, we found nuclear and cytoplasmic foci containing mutant RNA accompanied by subtle alternative splicing defects in MIO-M1 cells. RNA foci were also observed in cells from different lineages, including peripheral mononuclear leukocytes derived from SCA7 patient, suggesting that this molecular mark could be used as a blood biomarker for SCA7. Collectively, our data showed that our glial cell model exhibits the molecular features of SCA7, which makes it a suitable model to study the RNA toxicity mechanisms, as well as to explore therapeutic strategies aiming to alleviate glial dysfunction.

Transcriptome-wide analysis of pseudouridylation in Drosophila melanogaster.

Pseudouridine (Psi) is one of the most frequent post-transcriptional modification of RNA. Enzymatic Psi modification occurs on rRNA, snRNA, snoRNA, tRNA, and non-coding RNA and has recently been discovered on mRNA. Transcriptome-wide detection of Psi (Psi-seq) has yet to be performed for the widely studied model organism Drosophila melanogaster. Here, we optimized Psi-seq analysis for this species and have identified thousands of Psi modifications throughout the female fly head transcriptome. We find that Psi is widespread on both cellular and mitochondrial rRNAs. In addition, more than a thousand Psi sites were found on mRNAs. When pseudouridylated, mRNAs frequently had many Psi sites. Many mRNA Psi sites are present in genes encoding for ribosomal proteins, and many are found in mitochondrial encoded RNAs, further implicating the importance of pseudouridylation for ribosome and mitochondrial function. The 7SLRNA of the signal recognition particle is the non-coding RNA most enriched for Psi. The 3 mRNAs most enriched for Psi encode highly expressed yolk proteins (Yp1, Yp2, and Yp3). By comparing the pseudouridine profiles in the RluA-2 mutant and the w1118 control genotype, we identified Psi sites that were missing in the mutant RNA as potential RluA-2 targets. Finally, differential gene expression analysis of the mutant transcriptome indicates a major impact of loss of RluA-2 on the ribosome and translational machinery.

ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non‑redundantly to ADAM17.

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme participating in adaptive responses of tumors to hypoxia and acidosis. CA IX regulates pH, facilitates metabolic reprogramming, and supports migration, invasion and metastasis of cancer cells. Extracellular domain (ECD) of CA IX can be shed to medium and body fluids by a disintegrin and metalloproteinase (ADAM) 17. Here we show for the first time that CA IX ECD shedding can be also executed by ADAM10, a close relative of ADAM17, via an overlapping cleavage site in the stalk region of CA IX connecting its exofacial catalytic site with the transmembrane region. This finding is supported by biochemical evidence using recombinant human ADAM10 protein, colocalization of ADAM10 with CA IX, ectopic expression of a dominant‑negative mutant of ADAM10 and RNA interference‑mediated suppression of ADAM10. Induction of the CA IX ECD cleavage with ADAM17 and/or ADAM10 activators revealed their additive effect. Similarly, additive effect was observed with an ADAM17‑inhibiting antibody and an ADAM10‑preferential inhibitor GI254023X. These data indicated that ADAM10 is aCA IX sheddase acting on CA IX non‑redundantly to ADAM17.

RNAi machinery regulates nutrient metabolism and fluconazole resistance in the pathogenic fungus Cryptococcus deneoformans.

The RNAi machinery has been extensively studied in plant and animal cells for their crucial roles in the regulation of genome function. However, the potential roles of RNAi in controlling fungal growth and development have been poorly studied, especially in the basidiomycetous yeast Cryptococcus deneoformans. To characterize the biological functions of RNAi in the pathogenic fungus, a comparative analysis of mRNA profiles using high-throughput sequencing technology was performed for the wild type and the RNAi mutants of C. deneoformans. The results revealed a clear difference in the expression of genes associated with metabolic processes in the RNAi mutants. Besides, the growth under nutrient-limited conditions was significantly reduced in the ago2Δ mutant, suggesting the essential roles of Ago2 in nutrient metabolism. Further investigations revealed the differentially expressed transporters in the RNAi mutants, in which transporters involved in fluconazole efflux were significantly up-regulated. More importantly, on account of the upregulated transporters, RNAi mutant strains developed resistance to fluconazole. By disrupting AFR1 gene using the 'suicide' CRISPR-Cas9 system, we verified that the upregulated ABC transporter Afr1 in the RNAi mutants contributed to the fluconazole resistance. In summary, our data demonstrate that in C. deneoformans the RNAi pathway participates in nutrient metabolism and plays a role in the repression of fluconazole resistance, which provides a deep insight into RNAi mechanisms in Cryptococcus and brings great hints for the clinical treatment of cryptococcosis.

Computationally-guided design and selection of high performing ribosomal active site mutants.

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selectionmay serve as a powerful tool for rRNA engineering.

Single-Cell Analysis in Lung Adenocarcinoma Implicates RNA Editing in Cancer Innate Immunity and Patient Prognosis.

RNA editing analysis in single lung adenocarcinoma cells uncovers RNA mutations that correlate with tumor mutation burden and cancer innate immunity and reveals the amount of RNA mutations that strongly predicts patient survival. See related commentary by Luo and Liang, p. 351.

Evaluation of clarithromycin and levofloxacin resistance of Helicobacter pylori strains isolated from patients at the Hospital for Tropical Diseases, Ho Chi Minh City

Clarithromycin (CLA) and levofloxacin (LFX) have been recommended as the most effective antibiotics for treating Helicobacter pylori infection. However, the increase in H. pylori’s resistance to antibiotics is an alarming and growing challenge. The study aimed to determine the antimicrobial resistance profiles of H. pylori strains isolated from patients at the Hospital for Tropical Diseases and to detect point mutations in 23S ribosomal RNA (23S rRNA) and gyraseA (gyrA) genes. Point mutations in 23S rRNA and gyrA were detected using Sanger sequencing. Antibiotic resistance was tested by the microdilution method. Out of the 45 isolates, 44 (97.8%) were found to be resistant to at least one antibiotic, 38 (84.4%) resistant to metronidazole (MTZ), and all were sensitive to amoxicillin (AMX) and tetracycline (TET). Sixty percent of the isolated strains were resistant to 3-6 antibiotics; among them, multiple drug resistance (MDR) strains presented twenty percent resistance to more than two classes of antibiotics. Eleven strains (24.4%) carried two mutations associated with CLA and LFX but only nine of them were dual resistant to CLA and LFX, and twenty-four (53.3%) carried either CLA or LFX mutation. The point mutations A2143G in the 23S rRNA and N87K (Asn87Lys) in the gyrA were detected in the CLA and LFX resistant strains, respectively. The prevalence of MDR, especially CLA, MTZ, and LFX resistance, in the H. pylori isolates suggests that the use of these antibiotics need to be more considerable and cautious.

Parental mutations influence wild-type offspring via transcriptional adaptation.

Transgenerational epigenetic inheritance (TEI) is mostly discussed in the context of physiological or environmental factors. Here, we show intergenerational and transgenerational inheritance of transcriptional adaptation (TA), a process whereby mutant messenger RNA (mRNA) degradation affects gene expression, in nematodes and zebrafish. Wild-type offspring of animals heterozygous for mRNA-destabilizing alleles display increased expression of adapting genes. Notably, offspring of animals heterozygous for nontranscribing alleles do not display this response. Germline-specific mutations are sufficient to induce TA in wild-type offspring, indicating that, at least for some genes, mutations in somatic tissues are not necessary for this process. Microinjecting total RNA from germ cells of TA-displaying heterozygous zebrafish can trigger TA in wild-type embryos and in their progeny, suggesting a model whereby mutant mRNAs in the germline trigger a TA response that can be epigenetically inherited. In sum, this previously unidentified mode of TEI reveals a means by which parental mutations can modulate the offspring's transcriptome.

Hematopoietic and Immunological Assessment in Fanconi Anemia after Ex Vivo Lentiviral FANCA Gene Therapy with RP-L102

Introduction: Fanconi anemia (FA) is the most frequent cause of genetic bone marrow failure (BMF) resulting from inherited defects in the FA pathway, one of several essential deoxyribonucleic acid (DNA) repair mechanisms. Although allogeneic hematopoietic stem cell transplantation (alloHSCT) offers hematolymphoid cure, it confers significant morbidity and mortality and is thereby indicated only after development of severe BMF or dysplasia. Ex vivo hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) offers an alternative strategy for earlier and potentially safer intervention with the goal of preventing BMF and the need for alloHSCT. Core assessments of safety and efficacy are conducted as part of ongoing GT clinical trials for FA-type A patients who have received ex vivo lentiviral FANCA GT. In these additional exploratory research studies we seek to better understand how GT may affect HSPC behavior, mutational acquisition and immune function. Methods: To deepen our understanding of the effects of ex vivo lentiviral FANCA GT on FA biology, we collected serial bone marrow (BM) and peripheral blood (PB) samples from FA-Type A patients who were enrolled at Stanford University on the Phase I and II RP-L102-0418 and RP-L102-0319 clinical trials. We initiated the assessment of Phase I subjects at 18 months post treatment and the assessment of Phase II subjects at study enrollment. BM was assessed to characterize HSPC distribution by flow cytometry, proliferative capacity by colony-forming count (CFC) assays, transcriptomics by single-cell RNA sequencing (scRNASeq) and mutation acquisition by targeted next generation sequencing (NGS). Immune system composition and function were assessed using PB flow cytometry, immunoglobulin levels and T cell proliferative assays. Results: Of the 8 enrolled patients, 5 subjects met eligibility criteria and were treated with RP-L102 GT. As of April 2022, 4 remain on study, now 18 to 36 months post-treatment. Of these 4 subjects, 3 have demonstrated increasing vector copy number and MMC-resistance. At time of enrollment, HSCs were markedly reduced in these patients compared to healthy controls, even in patients with no or mild cytopenia, and to-date have remained decreased post-GT. Functional HSPC proliferation capacity, as assessed by CFCs, was reduced in all patients at screening but showed evidence of improvement or stabilization in 3 of the 4 post-GT patients, as did BM total cellularity and CD34+ counts. scRNASeq showed reduced proportions of HSCs pre- and post-GT relative to healthy controls. Expression of ribosome biogenesis genes, an indicator of cancer risk, was downregulated post-GT. Targeted bulk-NGS revealed that none of the patients evaluated on these studies had mutations known to be associated with myeloid malignancies pre- or post-GT treatment. Detailed immune monitoring revealed that 3 patients had B-cell lymphopenia at time of enrollment. Additionally, 1 patient showed an increase in B-cell numbers post-GT. IgG levels were normal in all 8 screened patients pre-GT and remained stable post-GT. Interestingly, among 7 vaccinated patients evaluated at enrollment, 3 did not have protective IgG levels to Hepatitis B, 1 did not have protective IgG levels to Varicella Zoster Virus (VZV), and 3 showed equivocal levels of anti-VZV IgG. Conclusions: Although our results are based on a small cohort and are interim within ongoing clinical trials, they indicate the need for more thorough assessment of BM and immune characterization in FA patients beyond reliance on blood counts. Importantly, the paucity of HSCs in FA patients even prior to the development of cytopenias supports the need for early intervention for HSC preservation which may be possible with GT upon diagnosis. Additionally, these studies show that RP-L102 GT appears to be a safe approach for hematopoietic preservation in FA without acquisition of detectable mutations in HSPCs to-date. Moreover, downregulation of ribosome biogenesis genes can potentially lead to decreased leukemogenesis which could decrease cancer risk post-GT. Finally, we show laboratory evidence of baseline immune dysfunction in FA patients mainly consisting of B-cell lymphopenia and poor response to certain vaccines. Given this, post-GT re-vaccination of patients may be clinically indicated and would enable assessment of functional immune recovery potentially associated with the GT treatment.

Sustainable recovery of MBNL activity in autoregulatory feedback loop in myotonic dystrophy

Muscleblind-like proteins (MBNLs) are RNA-binding proteins essential for the developmental regulation of various processes including alternative splicing. Their activity is misregulated in myotonic dystrophy type 1 (DM1), an incurable genetic, neuro-muscular disorder caused by uncontrolled expansion of CTG repeats. Mutant RNAs containing hundreds or thousands of repeats efficiently sequester MBNL proteins. As a consequence, global alternative splicing abnormalities are induced. Importantly, the size of expansion differs significantly not only between patients but also between different parts of the same muscle as a consequence of somatic expansion. One of the potential therapeutic strategies in DM is overexpression of MBNLs. However, gene therapy tools might induce excessive activity of MBNLs, what in turn might change the metabolism of many RNAs. To overcome these limitations, we designed an autoregulated MBNL1 overexpression system. The genetic construct contains an MBNL1-coding sequence separated by the fragment of ATP2A1 pre-mRNA with an MBNL-sensitive alternative exon containing stop codon in the reading frame of MBNL1. Inclusion of this exon leads to the arrangement of an inactive form of the protein, but exclusion gives rise to fully active MBNL1. This approach enables the autoregulation of the amount of overexpressed MBNL1 with high dynamic range which ensures a homogeneous level of this protein in cells treated with the genetic construct. We demonstrated beneficial effects of an autoregulated construct on alternative splicing patterns in DM1 models and cells derived from patients with DM1.