Splicing Modifications Research Articles

The Rise of RNA-Based Therapeutics: Recent Advances and Therapeutic Potential

In recent years, RNA-based therapeutics have emerged as a groundbreaking field, offering innovative approaches for drug development and therapeutic interventions. This review article presents a comprehensive exploration of the advancements in RNA-based therapeutics, focusing on key modalities such as RNA interference (RNAi), antisense oligonucleotides (ASOs), messenger RNA (mRNA) vaccines, and other emerging RNA-based therapies. The introduction provides an insightful overview of the potential of RNA as a therapeutic target, highlighting its unique mechanisms of action and its transformative role in precision medicine. Subsequently, the review delves into the intricacies of RNAi, explaining the function of small interfering RNAs (siRNAs) and microRNAs (miRNAs) in selectively silencing disease-associated genes, thereby opening new avenues for therapeutic interventions. Antisense oligonucleotides (ASOs) are discussed in detail, elucidating how they target mRNA for degradation or modulation of splicing, offering promising solutions for treating genetic disorders, neurodegenerative diseases, and viral infections. Additionally, the groundbreaking success of mRNA vaccines is explored, with an emphasis on their role in combatting infectious diseases like COVID-19 and their potential application in cancer immunotherapy and other therapeutic areas. Addressing the critical issue of delivery challenges in RNA-based therapeutics, the review presents various strategies to enhance stability, cellular uptake, and minimize immunogenicity, thereby improving the effectiveness of these therapies in reaching their intended targets. Clinical successes and challenges of RNA-based therapeutics are critically evaluated, providing insights into ongoing clinical trials and approved therapies. Success stories underscore the transformative potential of RNA-based treatments, while safety concerns are addressed, paving the way for safer and more efficient therapeutic applications. The review concludes by exploring future prospects and innovations in the field, highlighting novel delivery strategies, advancements in RNA editing technologies, and the promise of combination therapies to augment therapeutic outcomes. Regulatory considerations and commercialization challenges are also discussed, offering an understanding of the regulatory landscape for RNA-based therapeutics and the potential for market growth. In conclusion, this review article serves as an informative resource for researchers, clinicians, and pharmaceutical professionals, shedding light on the rapid progress in RNA-based therapeutics and their potential to revolutionize disease treatment. By integrating knowledge from diverse sources, this review contributes to advancing the field and underscores the exciting possibilities of RNA-based interventions in improving patient outcomes and addressing unmet medical needs.

Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters thepre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

Modulation of D3R Splicing, Signaling, and Expression by D1R through PKA→PTB Phosphorylation.

The D1R and D3R receptors functionally and synergistically interact in striatonigral neurons. Dopaminergic denervation turns this interaction antagonistic, which is correlated with a decrement in D3nf isoform and an increment in D3R membranal expression. The mechanisms of such changes in D3R are attributed to the dysregulation of the expression of their isoforms. The cause and mechanism of this phenomenon remain unknown. Dopaminergic denervation produces a decrement in D1R and PKA activity; we propose that the lack of phosphorylation of PTB (regulator of alternative splicing) by PKA produces the dysregulation of D3R splicing and changes D3R functionality. By using in silico analysis, we found that D3R mRNA has motifs for PTB binding and, by RIP, co-precipitates with PTB. Moreover, D1R activation via PKA promotes PTB phosphorylation. Acute and 5-day D1R blockade decreases the expression of D3nf mRNA. The 5-day treatment reduces D3R, D3nf, and PTB protein in the cytoplasm and increases D3R in the membrane and PTB in the nucleus. Finally, the blockade of D1R mimics the effect of dopaminergic denervation in D1R and D3R signaling. Thus, our data indicate that through PKA→PTB, D1R modulates D3R splicing, expression, and signaling, which are altered during D1R blockade or the lack of stimulation in dopaminergic denervation.

Abstract C075: Altered RNA splicing causes pancreatic cancer and exposes a therapeutic vulnerability

Abstract Recently we reported that pancreatic ductal adenocarcinomas (PDACs) are exquisitely susceptible to a range of anti-RNA splicing therapies. Thus, altered RNA splicing is a non-mutational dependency of PDACs and represents a novel therapeutic target. However, how these alterations drive tumorigenesis and determine therapeutic responses remains unknown. Using unbiased and quantitative methods to evaluate the effects of all somatic mutations on cancer, we found that mutations in RNA splicing factors SF3B1 and RBM10 (~15% of cases) were among the most significant and mutually exclusive to mutations in p53, suggesting that mutations in these splicing factors act as “second-hit” driver mutations in PDAC. To test this hypothesis, we engineered mice to co-express KrasG12D with either Sf3b1 K700E mutation or Rbm10 loss in acinar cells. Importantly, mice harboring KrasG12D and Sf3b1K700E or Rbm10 loss developed autochthonous PDACs in mice. Mechanistically, we found that both mutant splicing factors, in PDACs from mice and patients, induce alterations in exon processing in common mRNAs. Of these target mRNAs, ~67% were found to be degraded by nonsense-mediated mRNA decay (NMD)- a surveillance process that eliminates mRNAs that harbor sudden stops from splicing alterations. Interestingly, NMD-inducing splicing changes by both SF3B1K700E and RBM10 loss mapped to chromosomes 7 and 11 (chromosomes 17 and 19 in patients, respectively), which are home to several tumor-suppressor genes, including p53. This suggests that mutant splicing factors decrease the expression of specific tumor suppressors by promoting altered RNA splicing and downstream RNA degradation for PDAC pathogenesis. Supporting these findings, it was previously reported that loss of p53 in PDAC, the most common “second-hit” mutation in PDAC, leads to deletions in chromosomes 7 and 11 for PDAC pathogenesis (Nature 2022). Given that 70% of PDACs carry mutations in p53, interestingly however, PDACs with mutant splicing factors have wild-type (WT) p53. We determined that SF3B1K700E and RBM10 loss promote increased degradation of p53 compared to WT counterparts but stabilize p53 protein upon DNA damage and RNA splicing perturbations. Forced expression of gain-of-function mutant p53 in PDACs with either SF3B1K700E or RBM10 loss led to synthetic lethality, explaining the mutual exclusivity found in patient samples (described above). Lastly, splicing factor mutant PDACs are more sensitive to Gemcitabine than 5FU, and combination of Gemcitabine with splicing factor modulator, H3B-8800 (in phase 2 clinical trials) synergized to selectively kill mutant splicing cells. Animal testing of this combination therapy is ongoing using patient-derived organoids implanted in the pancreata of immunocompromised mice. These studies uncover novel driver mutations of PDAC that can bypass the effect of WT p53 and highlights a common mechanism of PDAC pathogenesis that depends on decreased expression of tumor suppressors, irrespective of second-hit mutations, exposing a therapeutic vulnerability to splicing modulators. Citation Format: Natasha Pinto Medici, Diana Martinez Saucedo, Danny Lee, Li-Ting Ku, Robert Tseng, Xinning Shan, Tianyi Chu, Pablo Perez-Piñera, Vincent Cannataro, Jeffrey Townsend, Christine Iacobuzio-Donahue, Marie Robert, Omar Abdel-Wahab, Steven D. Leach, Luisa Escobar-Hoyos. Altered RNA splicing causes pancreatic cancer and exposes a therapeutic vulnerability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C075.

Splicing modulators impair DNA damage response and induce killing of cohesin-mutant MDS and AML.

Splicing modulation is a promising treatment strategy pursued to date only in splicing factor-mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in cancer, including myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. Here, we showed that cohesin mutations are biomarkers of sensitivity to drugs targeting the splicing factor 3B subunit 1 (SF3B1) H3B-8800 and E-7107. We identified drug-induced alterations in splicing, and corresponding reduced gene expression, of a number of DNA repair genes, including BRCA1 and BRCA2, as the mechanism underlying this sensitivity in cell line models, primary patient samples and patient-derived xenograft (PDX) models of AML. We found that DNA damage repair genes are particularly sensitive to exon skipping induced by SF3B1 modulators due to their long length and large number of exons per transcript. Furthermore, we demonstrated that treatment of cohesin-mutant cells with SF3B1 modulators not only resulted in impaired DNA damage response and accumulation of DNA damage, but it sensitized cells to subsequent killing by poly(ADP-ribose) polymerase (PARP) inhibitors and chemotherapy and led to improved overall survival of PDX models of cohesin-mutant AML in vivo. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS and AML.

Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification–mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.

The importance of having a relevant model system to investigate splicing modulation in the retina: The unforeseen cases of the intronic variants in RPE65 and IMPG2 genes

Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous disorders that can be caused by pathogenic variants in one of the more than 270 genes associated with IRDs. Among them, mutations in RPE65 can lead to Leber congenital amaurosis (LCA) whereas mutations in IMPG2 to vitelliform macular dystrophy (VMD).During my talk, I will provide evidence of the importance of employing the right cellular models to identify disease mechanism(s) and eventually evaluate suitable treatments. For this, I will highlight two intriguing examples of potential splicing defects studied by our group. Such defects are attractive for splicing modulation therapies using antisense oligonucleotides (AONs), a group of versatile molecules that can alter splicing by interacting with the target sequence(s) at pre‐mRNA level. Thus, characterizing the effect of a variant at the RNA level is instrumental to assess the applicability of AON‐based therapeutic intervention.We explored the effect of the homozygous variant c.11 + 5G>A in RPE65 and a heterozygous complex allele consisting of c.3023‐15T>A and c.3023G>A (p.(Gly1008Asp)) in IMPG2. In silico predictions suggested that these variants lead to a splicing defect. To assess the pathogenic effect of these variants, splice reporter assays using midigenes were conducted in HEK293T cells. Initial results pointed towards a 124‐nt exon elongation for the RPE65 variant and an in‐frame deletion of 48‐nt at the beginning of exon 15 for the complex allele in IMPG2.To validate these results, blood cells from the patients harbouring these defects were reprogrammed to induced pluripotent stem cells (iPSCs). Subsequently, iPSCs were differentiated to the retinal cells in which these genes are endogenously expressed. For RPE65, iPSCs were differentiated into retinal pigment epithelium (RPE) cells (>90 days differentiation) or, for IMPG2, into photoreceptor precursor cells (PPCs, 30 days differentiation). For the RPE65 variant, a cell specific effect was observed: while a clear exon elongation was observed in the midigene system, in RPE, the expression of the gene was highly reduced. In addition, no protein was detected and the patient‐derived cells showed differences compared to the control line. For IMPG2, the 48‐nt deletion was detected in a lower percentage in patient‐derived PPCs compared to the levels observed in the midigene system. However, aberrantly spliced IMPG2 transcripts were highly elevated in patient‐derived PPCs in comparison to control PPCs.Overall, our work shows the importance of having the right cellular context to determine the effect of genetic variants on splicing in the retina.

Antisense Oligonucleotide-Based Rescue of Complex Intronic Splicing Defects in ABCA4.

The ABCA4 gene, involved in Stargardt disease, has a high percentage of splice-altering pathogenic variants, some of which cause complex RNA defects. Although antisense oligonucleotides (AONs) have shown promising results in splicing modulation, they have not yet been used to target complex splicing defects. Here, we performed AON-based rescue studies on ABCA4 complex splicing defects. Intron 13 variants c.1938-724A>G, c.1938-621G>A, c.1938-619A>G, and c.1938-514A>G all lead to the inclusion of different pseudo-exons (PEs) with and without an upstream PE (PE1). Intron 44 variant c.6148-84A>T results in multiple PE inclusions and/or exon skipping events. Five novel AONs were designed to target these defects. AON efficacy was assessed by in vitro splice assays using midigenes containing the variants of interest. All screened complex splicing defects were effectively rescued by the AONs. Although varying levels of efficacy were observed between AONs targeting the same PEs, for all variants at least one AON restored splicing to levels comparable or better than wildtype. In conclusion, AONs are a promising approach to target complex splicing defects in ABCA4.

Inhibition of survivin by 2'-O-methyl phosphorothioate-modified steric-blocking antisense oligonucleotides.

Chemically modified antisense oligonucleotide (ASO) has been established as a successful therapeutic strategy for treating various human diseases. To date, ten ASO drugs, which are capable of either inducing mRNA degradation via RNase H recruitment (fomivirsen, mipomersen, inotersen, volanesorsen and tofersen) or splice modulation (eteplirsen, nusinersen, golodirsen, viltolarsen and casimersen), have been approved by the regulatory agencies for market entry. Nonetheless, none of these approved drugs are prescribed as cancer therapy. Towards this, we have developed steric-blocking ASOs targeting BIRC5 - a well-validated oncogene. Initial screening was performed by transfection of HepG2 cells with seven BIRC5 exon-2 targeting, uniformly 2'-OMe-PS modified ASOs at 400 nM respectively, leading to the identification of two best-performing candidates ASO-2 and ASO-7 in reducing the production of BIRC5 mRNA. Subsequent dose-response assay was conducted via transfection of HepG2 cells by different concentrations (400, 200, 100, 50, 25 nM) of ASO-2 and ASO-7 respectively, showing that both ASOs consistently and efficiently inhibited BIRC5 mRNA expression in a dose-dependent manner. Furthermore, western blot analysis confirmed that ASO-7 could significantly repress survivin production on protein level. Based on our preliminary results, we believe that ASO-7 could be a useful BIRC5 inhibitor for both research purpose and therapeutic development.

Mass spectrometry-intensive top-down proteomics: an update on technology advancements and biomedical applications.

Proteoforms are all forms of protein molecules from the same gene because of variations at the DNA, RNA, and protein levels, e.g., alternative splicing and post-translational modifications (PTMs). Delineation of proteins in a proteoform-specific manner is crucial for understanding their biological functions. Mass spectrometry (MS)-intensive top-down proteomics (TDP) is promising for comprehensively characterizing intact proteoforms in complex biological systems. It has achieved substantial progress in technological development, including sample preparation, proteoform separations, MS instrumentation, and bioinformatics tools. In a single TDP study, thousands of proteoforms can be identified and quantified from a cell lysate. It has also been applied to various biomedical research to better our understanding of protein function in regulating cellular processes and to discover novel proteoform biomarkers of diseases for early diagnosis and therapeutic development. This review covers the most recent technological development and biomedical applications of MS-intensive TDP.

Exploring Splicing Modulation as an Innovative Approach to Combat Pancreatic Cancer: SF3B1 Emerges as a Prognostic Indicator and Therapeutic Target.

Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in terms of prognosis and treatment. Recent research has identified splicing deregulation as a new cancer hallmark. Herein, we investigated the largely uncharacterized alternative splicing profile and the key splicing factor SF3B1 in PDAC pancreatic cells and tissues as a potential discovery source of plausible drug targets and new predictive biomarkers of clinical outcome. The research involved a transcriptome-wide analysis, comparing profiles of splicing profiles in PDAC primary cells with normal ductal cells. This revealed more than 400 significant differential splicing events in genes involved in regulation of gene expression, primarily related to mRNA splicing, and metabolism of nucleic acids. PDAC cultures were highly sensitive to the SF3B1 modulators, E7107 and Pladienolide-B, showing IC50s in the low nanomolar range. These compounds induced apoptosis, associated to induction of the MCL-1/S splice variant. and reduced cell migration, associated to RON mis-splicing. In an orthotopic mouse model, E7107 showed promising results. Furthermore, we evaluated SF3B1 expression in specimens from 87 patients and found a significant association of SF3B1 expression with progression-free and overall survival. In conclusion, SF3B1 emerges as both a potential prognostic factor and therapeutic target in PDAC, impacting cell proliferation, migration, and apoptosis. These findings warrant future studies on this new therapeutic strategy against PDAC.

Spinal Muscular Atrophy, Approved Therapies and Future Prospects

Spinal muscular atrophy (SMA) is a complex congenital myoneural disorder, noted for gradual muscle atrophy leading to increasing muscular weakness and significant disability. SMA manifests broad array of clinical severity and its clinical traits can be graded into four primary phenotypes -SMA type I, II, III and IV mainly based on the age of onset of symptoms and highest motor milestones attained. Over the past few years, numerous feasible interventions found on the commonly accepted precept of augmenting the translation of the survival motor neuron (SMN) protein have been tried. Gene therapies focusing on SMN include splicing modulation of SMN2 gene (nusinersen) and replacing SMN1 gene (onasemnogene abeparvovec), whereas therapies focusing SMN-independent factors involve treatments improving muscular function, treatment of spinal deformities and neuroprotection. A noted milestone in history of SMA was the approval of Nusinersen in 2016. It was followed by approval in 2019 and in 2020 to onasemnogene abeparvovec and risdiplam respectively. These three approved therapies offered hope to the patients and their families globally. But some of the weaknesses are also associated with these therapies. Deficiency of phase 4 clinical trial data, high price tag, nonavailability of guidelines for the selection of therapies for specific phenotypes and lack of data form clinical trial targeted to compare safety and efficacy of currently approved therapies are some of the issues that licensed therapies for SMA are suffering. In the face such challenges, initiation of intervention at an early age along with the utilization of adjunct therapies seems to be a sound approach to treat SMA patients. The review discusses the classification, clinical description, genetics, diagnostics and treatment of SMA along with SMN independent therapeutic targets that can utilised for the development of new therapies.

Combined therapy of valproic acid and an SMN2 splicing modifier for an adult patient with SMA type II

Combined therapy of valproic acid and an SMN2 splicing modifier for an adult patient with SMA type II

Assessing the Binding Mode of a Splicing Modulator Stimulating Pre-mRNA Binding to the Plastic U2AF2 Splicing Factor.

RNA recognition motifs (RRMs) play a pivotal role in RNA metabolism and the regulation of gene expression. Owing to their plasticity and fuzziness, targeting RRM/RNA interfaces with small molecules is a daunting challenge for drug discovery campaigns. The U2AF2 splicing factor, which recognizes the polypyrimidine (polyPy) sequence of premature messenger (pre-m)RNA, exhibits a dynamic architecture consisting of two RRMs joined by a disordered linker. An inhibitor, NSC-194308, was shown to enhance the binding of pre-mRNA to U2AF2, selectively triggering cell death in leukemia cell lines containing spliceosome mutations. The NSC-194308 binding mode remains elusive; yet, unraveling its knowledge may offer intriguing insights for effectively targeting U2AF2 and other flexible protein/protein/RNA interfaces with small molecules. To infer plausible NSC-194308 binding poses to U2AF2, here, we applied and benchmarked the performance of static and dynamic docking approaches, elucidating the molecular basis of the NSC-194308-induced pre-mRNA stabilization on U2AF2. We demonstrate that introducing dynamic effects is mandatory to assess the binding mode of the inhibitors when they target plastic and modular architectures, such as those formed by interacting RRMs. The latter are widespread across RNA binding proteins; therefore, this mechanism may be broadly applicable to discover new therapeutics aimed at selectively modulating the RNA function by targeting protein/protein/RNA interfaces.

The eukaryotic translation initiation factor eIF4E unexpectedly acts in splicing thereby coupling mRNA processing with translation: eIF4E induces widescale splicing reprogramming providing system-wide connectivity between splicing, nuclear mRNA export and translation.

Recent findings position the eukaryotic translation initiation factor eIF4E as a novel modulator of mRNA splicing, a process that impacts the form and function of resultant proteins. eIF4E physically interacts with the spliceosome and with some intron-containing transcripts implying a direct role in some splicing events. Moreover, eIF4E drives the production of key components of the splicing machinery underpinning larger scale impacts on splicing. These drive eIF4E-dependent reprogramming of the splicing signature. This work completes a series of studies demonstrating eIF4E acts in all the major mRNA maturation steps whereby eIF4E drives production of the RNA processing machinery and escorts some transcripts through various maturation steps. In this way, eIF4E couples the mRNA processing-export-translation axis linking nuclear mRNA processing to cytoplasmic translation. eIF4E elevation is linked to worse outcomes in acute myeloid leukemia patients where these activities are dysregulated. Understanding these effects provides new insight into post-transcriptional control and eIF4E-driven cancers.

GDF11/SMAD Regulated Splicing of GATA1 Is Associated with Response to Luspatercept in Lower-Risk Myelodysplastic Syndromes (LR-MDS)

Introduction: Luspatercept is an erythroid maturation agent that promotes late-stage erythropoiesis in patients with LR-MDS. While it is known that luspatercept, a modified activin receptor ligand trap, binds to GDF11 and Activin B, the mechanism by which these ligands trigger a decrease in red blood cell (RBC) formation is unknown. Luspatercept was approved by the FDA following the phase 3 MEDALIST study, but biomarkers of response are currently unknown. Results:In a large cohort of primary MDS CD34+ samples, GDF11 expression was significantly higher in MDS samples than in age-matched healthy controls (183 MDS samples vs 17 controls, P < 0.05). GDF11 was detected in primary MDS sera and was substantially elevated in the refractory anemia (RA) and RA with ring sideroblasts (RARS) MDS subgroups. Activin receptor 2B (ACVR2B), the receptor of GDF11, was also overexpressed in MDS samples, and its expression was confirmed by immunohistochemistry (IHC) in the bone marrows of primary MDS patients. GDF11/ACVR2B activates SMAD2, and we found that the overexpression of SMAD2 in CD34+ cells was associated with more severe anemia in MDS patients. RNA-seq and SMAD2 ChIP-seq analyses were then performed to determine the downstream effects of GDF11 in erythroid progenitors. Human CD34+ primary cells were utilized to generate BFU-E stage erythroid progenitors. We discovered that GDF11 promotes an abnormal erythroid gene expression program characterized by increased expression of pro-apoptotic and early erythroid differentiation-related genes. SMAD2 binding was detected in regulatory regions of early erythroid (including GATA1), proinflammatory, and apoptotic transcripts using ChIP-seq analysis with p-SMAD2. In a zebrafish model, administration of GDF11 caused a decrease in erythroid cells, which was rescued by treatment with luspatercept (Wobus et al. Leukemia 2021 35(10):2936-47). Luspatercept inhibited the phosphorylation and activation of SMAD2 in response to GDF11 stimulation in primary hematopoietic cells and leukemia cell lines. Importantly, luspatercept could reverse the growth-inhibiting effects of MDS-derived sera on primary hematopoietic progenitors. Finally, to identify biomarkers associated with clinical responses, we analyzed phase 3 MEDALIST study samples collected before and after treatment with lupatercept. RNA-seq analysis was performed on bone marrow samples from responders (R; n = 14) and non-responders (NR; n = 9) (Figure). Analysis of pre-treatment/screening bone marrow transcriptomes revealed no significant differences between R and NR in global gene expression. Analysis of splice isoforms between the two cohorts revealed strikingly greater differences. Most differential splicing events were observed in NR (702 in R vs 5232 in NR). The most prevalent splicing modifications were exon skipping events, and most functional pathways affected by differential exon usage were RNA splicing related. GATA1, the master erythroid transcription factor, was spliced differently in R at baseline, which was an intriguing observation. Response to luspatercept was found to be associated with exon 2 skipping, resulting in a shortened isoform of GATA1. R had a greater proportion of the short GATA1 isoform than NR (12% in R versus 6% in NR; P = 0.04). We analyzed ChIP-seq data from primary erythroid cells to determine the effect of GDF11/SMAD2 activation on GATA1 splicing. GDF11 stimulation resulted in SMAD2 binding to the second intron of GATA1. CRISPR-mediated deletion of the SMAD2 binding region in primary erythroid progenitors increased expression of the GATA1 long isoform. Next, we performed a CRISPR-mediated deletion of exon 2 of GATA1, resulting in a predominance of GATA1 short isoform expression and a decrease in erythroid output. In CRISPR-modified primary erythroid cells with a predominant short GATA1 isoform (exon 2-deleted cells), luspatercept was able to reverse the inhibition induced by GDF11. Conclusions: These results demonstrate that MDS cases that respond to luspatercept are associated with a relatively high level of expression of functionally deficient GATA1 short isoform. We further demonstrate that GDF11 stimulates the production of the GATA1 short isoform via SMAD2 binding to intron 2 of the GATA1 gene. The regulation of a GATA1 splicing isoform by GDF11/SMAD2 is associated with clinical responses to luspatercept, according to these novel findings.

Noncoding rules of survival: epigenetic regulation of normal and malignant hematopoiesis.

Hematopoiesis is an essential process for organismal development and homeostasis. Epigenetic regulation of gene expression is critical for stem cell self-renewal and differentiation in normal hematopoiesis. Increasing evidence shows that disrupting the balance between self-renewal and cell fate decisions can give rise to hematological diseases such as bone marrow failure and leukemia. Consequently, next-generation sequencing studies have identified various aberrations in histone modifications, DNA methylation, RNA splicing, and RNA modifications in hematologic diseases. Favorable outcomes after targeting epigenetic regulators during disease states have further emphasized their importance in hematological malignancy. However, these targeted therapies are only effective in some patients, suggesting that further research is needed to decipher the complexity of epigenetic regulation during hematopoiesis. In this review, an update on the impact of the epigenome on normal hematopoiesis, disease initiation and progression, and current therapeutic advancements will be discussed.

Reduction of retinal ganglion cell death in mouse models of familial dysautonomia using AAV-mediated gene therapy and splicing modulators

Familial dysautonomia (FD) is a rare neurodevelopmental and neurodegenerative disease caused by a splicing mutation in the Elongator Acetyltransferase Complex Subunit 1 (ELP1) gene. The reduction in ELP1 mRNA and protein leads to the death of retinal ganglion cells (RGCs) and visual impairment in all FD patients. Currently patient symptoms are managed, but there is no treatment for the disease. We sought to test the hypothesis that restoring levels of Elp1 would thwart the death of RGCs in FD. To this end, we tested the effectiveness of two therapeutic strategies for rescuing RGCs. Here we provide proof-of-concept data that gene replacement therapy and small molecule splicing modifiers effectively reduce the death of RGCs in mouse models for FD and provide pre-clinical foundational data for translation to FD patients.

Synthesis and Conformational Analysis of FR901464-Based RNA Splicing Modulators and Their Synergism in Drug-Resistant Cancers.

FR901464 is a cytotoxic natural product that binds splicing factor 3B subunit 1 (SF3B1) and PHD finger protein 5A (PHF5A), the components of the human spliceosome. The amide-containing tetrahydropyran ring binds SF3B1, and it remains unclear how the substituents on the ring contribute to the binding. Here, we synthesized meayamycin D, an analogue of FR901464, and three additional analogues to probe the conformation through methyl scanning. We discovered that the amide-containing tetrahydropyran ring assumes only one of the two possible chair conformations and that methylation of the nitrogen distorts the chair form, dramatically reducing cytotoxicity. Meayamycin D induced alternative splicing of MCL-1, showed strong synergism with venetoclax in drug-resistant lung cancer cells, and was cancer-specific over normal cells. Meayamycin D incorporates an alkyl ether and shows a long half-life in mouse plasma. The characteristics of meayamycin D may provide an approach to designing other bioactive L-shaped molecules.

CD44 Intracellular Domain: A Long Tale of a Short Tail.

CD44 is a single-chain transmembrane receptor that exists in multiple forms due to alternative mRNA splicing and post-translational modifications. CD44 is the main cell surface receptor of hyaluronan as well as other extracellular matrix molecules, cytokines, and growth factors that play important roles in physiological processes (such as hematopoiesis and lymphocyte homing) and the progression of various diseases, the predominant one being cancer. Currently, CD44 is an established cancer stem cell marker in several tumors, implying a central functional role in tumor biology. The present review aims to highlight the contribution of the CD44 short cytoplasmic tail, which is devoid of any enzymatic activity, in the extraordinary functional diversity of the receptor. The interactions of CD44 with cytoskeletal proteins through specific structural motifs within its intracellular domain drives cytoskeleton rearrangements and affects the distribution of organelles and transport of molecules. Moreover, the CD44 intracellular domain specifically interacts with various cytoplasmic effectors regulating cell-trafficking machinery, signal transduction pathways, the transcriptome, and vital cell metabolic pathways. Understanding the cell type- and context-specificity of these interactions may unravel the high complexity of CD44 functions and lead to novel improved therapeutic interventions.