Biology subjects

Hanifi, M.

Publications and source records attributed to Hanifi, M..

4 recordsLinked to original sources

bioRxiv2024

Robust CRISPR/dCas13 RNA blockers specifically perturb miRNA-target interactions and rescue type 1 myotonic dystrophy pathology

While RNA-targeting strategies are powerful tools for disease therapy, challenges, including low target engagement and off-target collateral effects, currently limit their efficacy. Here, we report the engineering and optimisation of a CRISPR/dCas13 RNA steric blocker (CRISPR-Lock) that prevents mRNA translation, shields mRNAs from miRNA-mediated silencing, and blocks RNA-protein interactions. By tuning the spatial resolution and mismatch tolerance of CRISPR-Lock, we develop a high-resolution perturbation approach that employs genetically encoded CRISPR-Lock as a miRNA target protector. This system enables precise spatiotemporal control of miRNA:mRNA interactions, offering broader applicability compared to phosphorodiamidate mor-pholino (PMO) target protectors. Moreover, we demonstrate the potential therapeutic application of CRISPR-Lock for blocking pathological RNA-protein interactions in type 1 myotonic dystro-phy (DM1). Optimising CRISPR-Lock to target expanded repeat RNAs corrects approximately 85% of clinically relevant splicing biomarkers in patient-derived myotubes and significantly out-performs third-generation PMO antisense oligonucleotides. Finally, by delivering a miniaturised AAV-encoded CRISPR-Lock system into an established DM1 mouse model, we demonstrate the dose-dependent correction of intranuclear foci and splicing dysregulation, underscoring the potential therapeutic application of this technology.

bioengineering↗

bioRxiv2023

Ancient genomic linkage couples metabolism with erythroid development

Generation of mature cells from progenitors requires tight coupling of differentiation and metabolism. During erythropoiesis, erythroblasts are required to massively upregulate globin synthesis then clear extraneous material and enucleate to produce erythrocytes1-3. Nprl3 has remained in synteny with the -globin genes for >500 million years4, and harbours the majority of the -globin enhancers5. Nprl3 is a highly conserved inhibitor of mTORC1, which controls cellular metabolism. However, whether Nprl3 itself serves an erythroid role is unknown. Here, we show that Nprl3 is a key regulator of erythroid metabolism. Using Nprl3-deficient fetal liver and adult competitive bone marrow - fetal liver chimeras, we show that NprI3 is required for sufficient erythropoiesis. Loss of Nprl3 elevates mTORC1 signalling, suppresses autophagy and disrupts erythroblast glycolysis and redox control. Human CD34+ progenitors lacking NPRL3 produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Finally, we show that the -globin enhancers upregulate NprI3 expression, and that this activity is necessary for optimal erythropoiesis. Therefore, the anciently conserved linkage of NprI3, -globin and their associated enhancers has enabled coupling of metabolic and developmental control in erythroid cells. This may enable erythropoiesis to adapt to fluctuating nutritional and environmental conditions.

cell biology↗

bioRxiv2023

The characteristics of CTCF binding sequences contribute to enhancer blocking activity

While the elements encoding enhancers and promoters have been relatively well studied, the full spectrum of insulator elements which bind the CCCTC binding factor (CTCF), is relatively poorly characterised. This is partly due to the genomic context of CTCF sites greatly influencing their roles and activity. Here we have developed an experimental system to determine the ability of consistently sized, individual CTCF elements to interpose between enhancers and promoters and thereby reduce gene expression during differentiation. Importantly, each element is tested in the identical location thereby minimising the effect of genomic context. We found no correlation between the ability of CTCF elements to block enhancer-promoter activity with the amount of CTCF or cohesin bound at the natural genomic sites of these elements; the degree of evolutionary conservation; or their resemblance to the consensus core sequences. Nevertheless, we have shown that the strongest enhancer-promoter blockers include a previously described bound element lying upstream of the CTCF core motif. In addition, we found other uncharacterised DNaseI footprints located close to the core motif that may affect function. We have developed an assay of CTCF sequences which will enable researchers to sub-classify CTCF elements in a uniform and unbiased way.

molecular biology↗

bioRxiv2022

Type I PRMT inhibitor MS023 promotes SMN2 exon 7 inclusion and synergizes with nusinersen to rescue the phenotype of SMA mice

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed SMA patients life expectancy and quality of life. Nevertheless, these are not without limitations, and research efforts are underway to develop new approaches to be used alone and in combination, to ensure improved and long-lasting benefits for SMA patients. Protein arginine methyltransferases (PRMT) are emerging as druggable epigenetic targets, with several small molecule PRMT inhibitors already in clinical trial stage. From a screen of highly potent and selective next generation epigenetic small molecules, we have identified MS023, a potent and selective type I PRMT inhibitor, able to promote SMN2 exon 7 inclusion and increase SMN protein levels in preclinical SMA model, by inhibiting the binding of splicing factor hnRNPA1 to SMN2 pre-mRNA. Treatment of SMA mice with MS023 results in amelioration of the disease phenotype, with strong synergistic amplification of the positive effect when delivered in combination with the SMN2-targeting antisense oligonucleotide nusinersen. Moreover, transcriptomic analysis revealed that MS023 treatment has very minimal off-target effects and that the added benefit of the combination therapy is mainly attributable to targeting neuroinflammation. Our study warrants further clinical investigation of PRMT inhibition both as a stand-alone and add-on therapy for SMA patients.

neuroscience↗