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Sickle cell disease (SCD) is an autosomal recessive genetic disorder that occurs due to the point mutation in the ß-globin gene, which results in the formation of sickle hemoglobin (HbS) in the red blood cells (RBCs). When HbS is exposed to an oxygen-depleted environment, it polymerizes, resulting in hemolysis, vaso-occlusion pain, and impaired blood flow. Still, there is no affordable cure for this inherited disease. Approved medications held promise but were met with challenges due to limited patient tolerance and undesired side effects, thereby inhibiting their ability to enhance the quality of life across various individuals with SCD. Progress has been made in understanding the pathophysiology of SCD during the past few decades, leading to the discovery of novel targets and therapies. However, there is a compelling need for research to discover medications with improved efficacy and reduced side effects. Also, more clinical investigations on various drug combinations with different mechanisms of action are needed. This review comprehensively presents therapeutic approaches for SCD, including those currently available or under investigation. It covers fundamental aspects of the disease, such as epidemiology and pathophysiology, and provides detailed discussions on various disease-modifying agents. Additionally, expert insights are offered on the future development of pharmacotherapy for SCD.

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Rubinstein Taybi Syndrome (RSTS) is a rare genetic disorder which is caused by mutations in either CREBBP or EP300. RSTS with mutations in CREBBP is known as RSTS-1. We have generated an induced pluripotent stem cell (iPSC) line, IGIBi018-A from an Indian RSTS-patient using the episomal reprogramming method. The CREBBP gene in the patient harbours a nonsense mutation at position NM_004380.3(c.6876 del C). IGIBi018-A iPSC showed expression of pluripotent stem cell markers, has a normal karyotype and could be differentiated into three germ layers. This iPSC line will help to explore the role of CREBBP in RSTS associated developmental defects.

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It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20-42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice.

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Eye-related diseases, specifically retinal dystrophy (RD) conditions, are the leading cause of blindness worldwide. Gene addition, regulation, or editing could potentially treat such diseases through gene expression regulation. CRISPR/Cas9 gene editing is one of the most prominent and precise gene editing tools which could be employed to edit genes related to the dystrophic condition. However, CRISPR/Cas9 faces in vivo delivery challenges due to its high molecular weight, negative charge, prone to degradation in the presence of nucleases and proteases, poor cellular degradation, etc., which makes it challenging to adopt for therapeutic applications. We developed cRGD-modified lipopolymeric nanoplexes loaded with Cas9 RNPs with a particle size and zeta potential of 175 ±â€¯20 nm and 2.15 ±â€¯0.9 mV, respectively. The cRGD-modified lipopolymeric nanoplexes were stable for 194 h and able to transfect >70 % ARPE-19 and NIH3T3 cells with an Indel frequency of ~40 % for the VEGF-A gene. The cRGD-modified lipopolymeric nanoplexes found good vitreous mobility and could transfection retinal cells in vivo after 48 h of intravitreal injection in Wistar Rats. Moreover, in vivo VEGFA gene editing was ~10 % with minimal toxicities. Collectively, the cRGD-modified lipopolymeric nanoplexes were found to have extreme potential in delivering CRISPR/Cas9 RNPs payload to the retinal tissues for therapeutic applications.

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Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here, we report a novel antigen Plasmodium prohibitin "PfPHB2" and identify its cognate partner "Hsp70A1A" in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach, we show that loss of Hsp70A1A in red blood cells (RBCs) or PfPHB2 in infected red blood cells (iRBCs), respectively, inhibit PfPHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting PfPHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant PfPHB2 binds to RBCs which is inhibited by anti-PfPHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of PfPHB2 to serve as antigen is further supported by detection of anti-PfPHB2 antibody in patient sera. Overall, this study proposes PfPHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment.

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Ex vivo cellular system that accurately replicates sickle cell disease and ß-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and ß-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications.

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Sickle cell disease (SCD) is an autosomal recessive genetic disorder affecting millions of people worldwide. A reversible and selective DNMT1 inhibitor, GSK3482364, has been known to decrease the overall methylation activity of DNMT1, resulting in the increase of HbF levels and percentage of HbF-expressing erythrocytes in an in vitro and in vivo model. In this study, a structure-based virtual screening was done with GSK3685032, a co-crystalized ligand of DNMT1 (PDB ID: 6X9K) with an IC50 value of 0.036 µM and identified 3988 compounds from three databases (ChEMBL, PubChem and Drug Bank). Using this screening method, we identified around 15 compounds with XP docking scores greater than -8 kcal/mol. Further, prime MM-GBSA calculations have been performed and found compound SCHEMBL19716714 with the highest binding free energy of -83.31 kcal/mol. Finally, four compounds were identified based on glide energy and ΔG bind scores that have the most binding with DG7, DG19, DG20 bases and Lys1535, His1507, Trp1510, Ser1230, which were required for the target enzyme inhibition. Furthermore, molecular dynamics simulation studies of top ligands validate the stability of the docked complexes by examining root mean square deviations, root mean square fluctuations, solvent accessible surface area, and radius of gyration graphs from simulation trajectories. These findings suggest that the top four hit compounds may be capable of inhibiting DNMT1 and that additional in vitro and in vivo studies will be essential to prove the clinical effectiveness of the selected lead compounds.Communicated by Ramaswamy H. Sarma.

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ß-hemoglobinopathies such as ß-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire ß-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline.

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We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

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We remember Dr Ajay Parida, a leading plant biotechnologist, whose premature passing has deprived the Indian plant science community of a committed scientist and an able administrator. Born on 12 December 1963 in Bhagabanpur, Cuttack District (now Jajpur district), Odisha, he passed away in Guwahati on 19 July 2022. A collegial scientist, his down-to-earth and approachable nature, as well as his resourcefulness were instrumental in advancing the cause of Indian science and harnessing frontier biotechnological tools as vehicles of social consciousness. His expertise in quantitative DNA variation and molecular marker analysis, paved the way for subsequent research on mangrove molecular diversity at the M. S. Swaminathan Research Foundation (MSSRF), Chennai. His contributions to mangrove biology, genetics and genomics as well as extremophile plant species in the Indian context over two decades are a benchmark in his field. He also provided commendable leadership in his capacity as Director, Institute of Life Sciences (ILS), Bhubaneshwar during the COVID-19 pandemic.

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CRISPR/Cas9 has proven its accuracy and precision for gene editing by making a double-strand break at the predetermined site. Despite being a mainstream gene editing tool, CRISPR/Cas9 has limitations for its in vivo delivery due to the physico-chemical properties such as high molecular weight, supranegative charge, degradation in the presence of nucleases, etc. Hereby, a cationic lipopolymer is explored for its efficiency in delivering CRISPR/Cas9 plasmid (pCas9) in vitro and in vivo. The lipopolymer is utilized to form blank cationic nanoplexes having a zeta potential of +15.8 ± 0.7 mV. Being cationic, the blank nanoplexes are able to condense the pCas9 plasmid at a ratio of 1:20 with a complexation efficiency of ≈98% and show a size and zeta potential of ≈141 ± 16 nm and 4.2 mV ± 0.7, respectively. The pCas9-loaded nanoplexes show a transfection efficiency of ≈69% in ARPE-19 cells and show ≈22% of indel frequency, indicating the successful translation of Cas9 protein and guide RNA in the cytosol. Further, they are found to be stable under in vivo environment when given intravenously in Swiss albino mice. These lipopolymeric nanoplexes can be a potential carrier for CRISPR plasmids for genome editing applications.

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Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the ß-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA-/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA-/-, GBA+/- and GBA+/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis.

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Plant precursor miRNAs (pre-miRNA) have conserved evolutionary footprints that correlate with mode of miRNA biogenesis. In plants, base to loop and loop to base modes of biogenesis have been reported. Conserved structural element(s) in pre-miRNA play a major role in turn over and abundance of mature miRNA. Pre-miR396c sequences and secondary structural characteristics across Oryza species are presented. Based on secondary structure, twelve Oryza pre-miR396c sequences are divided into three groups, with the precursor from halophytic Oryza coarctata forming a distinct group. The miRNA-miRNA* duplex region is completely conserved across eleven Oryza species as are other structural elements in the pre-miRNA, suggestive of an evolutionarily conserved base-to-loop mode of miRNA biogenesis. SNPs within O. coarctata mature miR396c sequence and miRNA* region have the potential to alter target specificity and association with the RNA-induced silencing complex. A conserved SNP variation, rs10234287911 (G/A), identified in O. sativa pre-miR396c sequences alters base pairing above the miRNA-miRNA* duplex. The more stable structure conferred by the 'A10234287911' allele may promote better processing vis-à-vis the structure conferred by 'G10234287911' allele. We also examine pri- and pre-miR396c expression in cultivated rice under heat and salinity and their correlation with miR396c expression.

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Trichophyton rubrum is the most prevalent causative agent responsible for 80-90% of all known superficial fungal infections in humans, worldwide. Limited available methods for genetic manipulations have been one of the major bottlenecks in understanding relevant molecular mechanisms of disease pathogenesis in T. rubrum. Here, a dual-plasmid-based CRISPR/Cas9 strategy to edit pH regulatory transcription factor, pacC, of a clinical isolate of T. rubrum by non-homologous end joining (NHEJ) repair is presented. A cas9-eGFP fusion that aids pre-screening of primary transformants through detection of GFP fluorescence is expressed from one plasmid while target-specific sgRNA from the other brings about mutagenesis of pacC with an overall efficiency of 33.8-37.3%. The mutants had reduced transcript levels of pacC at both acidic and alkaline pH with several morphological abnormalities. We believe this dual-plasmid-based CRISPR/Cas9 strategy will aid functional genomics studies, especially in non-lab-adapted clinical strains of T. rubrum.

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In addition to their role in cellular energy production, mitochondria are increasingly recognized as regulators of the innate immune response of phagocytes. Here, we demonstrate that altering expression levels of the mitochondria-associated enzyme, cytidine monophosphate kinase 2 (CMPK2), disrupts mitochondrial physiology and significantly deregulates the resting immune homeostasis of macrophages. Both CMPK2 silenced and constitutively overexpressing macrophage lines portray mitochondrial stress with marked depolarization of their membrane potential, enhanced reactive oxygen species (ROS), and disturbed architecture culminating in the enhanced expression of the pro-inflammatory genes IL1ß, TNFα, and IL8. Interestingly, the long-term modulation of CMPK2 expression resulted in an increased glycolytic flux of macrophages akin to the altered physiological state of activated M1 macrophages. While infection-induced inflammation for restricting pathogens is regulated, our observation of a total dysregulation of basal inflammation by bidirectional alteration of CMPK2 expression only highlights the critical role of this gene in mitochondria-mediated control of inflammation.

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sgRNA/Cas9 ribonucleoproteins (RNPs) provide a site-specific robust gene-editing approach avoiding the mutagenesis and unwanted off-target effects. However, the high molecular weight (∼165 kDa), hydrophilicity and net supranegative charge (∼-20 mV) hinder the intracellular delivery of these RNPs. In the present study, we have prepared cationic RNPs lipopolymeric nanoplexes that showed a size of 117.3 ± 7.64 nm with +6.17 ± 1.04 mV zeta potential and >90% entrapment efficiency of RNPs. Further, these RNPs lipopolymeric nanoplexes showed good complexation efficiency and were found to be stable for 12 h with fetal bovine serum. These RNPs lipopolymeric nanoplexes did not induce any significant cytotoxicity in HEK293T cells, and were efficiently uptaken via a clathrin-mediated pathway with optimal transfection efficiency and nuclear localization after 48 h. Further, HEK293T cells having the mGFP insert were used as a cell line model for gene editing, wherein the loss of the mGFP signal was observed as a function of gene editing after transfection with mGFP targeting RNPs lipopolymeric nanoplexes. Further, the T7 endonuclease and TIDE assay data showed a decent gene editing efficiency. Additionally, the lipopolymeric nanoplexes were able to transfect muscle cells in vivo, when injected intra-muscularly. Collectively, this study explored the potential of cationic lipopolymeric nanoplexes for delivering gene-editing endonucleases.

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Phosphorylation and other post-translational modifications of red blood cell (RBC) proteins govern membrane function and have a role in the invasion of RBCs by the malaria parasite, Plasmodium falciparum. Furthermore, a percentage of RBC proteins are palmitoylated, although the functional consequences are unknown. We establish dynamic palmitoylation of 118 RBC membrane proteins using click chemistry and acyl biotin exchange (ABE)-coupled LC-MS/MS and characterize their involvement in controlling membrane organization and parasite invasion. RBCs were treated with a generic palmitoylation inhibitor, 2-bromopalmitate (2-BMP), and then analyzed using ABE-coupled LC-MS/MS. Only 42 of the 118 palmitoylated proteins detected were palmitoylated in the 2-BMP-treated sample, indicating that palmitoylation is dynamically regulated. Interestingly, membrane receptors such as semaphorin 7A, CR1, and ABCB6, which are known to be involved in merozoite interaction with RBCs and parasite invasion, were found to be dynamically palmitoylated, including the blood group antigen, Kell, whose antigenic abundance was significantly reduced following 2-BMP treatment. To investigate the involvement of Kell in merozoite invasion of RBCs, a specific antibody to its extracellular domain was used. The antibody targeting Kell inhibited merozoite invasion of RBCs by 50%, implying a role of Kell, a dynamically palmitoylated potent host-derived receptor, in parasite invasion. Furthermore, a significant reduction in merozoite contact with the RBC membrane and a consequent decrease in parasite invasion following 2-BMP treatment demonstrated that palmitoylation does indeed regulate RBC susceptibility to parasite invasion. Taken together, our findings revealed the dynamic palmitoylome of RBC membrane proteins and its role in P. falciparum invasion.

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Sickle cell disease (SCD) is the most prevalent life-threatening blood monogenic disorder. Currently, there is no cure available, apart from bone marrow transplantation. Early and efficient diagnosis of SCD is key to disease management, which would make considerable strides in alleviating morbidity and reducing mortality. However, the cost and complexity of diagnostic procedures, such as the Sanger sequencing method, impede the early detection of SCD in a resource-limited setting. To address this, the current study demonstrates a simple and efficient proof-of-concept assay for the detection of patients and carriers using extraction-free non-invasive buccal swab samples by isothermal DNA Amplification coupled Restrictase-mediated cleavage (iDAR). This study is a first of its kind reporting the use of buccal swab specimens for iDA in molecular diagnosis of a genetic disease, all the while being cost effective and time saving, with the total assay time of around 150 min at a cost of USD 5. Further, iDAR demonstrates 91.5% sensitivity and 100% specificity for detecting all three alleles: SS, AS, and AA, having a 100% concordance with Sanger sequencing. The applicability of the iDAR assay is further demonstrated with its adaptation to a one-pot reaction format, which simplifies the assay system. Overall, iDAR is a simple, cost-effective, precise, and non-invasive assay for SCD screening, with the potential for use in a limited resource setting.

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Limited treatment options and research in understanding the pathomechanisms of rare diseases has raised concerns about their therapeutic development. One such poorly understood ultra-rare neuromuscular disorder is GNE Myopathy (GNEM) which is caused due to mutation in key sialic acid biosynthetic enzyme, GNE. Treatment with sialic acid or its derivatives/precursors slows the disease progression, but curative strategies need to be explored further. Pathologically, muscle biopsy samples of GNEM patients reveal rimmed vacuole formation due to aggregation of ß-amyloid, Tau, presenilin proteins with unknown mechanism. The present study aims to understand the mechanism of protein aggregate formation in GNE mutant cells to decipher role of chaperones in disease phenotype. The pathologically relevant GNE mutations expressed as recombinant proteins in HEK cells was used as a model system for GNEM to estimate extent of protein aggregation. We identified HSP70, a chaperone, as binding partner of GNE. Downregulation of HSP70 with altered BAG3, JNK, BAX expression levels was observed in GNE mutant cells. The cell apoptosis was observed in GNE mutation specific manner. An activator of HSP70 chaperone, BGP-15, rescued the phenotypic defects due to GNE mutation, thereby, reducing protein aggregation significantly. The results were further validated in rat skeletal muscle cell lines carrying single Gne allele. Our study suggests that HSP70 activators can be a promising therapeutic target in the treatment of ultra-rare GNE Myopathy disease.

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