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1دورية أكاديمية
المؤلفون: Abhirup Bagchi, Nivedhitha Devaraju, Karthik Chambayil, Vignesh Rajendiran, Vigneshwaran Venkatesan, Nilofer Sayed, Aswin Anand Pai, Aneesha Nath, Ernest David, Yukio Nakamura, Poonkuzhali Balasubramanian, Alok Srivastava, Saravanabhavan Thangavel, Kumarasamypet M. Mohankumar, Shaji R. Velayudhan
المصدر: Scientific Reports, Vol 12, Iss 1, Pp 1-13 (2022)
الوصف: Abstract Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel lineage-specific RNA interference (RNAi) lentiviral vectors, H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for any gene, and they also provide superior knockdown of the target gene expression with a single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A, two well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34+ cells with these vectors resulted in >80% reduction in the target protein levels and up to 40% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34+ cells transduced with H23B-Ery-Lin-shBCL11A LV in immunocompromised mice showed ~ 60% reduction in BCL11A protein expression with ~ 40% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34+ cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.
وصف الملف: electronic resource
العلاقة: https://doaj.org/toc/2045-2322Test
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2دورية أكاديميةEfficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2
المؤلفون: Anila George, Nithin Sam Ravi, Kirti Prasad, Lokesh Panigrahi, Sanya Koikkara, Vignesh Rajendiran, Nivedhitha Devaraju, Joshua Paul, Aswin Anand Pai, Yukio Nakamura, Ryo Kurita, Poonkuzhali Balasubramanian, Saravanabhavan Thangavel, Srujan Marepally, Shaji R. Velayudhan, Alok Srivastava, Kumarasamypet M. Mohankumar
المصدر: Frontiers in Genome Editing, Vol 4 (2022)
مصطلحات موضوعية: prime editing, sickle cell anaemia, CRISPR, haemoglobinopathies, beta-globin, Biotechnology, TP248.13-248.65, Genetics, QH426-470
الوصف: Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.
وصف الملف: electronic resource
العلاقة: https://www.frontiersin.org/articles/10.3389/fgeed.2022.1085111/fullTest; https://doaj.org/toc/2673-3439Test
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3دورية أكاديمية
المؤلفون: Nithin Sam Ravi, Beeke Wienert, Stacia K Wyman, Henry William Bell, Anila George, Gokulnath Mahalingam, Jonathan T Vu, Kirti Prasad, Bhanu Prasad Bandlamudi, Nivedhitha Devaraju, Vignesh Rajendiran, Nazar Syedbasha, Aswin Anand Pai, Yukio Nakamura, Ryo Kurita, Muthuraman Narayanasamy, Poonkuzhali Balasubramanian, Saravanabhavan Thangavel, Srujan Marepally, Shaji R Velayudhan, Alok Srivastava, Mark A DeWitt, Merlin Crossley, Jacob E Corn, Kumarasamypet M Mohankumar
المصدر: eLife, Vol 11 (2022)
مصطلحات موضوعية: base editing, globin regulation, HPFH mutations, beta-hemoglobinopathies, fetal hemoglobin, CRISPR/Cas9, Medicine, Science, Biology (General), QH301-705.5
الوصف: Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the –123 region. Base editing at –123 and –124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of –123T > C and –124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.
وصف الملف: electronic resource
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المؤلفون: Kirti Prasad, Nivedhitha Devaraju, Anila George, Nithin Sam Ravi, Gokulnath Mahalingam, Vignesh Rajendiran, Lokesh Panigrahi, Vigneshwaran Venkatesan, Kartik Lakhotiya, Yogapriya Moorthy, Aswin Anand Pai, Yukio Nakamura, Ryo Kurita, Poonkuzhali Balasubramanian, Saravanabhavan Thangavel, Shaji R Velayudhan, Srujan Marepally, Alok Srivastava, Kumarasamypet M Mohankumar
الوصف: β-thalassemia and HbE result from mutations in the β-globin locus that impedes the production of functional β-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of β-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::23e4cd3c8318f2001c9d7e4914d6c62fTest
https://doi.org/10.1101/2022.06.01.494256Test -
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المصدر: Methods in molecular biology (Clifton, N.J.). 2429
مصطلحات موضوعية: Gene Editing, Mice, Hematopoietic Stem Cell Transplantation, Animals, CRISPR-Cas Systems, Hematopoietic Stem Cells, Transplantation, Autologous
الوصف: Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=pmid________::f37000b493dbbf652092021a66bc6c91Test
https://pubmed.ncbi.nlm.nih.gov/35507170Test -
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المؤلفون: Nithin Sam Ravi, Beeke Wienert, Stacia K Wyman, Henry William Bell, Anila George, Gokulnath Mahalingam, Jonathan T Vu, Kirti Prasad, Bhanu Prasad Bandlamudi, Nivedhitha Devaraju, Vignesh Rajendiran, Nazar Syedbasha, Aswin Anand Pai, Yukio Nakamura, Ryo Kurita, Muthuraman Narayanasamy, Poonkuzhali Balasubramanian, Saravanabhavan Thangavel, Srujan Marepally, Shaji R Velayudhan, Alok Srivastava, Mark A DeWitt, Merlin Crossley, Jacob E Corn, Kumarasamypet M Mohankumar
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::10cdfe121f2613584ecf0e1f49c26c47Test
https://doi.org/10.7554/elife.65421.sa2Test -
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المصدر: Methods in Molecular Biology ISBN: 9781071619780
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::26757c77317797cf38f59de3e3c7bfd9Test
https://doi.org/10.1007/978-1-0716-1979-7_20Test -
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المؤلفون: Anila George (14281289), Nithin Sam Ravi (14281292), Kirti Prasad (14281295), Lokesh Panigrahi (14281298), Sanya Koikkara (14281301), Vignesh Rajendiran (14281304), Nivedhitha Devaraju (14281307), Joshua Paul (14281310), Aswin Anand Pai (14281313), Yukio Nakamura (39120), Ryo Kurita (393269), Poonkuzhali Balasubramanian (714388), Saravanabhavan Thangavel (4591855), Srujan Marepally (496823), Shaji R. Velayudhan (12291734), Alok Srivastava (290078), Kumarasamypet M. Mohankumar (12291731)
مصطلحات موضوعية: Genetics, Molecular Biology, Genetic Engineering, Molecular Medicine, Gene and Molecular Therapy, prime editing, sickle cell anaemia, CRISPR, haemoglobinopathies, beta-globin
الوصف: Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.
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المؤلفون: Anila George (14281289), Nithin Sam Ravi (14281292), Kirti Prasad (14281295), Lokesh Panigrahi (14281298), Sanya Koikkara (14281301), Vignesh Rajendiran (14281304), Nivedhitha Devaraju (14281307), Joshua Paul (14281310), Aswin Anand Pai (14281313), Yukio Nakamura (39120), Ryo Kurita (393269), Poonkuzhali Balasubramanian (714388), Saravanabhavan Thangavel (4591855), Srujan Marepally (496823), Shaji R. Velayudhan (12291734), Alok Srivastava (290078), Kumarasamypet M. Mohankumar (12291731)
مصطلحات موضوعية: Genetics, Molecular Biology, Genetic Engineering, Molecular Medicine, Gene and Molecular Therapy, prime editing, sickle cell anaemia, CRISPR, haemoglobinopathies, beta-globin
الوصف: Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.
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