%0 Journal Article %J Front Genome Ed %D 2022 %T Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2. %A George, Anila %A Ravi, Nithin Sam %A Prasad, Kirti %A Panigrahi, Lokesh %A Koikkara, Sanya %A Rajendiran, Vignesh %A Devaraju, Nivedhitha %A Paul, Joshua %A Pai, Aswin Anand %A Nakamura, Yukio %A Kurita, Ryo %A Balasubramanian, Poonkuzhali %A Thangavel, Saravanabhavan %A Marepally, Srujan %A Velayudhan, Shaji R %A Srivastava, Alok %A Mohankumar, Kumarasamypet M %X

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.

%B Front Genome Ed %V 4 %P 1085111 %8 2022 %G eng %R 10.3389/fgeed.2022.1085111 %0 Journal Article %J Sci Rep %D 2022 %T Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications. %A Bagchi, Abhirup %A Devaraju, Nivedhitha %A Chambayil, Karthik %A Rajendiran, Vignesh %A Venkatesan, Vigneshwaran %A Sayed, Nilofer %A Pai, Aswin Anand %A Nath, Aneesha %A David, Ernest %A Nakamura, Yukio %A Balasubramanian, Poonkuzhali %A Srivastava, Alok %A Thangavel, Saravanabhavan %A Mohankumar, Kumarasamypet M %A Velayudhan, Shaji R %K Animals %K Cell Line, Tumor %K Cell Lineage %K DNA-Binding Proteins %K Genetic Vectors %K Humans %K Lentivirus %K Mice %K RNA Interference %K RNA, Small Interfering %K Transcription Factors %K Transduction, Genetic %X

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.

%B Sci Rep %V 12 %P 14033 %8 2022 08 18 %G eng %N 1 %R 10.1038/s41598-022-13783-0 %0 Journal Article %J Elife %D 2022 %T Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. %A Ravi, Nithin Sam %A Wienert, Beeke %A Wyman, Stacia K %A Bell, Henry William %A George, Anila %A Mahalingam, Gokulnath %A Vu, Jonathan T %A Prasad, Kirti %A Bandlamudi, Bhanu Prasad %A Devaraju, Nivedhitha %A Rajendiran, Vignesh %A Syedbasha, Nazar %A Pai, Aswin Anand %A Nakamura, Yukio %A Kurita, Ryo %A Narayanasamy, Muthuraman %A Balasubramanian, Poonkuzhali %A Thangavel, Saravanabhavan %A Marepally, Srujan %A Velayudhan, Shaji R %A Srivastava, Alok %A DeWitt, Mark A %A Crossley, Merlin %A Corn, Jacob E %A Mohankumar, Kumarasamypet M %K Adenine %K Anemia, Sickle Cell %K beta-Globins %K beta-Thalassemia %K Cell Line %K Clustered Regularly Interspaced Short Palindromic Repeats %K CRISPR-Cas Systems %K Cytosine %K Fetal Hemoglobin %K gamma-Globins %K Gene Editing %K Hematopoietic Stem Cells %K Humans %K Point Mutation %K Promoter Regions, Genetic %X

Naturally occurring point mutations in the 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 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 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 promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

%B Elife %V 11 %8 2022 02 11 %G eng %R 10.7554/eLife.65421 %0 Journal Article %J Cell Death Dis %D 2019 %T Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia. %A Ganesan, Saravanan %A Palani, Hamenth Kumar %A Lakshmanan, Vairavan %A Balasundaram, Nithya %A Alex, Ansu Abu %A David, Sachin %A Venkatraman, Arvind %A Korula, Anu %A George, Biju %A Balasubramanian, Poonkuzhali %A Palakodeti, Dasaradhi %A Vyas, Neha %A Mathews, Vikram %X

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

%B Cell Death Dis %V 10 %P 736 %8 2019 Sep 30 %G eng %N 10 %R 10.1038/s41419-019-1964-8