%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 Methods Mol Biol %D 2022 %T Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System. %A Devaraju, Nivedhitha %A Rajendiran, Vignesh %A Ravi, Nithin Sam %A Mohankumar, Kumarasamypet M %K Animals %K CRISPR-Cas Systems %K Gene Editing %K Hematopoietic Stem Cell Transplantation %K Hematopoietic Stem Cells %K Mice %K Transplantation, Autologous %X

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.

%B Methods Mol Biol %V 2429 %P 307-331 %8 2022 %G eng %R 10.1007/978-1-0716-1979-7_20 %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 Hum Gene Ther %D 2022 %T Preferential Expansion of Human CD34CD133CD90 Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy. %A Christopher, Abisha Crystal %A Venkatesan, Vigneshwaran %A Karuppusamy, Karthik V %A Srinivasan, Saranya %A Babu, Prathibha %A Azhagiri, Manoj Kumar K %A Chambayil, Karthik %A Bagchi, Abhirup %A Rajendiran, Vignesh %A Ravi, Nithin Sam %A Kumar, Sanjay %A Marepally, Srujan Kumar %A Mohankumar, Kumarasamypet Murugesan %A Srivastava, Alok %A Velayudhan, Shaji R %A Thangavel, Saravanabhavan %K Animals %K Antigens, CD34 %K Fetal Blood %K Genetic Therapy %K Hematopoietic Stem Cell Transplantation %K Hematopoietic Stem Cells %K Humans %K Mice %K Mice, Inbred NOD %K Mice, SCID %X

CD34CD133CD90 hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34CD133CD90 HSCs over other subpopulations of adult HSPCs in culture. The preferential expansion enriches the HSCs in culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold . The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells . This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

%B Hum Gene Ther %V 33 %P 188-201 %8 2022 02 %G eng %N 3-4 %R 10.1089/hum.2021.089