%0 Journal Article %J J Vis Exp %D 2022 %T CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications. %A Venkatesan, Vigneshwaran %A Christopher, Abisha Crystal %A Karuppusamy, Karthik V %A Babu, Prathibha %A Alagiri, Manoj Kumar K %A Thangavel, Saravanabhavan %K Animals %K CRISPR-Cas Systems %K Gene Editing %K Genetic Therapy %K Hematopoietic Stem Cell Transplantation %K Hematopoietic Stem Cells %K Mice %X

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

%B J Vis Exp %8 2022 08 09 %G eng %N 186 %R 10.3791/64064 %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 ACS Appl Bio Mater %D 2022 %T Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing. %A Rapaka, Hithavani %A Manturthi, Shireesha %A Arjunan, Porkizhi %A Venkatesan, Vigneshwaran %A Thangavel, Saravanabhavan %A Marepally, Srujan %A Patri, Srilakshmi V %K alpha-Tocopherol %K Amino Acids %K Cations %K Gene Editing %K Gene Transfer Techniques %K Hydrophobic and Hydrophilic Interactions %K Lipids %K Nucleic Acids %K Phenylalanine %K Triazoles %K Tryptophan %X

Intracellular delivery of biomolecules using non-viral vectors critically depends on the vectors' ability to allow the escape and release of the contents from the endosomes. Prior findings demonstrated that aromatic/hydrophobic group-containing amino acids such as phenylalanine (F) and tryptophan (W) destabilize cellular membranes by forming pores in the lipid bilayer. Taking cues from these findings, we have developed four α-tocopherol-based cationic amphiphiles by varying the aromatic/hydrophobic amino acids such as glycine (G), proline (P), phenylalanine (F), and tryptophan (W) as head groups and triazole in the linker region to study their impact on endosomal escape for the enhanced transfection efficacy. The lipids tocopherol-triazole-phenylalanine (TTF) and tocopherol-triazole-tryptophan (TTW) exhibited similar potential to commercial transfecting reagents, Lipofectamine (LF) 3000 and Lipofectamine Messenger Max (LFMM), respectively, in transfecting plasmid DNA and messenger RNA in multiple cultured cell lines. The TTW liposome was also found to be effective in delivering Cas9 mRNA and demonstrated equal efficiency of gene editing AAVS1 locus compared to LFMM in CHO, Neuro-2a, and EA.HY926 cell lines. In this current investigation, it is shown that the synthesized cationic lipids with aromatic hydrophobic R group-containing amino acids are safe, economic, and actually more efficient in nucleic acid delivery and genome-editing applications. These findings can be further explored in the genome-editing approach for treating genetic disorders.

%B ACS Appl Bio Mater %V 5 %P 1489-1500 %8 2022 Apr 18 %G eng %N 4 %R 10.1021/acsabm.1c01226 %0 Journal Article %J Life Sci %D 2022 %T Integration of synthetic and natural derivatives revives the therapeutic potential of temozolomide against glioma- an in vitro and in vivo perspective. %A Daisy Precilla, S %A Kuduvalli, Shreyas S %A Angeline Praveena, E %A Thangavel, Saravanabhavan %A Anitha, T S %X

AIMS: Malignant gliomas constitute one of the deadly brain tumors with high degeneration rate. Though temozolomide (TMZ) is the first-line drug for glioma, its efficacy has decreased due to chemo-resistance. Repurposing synthetic and natural compounds have gained increasing interest in glioma. Hence, we combined chloroquine (CHL) a synthetic drug, naringenin (NAR) and phloroglucinol (PGL) (natural derivatives), to investigate whether the apoptotic effect of these drugs both alone and in combination, enhances the anti-tumor effects of TMZ in an in vitro and in vivo orthotopic xenograft glioma model.

MAIN METHODS: The cytotoxic effect of the drugs was assessed in C6 (murine) glioma cells, U-87 MG and LN229 (human) glioblastoma cells, primary astrocytes (isolated from rat brain tissues) and HEK-293 T cells. Mitochondrial depolarization and alterations in the cell cycle was determined by confocal imaging and flow cytometry. The expression of angiogenic and apoptotic markers was evaluated using qRT-PCR and ELISA. The efficacy of the combinatorial treatment was assessed in an orthotopic xenograft model using U-87 MG cells.

KEY FINDINGS: The combinatorial treatment inhibited cell proliferation, induced apoptosis and contributed to cell cycle arrest in glioma cells. The quadruple combinatorial cocktail down-regulated BCL-2 with a concomitant decrease in VEGF. As observed in vitro, the quadruple combinatorial treatment enhanced the median survival of glioma-induced rats with lower cellularity rate.

SIGNIFICANCE: The combination of CHL, NAR and PGL synergistically potentiated the efficacy of TMZ on glioma in vitro and in vivo. Hence, this combination may characterize an advanced strategy for glioma treatment, thereby providing a possible translation to clinical trial.

%B Life Sci %V 301 %P 120609 %8 2022 May 06 %G eng %R 10.1016/j.lfs.2022.120609 %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