%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 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