crisprcas9-gene-editing-hematopoietic-stem-progenitor-cells-for-gene

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

Christian Medical College Campus

Manipal Academy of Higher Education

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.

Preferential Expansion of Human CD34CD133CD90 Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy.

The advent of next-generation genome engineering tools like CRISPR-Cas9 has transformed the field of gene therapy, rendering targeted treatment for several incurable diseases. Hematopoietic stem and progenitor cells (HSPCs) continue to be the ideal target cells for gene manipulation due to their long-term repopulation potential. Among the gene manipulation strategies such as lentiviral gene augmentation, non-homologous end joining (NHEJ)-mediated gene editing, base editing and prime editing, only the homology-directed repair (HDR)-mediated gene editing provides the option of inserting a large transgene under its endogenous promoter or any desired locus. In addition, HDR-mediated gene editing can be applied for the gene knock-out, correction of point mutations and introduction of beneficial mutations. HSPC gene therapy studies involving lentiviral vectors and NHEJ-based gene-editing studies have exhibited substantial clinical progress. However, studies involving HDR-mediated HSPC gene editing have not yet progressed to the clinical testing. This suggests the existence of unique challenges in exploiting HDR pathway for HSPC gene therapy. Our review summarizes the mechanism, recent progresses, challenges, and the scope of HDR-based gene editing for the HSPC gene therapy.

Prathibha Babu

Centre for Stem Cell Research (CSCR),
A unit of InStem Bengaluru,
Centre for Stem Cell Research (CSCR), A unit of InStem Bengaluru

Christian Medical College Campus

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

Prathibha Babu

Centre for Stem Cell Research (CSCR),A unit of InStem Bengaluru,Centre for Stem Cell Research (CSCR), A unit of InStem Bengaluru

Christian Medical College Campus

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

Centre for Stem Cell Research (CSCR),
A unit of InStem Bengaluru,
Centre for Stem Cell Research (CSCR), A unit of InStem Bengaluru