Targeted genome editing in human repopulating haematopoietic stem cells

Abstract

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating Hematopoietic Stem Cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective cDNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked Severe Combined Immunodeficiency (SCID-X1). Gene edited HSCs sustained normal hematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open new avenues for treating SCID-X1 and other diseases.

Short-hairpin RNA against aberrant HBBIVSI-110(G>A) mRNA restores β-globin levels in a novel cell model and acts as mono- and combination therapy for β-thalassemia in primary hematopoietic stem cells

“…We note that current trends in gene-therapy development for β-globinopathies favor editing (for potentially improved biosafety) or universal treatment approaches (for R&D profitability). 24 However, improvements in the efficiency of targeted gene addition have started to address biosafety for integrating vectors, 23 and changes in regulatory requirements, manufacturing cost and reimbursement policies may in the future favor more effective, stratified gene-therapy applications, 25 such as that proposed in this study. Figure 3C (n=3), including raw values of band intensities as measured for the unsaturated original images using ImageJ, and including VCN measurements for the three independent biological replicates.…”

“…Besides higher vector yields and towards targeted gene addition for increased biosafety or exploitation of endogenous control elements, 22,23 the sequences required for shRNAmediated knockdown provide shorter and more efficient integration templates than the large fragments required for HBB gene addition. Moreover, mutation-specific shRNAs alone or combined with gene addition may achieve transfusion independence at lower VCN or at milder conditioning regimens, thus improving biosafety and tolerability of therapy.…”

Thalassemia 2016: Modern medicine battles an ancient disease

“…While this would be ideal, this approach has a low level of efficiency of transfecting hematopoietic stem cells in addition to low efficiency of the homologous recombination necessary for the correction of the mutation(s). Though gene editing will not be clinically applicable in the near future, a recent report suggests that these shortcomings can be overcome [85].…”

Lentiviral Hematopoietic Stem Cell Gene Therapy in Inherited Immune and Lysoso- mal Enzyme Deficiencies

“…Promoting engraftment by temporary mobilization of endogenous stem cells to open the stem cell niches in the bone marrow has been proposed [9] and applied successfully in the X-SCID mouse model [26]. An initial success has recently been reported in gene editing [20]. If the current clinical trials using lentiviral stem cell gene transfer prove efficacious and safe, its rapid clinical implementation in a variety of eligible inherited disorders will become within reach in the interest of the patients involved and thereby of health care and its costs.…”