Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of ß-hemoglobinopathies.

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate ß-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to ß-globin exon-1 (ßE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-ßE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells in vivo. Furthermore, PRR-ßE1 gene editing is feasible without ex vivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and ß-thalassemia major. These findings imply that PRR-ßE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for ß-hemoglobinopathy gene therapy.

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 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.

The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy.

Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.

Preferential Expansion of Human CD34+CD133+CD90+ Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy.

CD34+CD133+CD90+ 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 ex vivo 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 CD34+CD133+CD90+ HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo 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 in vivo. 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 in vivo. This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell 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.

The Strategies and Challenges of CCR5 Gene Editing in Hematopoietic Stem and Progenitor Cells for the Treatment of HIV.

HIV infection continues to be a serious health issue with an alarming global spread, owing to the fact that attempts at developing an effective vaccine or a permanent cure remains futile. So far, the only available treatment for the clinical management of HIV is the combined Anti-Retroviral Therapy (cART), but the long-term cART is associated with metabolic changes, organ damages, and development and transmission of drug resistant HIV strains. Thus, there is a need for the development of one-time curative treatment for HIV infection. The allogeneic transplantation with the Hematopoietic Stem and Progenitor cells (HSPCs) having 32 bp deletion in Chemokine receptor 5 gene (CCR5 Δ32) demonstrated successful HIV remission in the Berlin and London patients, and highlighted that transplantation of CCR5 null HSPCs is a promising approach for a long- term HIV remission. The advent of gene editing technologies offers a new choice of generating ex vivo CCR5 ablated allogeneic or autologous HSPCs for stem cell transplantation into HIV patients. Many groups are attempting CCR5 disruption in HSPCs using various gene-editing strategies. At least two such studies, involving CCR5 gene editing in HSPCs have entered the clinical trials. This review aims to outline the strategies taken for CCR5 gene editing and discuss the challenges associated with the development of CCR5 manipulated HSPCs for the gene therapy of HIV infection.

Manipulation of Developmental Gamma-Globin Gene Expression: an Approach for Healing Hemoglobinopathies.

ß-Hemoglobinopathies are the most common monogenic disorders, and a century of research has provided us with a better understanding of the attributes of these diseases. Allogenic stem cell transplantation was the only potentially curative option available for these diseases until the discovery of gene therapy. The findings on the protective nature of fetal hemoglobin in sickle cell disease (SCD) and thalassemia patients carrying hereditary persistence of fetal hemoglobin (HPFH) mutations has given us the best evidence that the cure for ß-hemoglobinopathies remains hidden in the hemoglobin locus. The detailed understanding of the developmental gene regulation of gamma-globin (γ-globin) and the emergence of gene manipulation strategies offer us the opportunity for developing a γ-globin gene-modified autologous stem cell transplantation therapy. In this review, we summarize different therapeutic strategies that reactivate fetal hemoglobin for the gene therapy of ß-hemoglobinopathies.

The Periostin/Integrin-αv Axis Regulates the Size of Hematopoietic Stem Cell Pool in the Fetal Liver.

We earlier showed that outside-in integrin signaling through POSTN-ITGAV interaction plays an important role in regulating adult hematopoietic stem cell (HSC) quiescence. Here, we show that Itgav deletion results in increased frequency of phenotypic HSCs in fetal liver (FL) due to faster proliferation. Systemic deletion of Postn led to increased proliferation of FL HSCs, albeit without any loss of stemness, unlike Vav-Itgav-/- HSCs. Based on RNA sequencing analysis of FL and bone marrow HSCs, we predicted the involvement of DNA damage response pathways in this dichotomy. Indeed, proliferative HSCs from Postn-deficient FL tissues showed increased levels of DNA repair, resulting in lesser double-strand breaks. Thus POSTN, with its expression majorly localized in the vascular endothelium of FL tissue, acts as a regulator of stem cell pool size during development. Overall, we demonstrate that the duality of response to proliferation in HSCs is developmental stage dependent and can be correlated with DNA damage responses.