Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models.

Sickle cell disease (SCD) is caused by a 20A > T mutation in the ß-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34+ cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact ß-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34+ cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus ß-to-ßs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34+ cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34+ HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.

Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease.

Gene editing with the CRISPR-Cas9 system could revolutionize hematopoietic stem cell (HSC)-targeted gene therapy for hereditary diseases, including sickle cell disease (SCD). Conventional delivery of editing tools by electroporation limits HSC fitness due to its toxicity; therefore, efficient and non-toxic delivery remains crucial. Integrating lentiviral vectors are established for therapeutic gene delivery to engraftable HSCs in gene therapy trials; however, their sustained expression and size limitation preclude their use for CRISPR-Cas9 delivery. Here, we developed a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). We demonstrated efficient one-time correction of the SCD mutation in the endogenous ßs-globin gene up to 42% at the protein level (p < 0.01) with the Cas9 protein delivery non-integrating lentiviral all-in-one system without electroporation. Our findings improve prospects for efficient and safe genome editing.

Sustained fetal hemoglobin induction in vivo is achieved by BCL11A interference and coexpressed truncated erythropoietin receptor.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)-targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A-based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A-expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.

Hematopoietic-Stem-Cell-Targeted Gene-Addition and Gene-Editing Strategies for ß-hemoglobinopathies.

Sickle cell disease (SCD) is caused by a well-defined point mutation in the ß-globin gene and therefore is an optimal target for hematopoietic stem cell (HSC) gene-addition/editing therapy. In HSC gene-addition therapy, a therapeutic ß-globin gene is integrated into patient HSCs via lentiviral transduction, resulting in long-term phenotypic correction. State-of-the-art gene-editing technology has made it possible to repair the ß-globin mutation in patient HSCs or target genetic loci associated with reactivation of endogenous γ-globin expression. With both approaches showing signs of therapeutic efficacy in patients, we discuss current genetic treatments, challenges, and technical advances in this field.

Immunoresponse to Gene-Modified Hematopoietic Stem Cells.

Gene transfer to and correction of hematopoietic stem cells (HSCs) are ideal strategies to cure a number of congenital and acquired disorders. However, transgene products may trigger immunological rejection of modified cells, limiting their therapeutic benefits. Preclinical and clinical data indicate that myeloablative total body irradiation (TBI) allows for efficient engraftment and tolerance to gene-modified HSCs. In contrast, myeloablative chemotherapy using busulfan or similar agents is only sufficient to induce tolerance to gene-modified HSCs producing no or non-immunogenic protein. If cells are modified to produce a protein that is xenogenic or congenitally absent in the patient, additional immunosuppression may be required to prevent an immunological reaction to the transduced cells. New gene editing and in vivo gene therapy techniques could pose additional immune concerns compared to ex vivo gene therapy methods. This review is intended to guide the design of conditioning and immunosuppression therapy in HSC-targeted gene therapy, as well as gene editing.

Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease.

BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear. METHODS: In this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients. RESULTS: In co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism. CONCLUSIONS: In summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.

Busulfan Combined with Immunosuppression Allows Efficient Engraftment of Gene-Modified Cells in a Rhesus Macaque Model.

Busulfan conditioning is utilized for hematopoietic stem cell (HSC) depletion in the context of HSC gene-therapy conditioning but may result in insufficient immunosuppression. In this study, we evaluated whether additional immunosuppression is required for efficient engraftment of gene-modified cells using a rhesus HSC lentiviral gene-therapy model. We transduced half of rhesus CD34+ cells with an enhanced green fluorescent protein (GFP)-encoding vector (immunogenic) and the other half with a γ-globin-encoding vector (no predicted immunogenicity). After autologous transplantation of both transduced cell populations following myeloablative busulfan conditioning (5.5 mg/kg/day for 4 days), we observed immunological rejection of GFP-transduced cells up to 3 months post-transplant and stable engraftment of γ-globin-transduced cells in two animals, demonstrating that ablative busulfan conditioning is sufficient for engraftment of gene-modified cells producing non-immunogenic proteins but insufficient to permit engraftment of immunogenic proteins. We then added immunosuppression with abatacept and sirolimus to busulfan conditioning and observed engraftment of both GFP- and γ-globin-transduced cells in two animals, demonstrating that additional immunosuppression allows for engraftment of gene-modified cells expressing immunogenic proteins. In conclusion, myeloablative busulfan conditioning should permit engraftment of gene-modified cells producing non-immunogenic proteins, while additional immunosuppression is required to prevent immunological rejection of a neoantigen.

High-Efficiency Lentiviral Transduction of Human CD34+ Cells in High-Density Culture with Poloxamer and Prostaglandin E2.

Hematopoietic stem cell (HSC) gene therapy is curative for various hereditary diseases; however, high-efficiency transduction in HSCs remains crucial to improve the prospects for hemoglobinopathies. We previously optimized lentiviral transduction in human CD34+ cells with serum-free medium containing minimal cytokines, allowing efficient transduction (∼50%) and robust xenograft engraftment. In this study, we further improved lentiviral transduction in human CD34+ cells. High-density culture conditions (4e6/mL) resulted in ∼5-fold more efficient transduction in CD34+ cells (p < 0.01) compared with standard cell density (1e5/mL). After co-culturing vector-exposed CD34+ cells with non-transduced CD34+ cells, high-density culture conditions enhanced lentiviral gene marking in the non-transduced population (p < 0.01) compared with low-density conditions, suggesting that increasing cell-to-cell contact allows more efficient transduction. Two adjuvants, poloxamer 407 (100 µg/mL) and prostaglandin E2 (10 µM), were added to high-density CD34+ cells, resulting in ∼4-fold more efficient transduction (p < 0.01) without significant toxicity compared with no adjuvant control. In summary, we developed a highly efficient lentiviral transduction method in high-density CD34+ cell culture with poloxamer 407 and prostaglandin E2, allowing overall ∼10-fold improvement in transduction efficiency and consistently achieving more than 90% transduction and an average vector copy number of ∼10. Our optimized transduction method should improve gene therapy approaches using lentiviral vectors targeting HSCs.

Low-Dose Busulfan Reduces Human CD34+ Cell Doses Required for Engraftment in c-kit Mutant Immunodeficient Mice.

Humanized animal models are central to efforts aimed at improving hematopoietic stem cell (HSC) transplantation with or without genetic modification. Human cell engraftment is feasible in immunodeficient mice; however, high HSC doses and conditioning limit broad use of xenograft models. We assessed human CD45+ chimerism after transplanting varying doses of human CD34+ HSCs (2 × 105 to 2 × 106 cells/mouse) with or without busulfan (BU) pretransplant conditioning in c-kit mutant mice that do not require conditioning (non-obese diabetic [NOD]/B6/severe combined immunodeficiency [SCID]/ interleukin-2 receptor gamma chain null (IL-2rγ-/-) KitW41/W41 [NBSGW]). We then tested a range of BU (5-37.5 mg/kg) using 2 × 105 human CD34+ cells. Glycophorin-A erythrocyte chimerism was assessed after murine macrophage depletion using clodronate liposomes. We demonstrated successful long-term engraftment of human CD34+ cells at all cell doses in this model, and equivalent engraftment using 10-fold less CD34+ cells with the addition of BU conditioning. Low-dose BU (10 mg/kg) was sufficient to allow human engraftment using 2 × 105 CD34+ cells, whereas higher doses (≥37.5 mg/kg) were toxic. NBSGW mice support human erythropoiesis in the bone marrow; however, murine macrophage depletion provided only minimal and transient increases in peripheral blood human erythrocytes. Our xenograft model is therefore useful in HSC gene therapy and genome-editing studies, especially for modeling in disorders, such as sickle cell disease, where access to HSCs is limited.