BCL11A +58/+55 enhancer-editing facilitates HSPC engraftment and HbF induction in rhesus macaques conditioned with a CD45 antibody-drug conjugate.

Editing the +58 region of the BCL11A erythroid enhancer has shown promise in treating β-globin disorders. To address variations in fetal hemoglobin (HbF) response, we investigated editing both +58 and +55 enhancers. Rhesus macaques transplanted with edited hematopoietic stem/progenitor cells (HSPCs) following busulfan conditioning exhibited durable, high-level (∼90%) editing frequencies post transplantation with sustained HbF reactivation over 4 years, without hematological perturbations.

Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model.

Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies.

CD3-immunotoxin mediated depletion of T cells in lymphoid tissues of rhesus macaques.

Selective T-cell depletion prior to cell or organ transplantation is considered a preconditioning regimen to induce tolerance and immunosuppression. An immunotoxin consisting of a recombinant anti-CD3 antibody conjugated with diphtheria toxin was used to eliminate T-cells. It showed significant T-cell depletion activity in the peripheral blood and lymph nodes in animal models used in previous studies.

Pre-existing immunity does not impair the engraftment of CRISPR-Cas9-edited cells in rhesus macaques conditioned with busulfan or radiation.

CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from (SpCas9) and (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system.

Forced enhancer-promoter rewiring to alter gene expression in animal models.

Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the β-globin locus (locus control region [LCR]) contacts the fetal-type () and adult-type () β-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and genes in cultured adult-stage erythroid cells can activate transcription.

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 β-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 β-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels.

Sustained fetal hemoglobin induction in vivo is achieved by 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.

Comparative engraftment and clonality of macaque HSPCs expanded on human umbilical vein endothelial cells versus non-expanded cells.

hematopoietic stem and progenitor cell (HSPC) expansion platforms are under active development, designed to increase HSPC numbers and thus engraftment ability of allogeneic cord blood grafts or autologous HSPCs for gene therapies. Murine and models have not correlated well with clinical outcomes of HSPC expansion, emphasizing the need for relevant pre-clinical models. Our rhesus macaque HSPC competitive autologous transplantation model utilizing genetically barcoded HSPC allows direct analysis of the relative short and long-term engraftment ability of lentivirally transduced HSPCs, along with additional critical characteristics such as HSPC clonal diversity and lineage bias.

Intrabone transplantation of CD34+ cells with optimized delivery does not enhance engraftment in a rhesus macaque model.

Intrabone (IB) injection of umbilical cord blood has been proposed as a potential mechanism to improve transplant engraftment and prevent graft failure. However, conventional IB techniques produce low retention of transplanted cells in the marrow. To overcome this barrier, we developed an optimized IB (OIB) injection method using low-volume, computer-controlled slow infusion that promotes cellular retention in the marrow.

NADPH oxidase correction by mRNA transfection of apheresis granulocytes in chronic granulomatous disease.

Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity.

BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer.

Impact of CMV Infection on Natural Killer Cell Clonal Repertoire in CMV-Naïve Rhesus Macaques.

Recent functional, gene expression, and epigenetic studies have suggested the presence of a subset of mature natural killer (NK) cells responsible for maintaining NK cell memory. The lack of endogenous clonal markers in NK cells impedes understanding the genesis of these cell populations. In humans, primates, and mice, this phenotype and memory or adaptive functions have been strongly linked to cytomegalovirus or related herpes virus infections.

Clonal tracking of erythropoiesis in rhesus macaques.

The classical model of hematopoietic hierarchies is being reconsidered on the basis of data from assays and single cell expression profiling. Recent experiments suggested that the erythroid lineage might differentiate directly from multipotent hematopoietic stem cells / progenitors or from a highly biased subpopulation of stem cells, rather than transiting through common myeloid progenitors or megakaryocyte-erythrocyte progenitors. We genetically barcoded autologous rhesus macaque stem and progenitor cells, allowing quantitative tracking of the clonal output of thousands of individual cells over time following transplantation.

Development of a forward-oriented therapeutic lentiviral vector for hemoglobin disorders.

Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing.

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).

Fetal hemoglobin and F-cell variance in mobilized CD34 cell-transplanted rhesus monkeys.

Elevated fetal hemoglobin (HbF) is associated with reduced severity of sickle cell disease. Therefore, γ-globin protein levels and F-cell (HbF-positive red blood cell) percentages are used for estimation of clinical benefit. Here, we monitored transplantation-related changes in HbF and F-cell percentages for rhesus macaques (Macaca mulatta) following total body irradiation or busulfan conditioning prior to CD34 cell transplantation.

Aberrant Clonal Hematopoiesis following Lentiviral Vector Transduction of HSPCs in a Rhesus Macaque.

Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR.

Barcoding of Macaque Hematopoietic Stem and Progenitor Cells: A Robust Platform to Assess Vector Genotoxicity.

Gene therapies using integrating retrovirus vectors to modify hematopoietic stem and progenitor cells have shown great promise for the treatment of immune system and hematologic diseases. However, activation of proto-oncogenes via insertional mutagenesis has resulted in the development of leukemia. We have utilized cellular bar coding to investigate the impact of different vector designs on the clonal behavior of hematopoietic stem and progenitor cells (HSPCs) during expansion, as a quantitative surrogate assay for genotoxicity in a non-human primate model with high relevance for human biology.

Clonal expansion and compartmentalized maintenance of rhesus macaque NK cell subsets.

Natural killer (NK) cells recognize and eliminate infected and malignant cells. Their life histories are poorly understood, particularly in humans, due to lack of informative models and endogenous clonal markers. Here, we apply transplantation of barcoded rhesus macaque hematopoietic cells to interrogate the landscape of NK cell production, expansion, and life histories at a clonal level long term and after proliferative challenge.

Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia.

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells.

The impact of aging on primate hematopoiesis as interrogated by clonal tracking.

Age-associated changes in hematopoietic stem and progenitor cells (HSPCs) have been carefully documented in mouse models but poorly characterized in primates and humans. To investigate clinically relevant aspects of hematopoietic aging, we compared the clonal output of thousands of genetically barcoded HSPCs in aged vs young macaques after autologous transplantation. Aged macaques showed delayed emergence of output from multipotent (MP) clones, with persistence of lineage-biased clones for many months after engraftment.

Geographic clonal tracking in macaques provides insights into HSPC migration and differentiation.

The geographic distribution of hematopoiesis at a clonal level is of interest in understanding how hematopoietic stem and progenitor cells (HSPCs) and their progeny interact with bone marrow (BM) niches during regeneration. We tagged rhesus macaque autologous HSPCs with genetic barcodes, allowing clonal tracking over time and space after transplantation. We found marked geographic segregation of CD34 HSPCs for at least 6 mo posttransplantation, followed by very gradual clonal mixing at different BM sites over subsequent months to years.

CD4+ levels control the odds of induction of humoral immune responses to tracer doses of therapeutic antibodies.

Rapidly increasing number of therapeutic antibodies are being repurposed to imaging probes for noninvasive diagnosis, as well as monitoring during treatment or disease recurrence. Though antibody-based imaging involves tracer doses (~3 log lower than therapeutic doses), and immune responses are severely reduced in patients with impaired immunity, formation of anti-tracer antibodies (ATA) has been observed hampering further diagnostic monitoring. Here, we explored the potential to develop humoral responses to intravenously administered tracer dose of a monoclonal antibody F(ab΄)2 fragment, and associated with host related immune measures in 49 rhesus macaques categorized into healthy (uninfected controls), SIV-progressors, SIV non-progressors, or total body irradiated (TBI).

Bone Marrow as a Hematopoietic Stem Cell Source for Gene Therapy in Sickle Cell Disease: Evidence from Rhesus and SCD Patients.

Steady state bone marrow (BM) is the preferred hematopoietic stem cell (HSC) source for gene therapy in sickle cell disease (SCD) due to the recognized risk of vaso-occlusive crisis during granulocyte colony-stimulating factor mobilization. We previously established clinically relevant HSC gene transfer in the rhesus model following transplantation of mobilized peripheral blood (PB) CD34 cells transduced with lentiviral vectors. In this study, we examined steady state bone marrow (BM) in the rhesus competitive repopulation model and demonstrate similar gene marking in vitro and in vivo, as compared with mobilized PB CD34 cells.