Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

In vivo measurement of RBC survival in patients with sickle cell disease before or after hematopoietic stem cell transplantation.

ABSTRACT: Stable, mixed-donor-recipient chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with sickle cell disease (SCD) is sufficient for phenotypic disease reversal, and results from differences in donor/recipient-red blood cell (RBC) survival. Understanding variability and predictors of RBC survival among patients with SCD before and after HSCT is critical for gene therapy research which seeks to generate sufficient corrected hemoglobin to reduce polymerization thereby overcoming the red cell pathology of SCD. This study used biotin labeling of RBCs to determine the lifespan of RBCs in patients with SCD compared with patients who have successfully undergone curative HSCT, participants with sickle cell trait (HbAS), and healthy (HbAA) donors. Twenty participants were included in the analysis (SCD pre-HSCT: N = 6, SCD post-HSCT: N = 5, HbAS: N = 6, and HbAA: N = 3). The average RBC lifespan was significantly shorter for participants with SCD pre-HSCT (64.1 days; range, 35-91) compared with those with SCD post-HSCT (113.4 days; range, 105-119), HbAS (126.0 days; range, 119-147), and HbAA (123.7 days; range, 91-147) (P<.001). RBC lifespan correlated with various hematologic parameters and strongly correlated with the average final fraction of sickled RBCs after deoxygenation (P<.001). No adverse events were attributable to the use of biotin and related procedures. Biotin labeling of RBCs is a safe and feasible methodology to evaluate RBC survival in patients with SCD before and after HSCT. Understanding differences in RBC survival may ultimately guide gene therapy protocols to determine hemoglobin composition required to reverse the SCD phenotype as it relates directly to RBC survival. This trial was registered at www.clinicaltrials.gov as #NCT04476277.

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.

Prdm16 is a critical regulator of adult long-term hematopoietic stem cell quiescence.

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene Prdm16 during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of Prdm16 in adult HSCs remain unclear. To investigate the role of Prdm16 in adult HSCs, we generated a novel conditional knockout mouse model and deleted Prdm16 in adult mouse hematopoietic system using the IFN-inducible Mx1-Cre Our results show that Prdm16 deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. Prdm16 deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of Prdm16 significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without Prdm16 deletion showed that Prdm16 loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which Cdkn1a and Egr1 were identified as direct targets of Prdm16 Our results suggest that Prdm16 preserves the function of adult LT-HSCs by promoting their quiescence.

Pogz deficiency leads to transcription dysregulation and impaired cerebellar activity underlying autism-like behavior in mice.

Several genes implicated in autism spectrum disorder (ASD) are chromatin regulators, including POGZ. The cellular and molecular mechanisms leading to ASD impaired social and cognitive behavior are unclear. Animal models are crucial for studying the effects of mutations on brain function and behavior as well as unveiling the underlying mechanisms. Here, we generate a brain specific conditional knockout mouse model deficient for Pogz, an ASD risk gene. We demonstrate that Pogz deficient mice show microcephaly, growth impairment, increased sociability, learning and motor deficits, mimicking several of the human symptoms. At the molecular level, luciferase reporter assay indicates that POGZ is a negative regulator of transcription. In accordance, in Pogz deficient mice we find a significant upregulation of gene expression, most notably in the cerebellum. Gene set enrichment analysis revealed that the transcriptional changes encompass genes and pathways disrupted in ASD, including neurogenesis and synaptic processes, underlying the observed behavioral phenotype in mice. Physiologically, Pogz deficiency is associated with a reduction in the firing frequency of simple and complex spikes and an increase in amplitude of the inhibitory synaptic input in cerebellar Purkinje cells. Our findings support a mechanism linking heterochromatin dysregulation to cerebellar circuit dysfunction and behavioral abnormalities in ASD.

ßT87Q-Globin Gene Therapy Reduces Sickle Hemoglobin Production, Allowing for Ex Vivo Anti-sickling Activity in Human Erythroid Cells.

Lentiviral addition of ßT87Q-globin, a modified ß-globin with an anti-sickling mutation, is currently being used in gene therapy trials for sickle cell disease (SCD) and ß-thalassemia patients. ßT87Q-globin interferes with sickle hemoglobin (HbS) polymerization. Here, we generated the SCD mutation in an immortalized human erythroid cell line (HUDEP-2) to investigate the anti-sickling activity of ßT87Q-globin. Sickle HUDEP-2 (sHUDEP-2) cells produced robust HbS after differentiation and sickled under deoxygenated conditions, comparable with SCD CD34+ progeny. Lentiviral transduction provided 9.5-26.8 pg/cell ßT87Q-globin (R2 = 0.83) in a vector copy number (VCN)-dependent manner, resulting in a significant reduction of sickling ratios (R2 = 0.92). Interestingly, ßT87Q-globin transduction markedly reduced endogenous ßS-globin (R2 = 0.84) to an undetectable level (0.4-16.8 pg/cell) in sHUDEP-2 cells, as well as endogenous ß-globin in human CD34+ cell-derived erythroid cells. RNA sequencing (RNA-seq) analysis with ßT87Q-transduced sHUDEP-2 and human CD34+-derived cells revealed activation of inflammation- and proliferation-related programs, suggesting minimal changes in background gene expression except for ßT87Q-globin expression and endogenous ß/ßS-globin suppression. In summary, using sHUDEP-2 and CD34+-derived cells, we demonstrated that lentiviral addition of ßT87Q-globin strongly reduced endogenous ß-/ßS-globin expression, resulting in an anti-sickling effect. Our findings should be helpful to understand the anti-sickling effects of therapeutic genes in SCD gene therapy.

POGZ Is Required for Silencing Mouse Embryonic ß-like Hemoglobin and Human Fetal Hemoglobin Expression.

Fetal globin genes are transcriptionally silenced during embryogenesis through hemoglobin switching. Strategies to derepress fetal globin expression in the adult could alleviate symptoms in sickle cell disease and ß-thalassemia. We identified a zinc-finger protein, pogo transposable element with zinc-finger domain (POGZ), expressed in hematopoietic progenitor cells. Targeted deletion of Pogz in adult hematopoietic cells in vivo results in persistence of embryonic ß-like globin expression without affecting erythroid development. POGZ binds to the Bcl11a promoter and erythroid-specific intragenic regulatory regions. Pogz+/- mice show elevated embryonic ß-like globin expression, suggesting that partial reduction of Pogz expression results in persistence of embryonic ß-like globin expression. Knockdown of POGZ in primary human CD34+ progenitor cell-derived erythroblasts reduces BCL11A expression, a known repressor of embryonic ß-like globin expression, and increases fetal hemoglobin expression. These findings are significant, since new therapeutic targets and strategies are needed to treat ß-globin disorders.