Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease.

BACKGROUND: Sickle cell disease is characterized by hemolytic anemia, pain, and progressive organ damage. A high level of erythrocyte fetal hemoglobin (HbF) comprising α- and γ-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor of γ-globin expression and HbF production in adult erythrocytes. Its down-regulation is a promising therapeutic strategy for induction of HbF. METHODS: We enrolled patients with sickle cell disease in a single-center, open-label pilot study. The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR), allowing erythroid lineage-specific knockdown. Patients were assessed for primary end points of engraftment and safety and for hematologic and clinical responses to treatment. RESULTS: As of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months (range, 7 to 29). All patients had engraftment, and adverse events were consistent with effects of the preparative chemotherapy. All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.3%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell. Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period. CONCLUSIONS: This study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk-benefit profile in sickle cell disease. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656).

F-erythrocytes promote Plasmodium falciparum proliferation in sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) remains prevalent because heterozygous carriers (HbAS) are partially resistant to Plasmodium falciparum malaria. Sickle hemoglobin (HbS) polymerization in low and intermediate oxygen (O2 ) conditions is the main driver of HbAS-driven resistance to P. falciparum malaria. However, epidemiological studies have reported mixed malaria morbidity and mortality outcomes in individuals with sickle cell disease (SCD). While maximum-tolerated dose hydroxyurea has been shown to lower malaria incidence, fetal hemoglobin (HbF), an inhibitor of HbS polymerization that is variably packaged in F-erythrocytes, might provide hemoglobin that is accessible to the parasite for feeding. METHODS: To explore that risk, we examined the effect of variable mean corpuscular fetal hemoglobin (MCHF) on P. falciparum proliferation, invasion, and development in HbSS RBCs. RESULTS: We found that greater MCHF in HbSS red blood cells (RBCs) is associated with increased P. falciparum proliferation in O2 environments comparable with the microcirculation. Moreover, both parasite invasion and intracellular growth, the major components of proliferation, occur predominantly in F-erythrocytes and are augmented with increasing MCHF. CONCLUSIONS: HbF modifies P. falciparum infection in HbSS RBCs, further highlighting the complexity of the molecular interactions between these two diseases. Other inhibitors of HbS polymerization that do not increase HbF or F-erythrocytes should be independently assessed for their effects on P. falciparum malaria proliferation in HbSS RBCs.

Perivascular deletion of murine Rac reverses the ratio of marrow arterioles and sinusoid vessels and alters hematopoiesis in vivo.

Hematopoietic stem cells (HSCs) are localized within specialized microenvironments throughout the BM. Nestin-expressing (Nestin(+)) mesenchymal stromal cells (MSCs) are important in the perivascular space. Rac is critical for MSC cell shape in vitro, whereas its function in MSCs in vivo remains poorly characterized. We hypothesized that deletion of Rac in the Nestin(+) cells would perturb the perivascular space, altering HSC localization and hematopoiesis. Nestin-Cre-directed excision of Rac1 in Rac3(-/-) mice reduces Nestin(+) cells in the marrow. We observed a 2.7-fold decrease in homing of labeled wild-type hematopoietic cells into Rac1(Δ/Δ)Rac3(-/-) mice compared with control mice. Rac1(Δ/Δ)Rac3(-/-) mice demonstrated a marked decrease in arterioles and an increase in the number and volume of venous sinusoids in the marrow that was associated with a reduction in the numbers of immunophenotypically and functionally-defined long-term HSCs in the marrow, a decrease in colony-forming cells and a reduction in circulating progenitors. Rac-deleted animals demonstrated a significant increase in trabecular bone. These data demonstrate that Rac GTPases play an important role in the integrity of perivascular space. Increased trabecular bone and sinusoidal space and decreased arteriolar volume in this model were associated with decreased HSC, underscoring the complexity of regulation of hematopoiesis in the perivascular space.

Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease.

Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms.

RhoH participates in a multi-protein complex with the zinc finger protein kaiso that regulates both cytoskeletal structures and chemokine-induced T cells.

RhoH is a haematopoietic -specific, GTPase-deficient Rho GTPase that plays an essential role in T lymphocyte development and haematopoietic cell migration. RhoH is known to interact with ZAP70 in T cell receptor (TCR) signaling and antagonize Rac GTPase activity. To further elucidate the molecular mechanisms of RhoH in T cell function, we carried out in vivo biotinylation and mass spectrometry analysis to identify new RhoH-interacting proteins in Jurkat T cells. We indentified Kaiso by streptavidin capture and confirmed the interaction with RhoH by co-immunoprecipitation. Kaiso is a 95 kDa dual-specific Broad complex, Trantrak, Bric-a-brac/Pox virus, Zinc finger (POZ-ZF) transcription factor that has been shown to regulate both gene expression and p120 catenin-associated cell-cell adhesions. We further showed that RhoH, Kaiso and p120 catenin all co-localize at chemokine-induced actin-containing cell protrusion sites. Using RhoH knockdown we demonstrated that Kaiso localization depends on RhoH function. Similar to the effect of RhoH deficiency, Kaiso down-regulation led to altered cell migration and actin-polymerization in chemokine stimulated Jurkat cells. Interestingly, RhoH and Kaiso also co-localized to the nucleus in a time-dependent fashion after chemokine stimulation and with T cell receptor activation where RhoH is required for Kaiso localization. Based on these results and previous studies, we propose that extracellular microenvironment signals regulate RhoH and Kaiso to modulate actin-cytoskeleton structure and transcriptional activity during T cell migration.

Retroviral transduction of murine and human hematopoietic progenitors and stem cells.

Genetic modification of cells using retroviral vectors is the method of choice when the cell population is difficult to transfect and/or requires persistent transgene expression in progeny cells. There are innumerable potential applications for these procedures in laboratory research and clinical therapeutic interventions. One paradigmatic example is the genetic modification of hematopoietic stem and progenitor cells (HSPCs). These are rare nucleated cells which reside in a specialized microenvironment within the bone marrow, and have the potential to self-renew and/or differentiate into all hematopoietic lineages. Due to their enormous regenerative capacity in steady state or under stress conditions these cells are routinely used in allogeneic bone marrow transplantation to reconstitute the hematopoietic system in patients with metabolic, inflammatory, malignant, and other hematologic disorders. For patients lacking a matched bone marrow donor, gene therapy of autologous hematopoietic stem cells has proven to be an alternative as highlighted recently by several successful gene therapy trials. Genetic modification of HSPCs using retrovirus vectors requires ex vivo manipulation to efficiently introduce the new genetic material into cells (transduction). Optimal culture conditions are essential to facilitate this process while preserving the stemness of the cells. The most frequently used retroviral vector systems for the genetic modifications of HSPCs are derived either from Moloney murine leukemia-virus (Mo-MLV) or the human immunodeficiency virus-1 (HIV-1) and are generally termed according to their genus gamma-retroviral (γ-RV) or lentiviral vectors (LV), respectively. This chapter describes in a step-by-step fashion some techniques used to produce research grade vector supernatants and to obtain purified murine or human hematopoietic stem cells for transduction, as well as follow-up methods for analysis of transduced cell populations.