An optimized SpCas9 high-fidelity variant for direct protein delivery.

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

Combination of lentiviral and genome editing technologies for the treatment of sickle cell disease.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene leading to polymerization of the sickle hemoglobin (HbS) and deformation of red blood cells. Autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically modified using lentiviral vectors (LVs) to express an anti-sickling ß-globin leads to some clinical benefit in SCD patients, but it requires high-level transgene expression (i.e., high vector copy number [VCN]) to counteract HbS polymerization. Here, we developed therapeutic approaches combining LV-based gene addition and CRISPR-Cas9 strategies aimed to either knock down the sickle ß-globin and increase the incorporation of an anti-sickling globin (AS3) in hemoglobin tetramers, or to induce the expression of anti-sickling fetal γ-globins. HSPCs from SCD patients were transduced with LVs expressing AS3 and a guide RNA either targeting the endogenous ß-globin gene or regions involved in fetal hemoglobin silencing. Transfection of transduced cells with Cas9 protein resulted in high editing efficiency, elevated levels of anti-sickling hemoglobins, and rescue of the SCD phenotype at a significantly lower VCN compared to the conventional LV-based approach. This versatile platform can improve the efficacy of current gene addition approaches by combining different therapeutic strategies, thus reducing the vector amount required to achieve a therapeutic VCN and the associated genotoxicity risk.

ABCG2 Is Overexpressed on Red Blood Cells in Ph-Negative Myeloproliferative Neoplasms and Potentiates Ruxolitinib-Induced Apoptosis.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.

Impact of hydroxycarbamide and interferon-α on red cell adhesion and membrane protein expression in polycythemia vera.

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within over-expressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms.

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies.

Sickle cell disease (SCD) is due to a mutation in the ß-globin gene causing production of the toxic sickle hemoglobin (HbS; α2ßS 2). Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling ß-globin (ßAS) is a promising treatment; however, it is only partially effective, and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing ßAS3-globin and an artificial microRNA (amiRNA) specifically downregulating ßS-globin expression with the aim of reducing HbS levels and favoring ßAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPCs by the bifunctional LV led to a substantial decrease of ßS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells, and effective correction of the sickling phenotype, outperforming ßAS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile, and neither HSPC viability, engraftment, and multilineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

CRISPRthripsis: The Risk of CRISPR/Cas9-induced Chromothripsis in Gene Therapy.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system has allowed the generation of disease models and the development of therapeutic approaches for many genetic and non-genetic disorders. However, the generation of large genomic rearrangements has raised safety concerns for the clinical application of CRISPR/Cas9 nuclease approaches. Among these events, the formation of micronuclei and chromosome bridges due to chromosomal truncations can lead to massive genomic rearrangements localized to one or few chromosomes. This phenomenon, known as chromothripsis, was originally described in cancer cells, where it is believed to be caused by defective chromosome segregation during mitosis or DNA double-strand breaks. Here, we will discuss the factors influencing CRISPR/Cas9-induced chromothripsis, hereafter termed CRISPRthripsis, and its outcomes, the tools to characterize these events and strategies to minimize them.

Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression.

Sickle cell disease and ß-thalassemia affect the production of the adult ß-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the -200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy - even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating ß-hemoglobinopathies.

Genome editing approaches to ß-hemoglobinopathies.

ß-hemoglobinopathies are the most common monogenic disorders worldwide and are caused by mutations in the ß-globin locus altering the production of adult hemoglobin (HbA). Transplantation of autologous hematopoietic stem cells (HSCs) corrected by lentiviral vector-mediated addition of a functional ß-like globin raised new hopes to treat sickle cell disease and ß-thalassemia patients; however, the low expression of the therapeutic gene per vector copy is often not sufficient to fully correct the patients with a severe clinical phenotype. Recent advances in the genome editing field brought new possibilities to cure ß-hemoglobinopathies by allowing the direct modification of specific endogenous loci. Double-strand breaks (DSBs)-inducing nucleases (i.e., ZFNs, TALENs and CRISPR-Cas9) or DSB-free tools (i.e., base and prime editing) have been used to directly correct the disease-causing mutations, restoring HbA expression, or to reactivate the expression of the fetal hemoglobin (HbF), which is known to alleviate clinical symptoms of ß-hemoglobinopathy patients. Here, we describe the different genome editing tools, their application to develop therapeutic approaches to ß-hemoglobinopathies and ongoing clinical trials using genome editing strategies.

Base and Prime Editing Technologies for Blood Disorders.

Nuclease-based genome editing strategies hold great promise for the treatment of blood disorders. However, a major drawback of these approaches is the generation of potentially harmful double strand breaks (DSBs). Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. The scope of base editing tools has been extensively broadened, allowing higher efficiency, specificity, accessibility to previously inaccessible genetic loci and multiplexing, while maintaining a low rate of Insertions and Deletions (InDels). Base editing is a promising therapeutic strategy for genetic diseases caused by point mutations, such as many blood disorders and might be more effective than approaches based on homology-directed repair, which is moderately efficient in hematopoietic stem cells, the target cell population of many gene therapy approaches. In this review, we describe the development and evolution of the base editing system and its potential to correct blood disorders. We also discuss challenges of base editing approaches-including the delivery of base editors and the off-target events-and the advantages and disadvantages of base editing compared to classical genome editing strategies. Finally, we summarize the recent technologies that have further expanded the potential to correct genetic mutations, such as the novel base editing system allowing base transversions and the more versatile prime editing strategy.

Endothelial Cells Harbouring the JAK2V617F Mutation Display Pro-Adherent and Pro-Thrombotic Features.

Thromboembolic events are the main cause of mortality in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) but their underlying mechanisms are largely unrecognized. The Janus kinase 2 (JAK2)V617F mutation is the most frequent genetic alteration leading to MPN. Usually found in haematopoietic progenitors and stem cells, this mutation has also been described in endothelial cells (ECs) of MPN patients. In this study, we have questioned the impact of the JAK2V617F mutation on EC phenotype and functions. We developed an induced pluripotent stem cells strategy to compare JAK2 mutant and wild-type ECs. Transcriptomic assays showed that several genes and pathways involved in inflammation, cell adhesion and thrombotic events were over-represented in JAK2V617F ECs and expression levels of von Willebrand factor and P-selectin (CD62P) proteins were increased. Finally, we found that leucocytes from MPN patients adhere more tightly to JAK2V617F ECs. Our results show that JAK2V617F ECs have a pro-inflammatory and pro-thrombotic phenotype and were functionally pro-adherent.

Abnormal adhesion of red blood cells in polycythemia vera: a prothrombotic effect?

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterised by the V617F activating mutation in the tyrosine kinase JAK2. PV patients exhibit increased haemoglobin levels and red cell mass because of uncontrolled proliferation of the erythroid lineage. Thrombosis and transformation to acute leukaemia are the major causes of morbidity and mortality in this disease. Increased thrombotic risk in PV patients is multifactorial and complex; it is associated with high levels of haemoglobin, impaired rheology and increased viscosity resulting from erythrocytosis. An additional parameter that might contribute to this risk was recently brought to light by work from our group showing abnormal activation of adhesion proteins in PV RBCs. In this review we provide an overview of these recent findings and discuss how the pro-adhesive features of JAK2V617F-positive red blood cells might initiate and contribute to the circulatory complications described in PV.