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.

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.

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.

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.

Corrigendum: Targeted Gene Delivery: Where to Land.

[This corrects the article DOI: 10.3389/fgeed.2020.609650.].

Recent Progress in Genome Editing for Gene Therapy Applications: The French Perspective.

Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.

Correction of ß-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells.

ß-thalassemias (ß-thal) are a group of blood disorders caused by mutations in the ß-globin gene (HBB) cluster. ß-globin associates with α-globin to form adult hemoglobin (HbA, α2ß2), the main oxygen-carrier in erythrocytes. When ß-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of ß-thal correlates with the number of inherited α-globin genes (HBA1 and HBA2), with α-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat ß-thal based on genome editing of the α-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) α-globin downregulation, by deleting the HBA2 gene to recreate an α-thalassemia trait, and (2) ß-globin expression, by targeted integration of a ß-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of ß-thalassemia (human erythroid progenitor cell [HUDEP-2] ß0). Then, we edited healthy donor HSPCs and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice. To assess the clinical potential of this approach, we next edited ß-thal HSPCs and achieved correction of α/ß globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve α/ß globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for ß-thal.

A combination of cyclophosphamide and interleukin-2 allows CD4+ T cells converted to Tregs to control scurfy syndrome.

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter's onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.

Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype.

Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) ß chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.

Author Correction: Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins.

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

GATA Factor-Mediated Gene Regulation in Human Erythropoiesis.

Erythroid commitment and differentiation are regulated by the coordinated action of a host of transcription factors, including GATA2 and GATA1. Here, we explored GATA-mediated transcriptional regulation through the integrative analysis of gene expression, chromatin modifications, and GATA factors' binding in human multipotent hematopoietic stem/progenitor cells, early erythroid progenitors, and late precursors. A progressive loss of H3K27 acetylation and a diminished usage of active enhancers and super-enhancers were observed during erythroid commitment and differentiation. GATA factors mediate transcriptional changes through a stage-specific interplay with regulatory elements: GATA1 binds different sets of regulatory elements in erythroid progenitors and precursors and controls the transcription of distinct genes during commitment and differentiation. Importantly, our results highlight a pivotal role of promoters in determining the transcriptional program activated upon erythroid differentiation. Finally, we demonstrated that GATA1 binding to a stage-specific super-enhancer sustains the expression of the KIT receptor in human erythroid progenitors.

Targeted Gene Delivery: Where to Land.

Genome-editing technologies have the potential to correct most genetic defects involved in blood disorders. In contrast to mutation-specific editing, targeted gene insertion can correct most of the mutations affecting the same gene with a single therapeutic strategy (gene replacement) or provide novel functions to edited cells (gene addition). Targeting a selected genomic harbor can reduce insertional mutagenesis risk, while enabling the exploitation of endogenous promoters, or selected chromatin contexts, to achieve specific transgene expression levels/patterns and the modulation of disease-modifier genes. In this review, we will discuss targeted gene insertion and the advantages and limitations of different genomic harbors currently under investigation for various gene therapy applications.

Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.

Editing the ß-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of ß-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the ß-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with ß-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9+ HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the ß-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.

Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human ß-globin locus.

Naturally occurring, large deletions in the ß-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and ß-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and ß-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the ß-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of ß-hemoglobinopathies.

The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension.

The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

Genome-wide maps of nuclear lamina interactions in single human cells.

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.

Nuclear lamins are not required for lamina-associated domain organization in mouse embryonic stem cells.

In mammals, the nuclear lamina interacts with hundreds of large genomic regions, termed lamina-associated domains (LADs) that are generally in a transcriptionally repressed state. Lamins form the major structural component of the lamina and have been reported to bind DNA and chromatin. Here, we systematically evaluate whether lamins are necessary for the LAD organization in murine embryonic stem cells. Surprisingly, removal of essentially all lamins does not have any detectable effect on the genome-wide interaction pattern of chromatin with emerin, a marker of the inner nuclear membrane. This suggests that other components of the lamina mediate these interactions.

Easy quantitative assessment of genome editing by sequence trace decomposition.

The efficacy and the mutation spectrum of genome editing methods can vary substantially depending on the targeted sequence. A simple, quick assay to accurately characterize and quantify the induced mutations is therefore needed. Here we present TIDE, a method for this purpose that requires only a pair of PCR reactions and two standard capillary sequencing runs. The sequence traces are then analyzed by a specially developed decomposition algorithm that identifies the major induced mutations in the projected editing site and accurately determines their frequency in a cell population. This method is cost-effective and quick, and it provides much more detailed information than current enzyme-based assays. An interactive web tool for automated decomposition of the sequence traces is available. TIDE greatly facilitates the testing and rational design of genome editing strategies.

Mechanisms and dynamics of nuclear lamina-genome interactions.

The nuclear lamina (NL) interacts with the genomic DNA and is thought to influence chromosome organization and gene expression. Both DNA sequences and histone modifications are important for NL tethering of the genomic DNA. These interactions are dynamic in individual cells and can change during differentiation and development. Evidence is accumulating that the NL contributes to the repression of transcription. Advances in mapping, genome-editing and microscopy techniques are increasing our understanding of the molecular mechanisms involved in NL-genome interactions.