Fu, B., Liao, J., Chen, S. et al. CRISPR–Cas9-mediated gene editing of the BCL11A enhancer for pediatric β0/β0 transfusion-dependent β-thalassemia. Nat Med (2022). https://doi.org/10.1038/s41591-022-01906-z
Gene editing to disrupt the GATA1-binding site at the +58 BCL11A erythroid enhancer could induce γ-globin expression, which is a promising therapeutic strategy to alleviate β-hemoglobinopathy caused by HBB gene mutation. In the present study, we report the preliminary results of an ongoing phase 1/2 trial (NCT04211480) evaluating safety and efficacy of gene editing therapy in children with blood transfusion-dependent β-thalassemia (TDT). We transplanted BCL11A enhancer-edited, autologous, hematopoietic stem and progenitor cells into two children, one carrying the β0/β0 genotype, classified as the most severe type of TDT. Primary endpoints included engraftment, overall survival and incidence of adverse events (AEs). Both patients were clinically well with multilineage engraftment, and all AEs to date were considered unrelated to gene editing and resolved after treatment. Secondary endpoints included achieving transfusion independence, editing rate in bone marrow cells and change in hemoglobin (Hb) concentration. Both patients achieved transfusion independence for >18 months after treatment, and their Hb increased from 8.2 and 10.8 g dl−1 at screening to 15.0 and 14.0 g dl−1 at the last visit, respectively, with 85.46% and 89.48% editing persistence in bone marrow cells. Exploratory analysis of single-cell transcriptome and indel patterns in edited peripheral blood mononuclear cells showed no notable side effects of the therapy.
Zhang, X., Zhu, B., Chen, L. et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nature Biotechnology (2020).
Although base editors are useful tools for precise genome
editing, current base editors can only convert either adenines
or cytosines. We developed a dual adenine and cytosine base
editor (A&C-BEmax) by fusing both deaminases with a Cas9
nickase to achieve C-to-T and A-to-G conversions at the same
target site. Compared to single base editors, A&C-BEmax’s
activity on adenines is slightly reduced, whereas activity on
cytosines is higher and RNA off-target activity is substan-
Yang L, Wang L, Huo Y, et al. Amelioration of an Inherited Metabolic Liver Disease through Creation of a De Novo Start Codon by Cytidine Base Editing [published online ahead of print, 2020 May 7].
Correction to: Increasing targeting scope
of adenosine base editors in mouse and rat
embryos through fusion of TadA deaminase
with Cas9 variants
Zhang X, Chen L, Zhu B, et al. Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain [published online ahead of print, 2020 May 11]. Nature Cell Biology. 2020.
Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci,
and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a
non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases,
serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window
towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively cata-
lysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with
eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in
the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of β-haemoglobinopathy,
suggesting the therapeutic potential of the improved base editors.
Wang L, Li L, Ma Y, Hu H, et al. Reactivation of γ-globin Expression through Cas9 or Base Editor to Treat β-Hemoglobinopathies. Cell Research, 2020,1
Mutations in the β-globin gene, the essential component of adult hemoglobin (HbA; α2β2), results in either a production of aberrant sickle hemoglobin (HbS) leading to sickle cell disease (SCD) or an insufficient β-globin synthesis leading to β-thalassemia. These two major forms of β-hemoglobinopathies cause impaired erythropoiesis and life-threatening anemia. Clinical evidence has suggested that re-activation of fetal γ-globin (HBG) gene expression which is normally silenced after birth by certain genetic mutations can ameliorate the clinical course of β- hemoglobinopathies 1, 2. In β-thalassemia, elevated levels of fetal γ-globin interact with α-globin to form fetal hemoglobin (HbF; α2γ2) restoring the α/β-like globin ratio and in SCD the γ-globin reduces HbS polymerization.
Wu Y, Zeng J, Roscoe B P, et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells[J]. Nature Medicine, 2019.
Re-expression of the paralogous γ-globin genes (HBG1/2 could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adultstage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9: sgRNA ribonucleoprotein(RNP)-mediated cleavage within a GATAL binding site at the +58 BCLTLA erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCLTLA expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express herapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HBF induction.
Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants[J]. Protein & Cell, 2018, 9(9): 814-819.
we demonstrated that ABE and its variants efficiently generate site-specific A:T>G:C conversions in cell lines, mouse and rat embryos. We found that the editing window of ABE7.10 in rodent embryos is from position 2–9. To the best of our knowledge, this is the first report to demonstrate efficient generation of point mutations through base editors in rats. The SaKKH-ABE and VQR-ABE system will be important tools to diversify the range of ABE targets in the genome. As A>G conversion may correct 48% of the pathogenic human SNPs (Gaudelli et al., 2017), in combination with BEs, these base editing systems have promising potential not only for generation of disease models, but more importantly for therapy of hereditary diseases caused by point substitutions.
Zhang T, Li J, He Y, et al. A small molecule targeting myoferlin exerts promising anti-tumor effects on breast cancer[J]. Nature Communications, 2018, 9(1).
Breast cancer is one of the most lethal cancers in women when it reaches the metastatic stage. Here, we screen a library of small molecules for inhibitors of breast cancer cell invasion, and use structure/activity relationship studies to develop a series of small molecules with improved activity. We find WJ460 as one of the lead compounds exerting anti-metastatic activity in the nanomolar range in breast cancer cells. Proteomic and biochemical studies identify myoferlin (MYOF) as the direct target of WJ460. In parallel, loss of MYOF or pharmacological inhibition of MYOF by WJ460 reduces breast cancer extravasation into the lung parenchyma in an experimental metastasis mouse model, which reveals an essential role of MYOF in breast cancer progression. Our findings suggest that MYOF can be explored as a molecular target in breast cancer metastasis and that targeting MYOF by WJ460 may be a promising therapeutic strategy in MYOF-driven cancers.
Tan B, Shi X, Zhang J, et al. Inhibition of RSPO-LGR4 facilitates checkpoint blockade therapy by switching macrophage polarization[J]. Cancer Research, 2018, 78(17): 4929-4942.
Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment(TME). As the most abundant immune cells in TME, tumor-associated macrophages(TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein–coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1–4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor micro-environment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4fl/flLyz2cre/+ mice, characterized by fewer protumoral M2 TAMs and increased CD8+ T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy.
Huang H, Xiong Q, Wang N, et al. Kisspeptin/GPR54 signaling restricts antiviral innate immune response through regulating calcineurin phosphatase activity[J]. Science Advances, 2018, 4(8).
G protein–coupled receptor 54 (GPR54), the key receptor for the neuropeptide hormone kisspeptin, plays essential roles in regulating puberty development and cancer metastasis. However, its role in the antiviral innate immune response is unknown. We report that virus-induced type I interferon (IFN-I) production was significantly enhanced in Gpr54-deficient cells and mice and resulted in restricted viral replication. We found a marked increase of kisspeptin in mouse serum during viral infection, which, in turn, impaired IFN-I production and antiviral immunity through the GPR54/calcineurin axis. Mechanistically, kisspeptin/GPR54 signaling recruited calcineurin and increased its phosphatase activity to dephosphorylate and deactivate TANK [tumor necrosis factor receptorassociated factor (TRAF) family member-associated NF-κB activator]–binding kinase 1 (TBK1) in a Ca2+-dependent manner. Thus, our data reveal a kisspeptin/GPR54/calcineurin-mediated immune evasion pathway exploited by virus through the negative feedback loop of TBK1 signaling. These findings also provide insights into the function and cross-talk of kisspeptin, a known neuropeptide hormone, in antiviral innate immune response.
Luo J, Yang Z, Ma Y, et al. LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption[J]. Nature Medicine, 2016, 22(5): 539-546.
Tumor necrosis factor (TNF) superfamily member 11 (TNFSF11, also known as RANKL) regulates multiple physiological or pathological functions, including osteoclast differentiation and osteoporosis. TNFRSF11A (also called RANK) is considered to be the sole receptor for RANKL. Herein we report that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL. LGR4 competes with RANK to bind RANKL and suppresses canonical RANK signaling during osteoclast differentiation. RANKL binding to LGR4 activates the Gαq and GSK3-β signaling pathway, an action that suppresses the expression and activity of nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (NFATC1) during osteoclastogenesis. Both whole-body (Lgr4-/-) and monocyte conditional knockout mice of Lgr4 (Lgr4 CKO) exhibit osteoclast hyperactivation (including elevation of osteoclast number, surface area, and size) and increased bone erosion. The soluble LGR4 extracellular domain (ECD) binds RANKL and inhibits osteoclast differentiation in vivo. Moreover, LGR4-ECD therapeutically abrogated RANKL-induced bone loss in three mouse models of osteoporosis. Therefore, LGR4 acts as a second RANKL receptor that negatively regulates osteoclast differentiation and bone resorption.
He Y, Peng S, Wang J, et al. Ailanthone targets p23 to overcome MDV3100 resistance in castration-resistant prostate cancer[J]. Nature Communications, 2016, 7(1).
Androgen receptor (AR) antagonist MDV3100 is the first therapeutic approach in treating castration-resistant prostate cancer (CRPC), but tumours frequently become drug resistant via multiple mechanisms including AR amplification and mutation. Here we identify the small molecule Ailanthone (AIL) as a potent inhibitor of both full-length AR (AR-FL) and constitutively active truncated AR splice variants (AR-Vs). AIL binds to the co-chaperone protein p23 and prevents AR's interaction with HSP90, thus resulting in the disruption of the AR-chaperone complex followed by ubiquitin/proteasome-mediated degradation of AR as well as other p23 clients including AKT and Cdk4, and downregulates AR and its target genes in PCa cell lines and orthotopic animal tumours. In addition, AIL blocks tumour growth and metastasis of CRPC. Finally, AIL possesses favourable drug-like properties such as good bioavailability, high solubility, lack of CYP inhibition and low hepatotoxicity. In general, AIL is a potential candidate for the treatment of CRPC.