CD19-immunoPET for noninvasive visualization of CD19 expression in B-cell lymphoma patients.

Cell- and antibody-based CD19-directed therapies have demonstrated great potential for treating B-cell non-Hodgkin lymphoma (B-NHL). However, all these approaches suffer from limited response rates and considerable toxicity. Until now, therapy decisions have been routinely based on histopathological CD19 staining of a single lesion at initial diagnosis or relapse, disregarding heterogeneity and temporal alterations in antigen expression. To visualize in vivo CD19 expression noninvasively, we radiolabeled anti-human CD19 monoclonal antibodies with copper-64 (64Cu-αCD19) for positron emission tomography (CD19-immunoPET). 64Cu-αCD19 specifically bound to subcutaneous Daudi xenograft mouse models in vivo. Importantly, 64Cu-αCD19 did not affect the anti-lymphoma cytotoxicity of CD19 CAR-T cells in vitro. Following our preclinical validation, 64Cu-αCD19 was injected into four patients with follicular lymphoma, diffuse large B-cell lymphoma or mantle zone lymphoma. We observed varying 64Cu-αCD19 PET uptake patterns at different lymphoma sites, both within and among patients, correlating with ex vivo immunohistochemical CD19 expression. Moreover, one patient exhibited enhanced uptake in the spleen compared to that in patients with prior B-cell-depleting therapy, indicating that 64Cu-αCD19 is applicable for identifying B-cell-rich organs. In conclusion, we demonstrated the specific targeting and visualization of CD19+ B-NHL in mice and humans by CD19-immunoPET. The intra- and interindividual heterogeneous 64Cu-αCD19 uptake patterns of lymphoma lesions indicate variability in CD19 expression, suggesting the potential of CD19-immunoPET as a novel tool to guide CD19-directed therapies.

CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Differential transcriptional control of hematopoiesis in congenital and cyclic neutropenia patients harboring ELANE mutations.

Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most CyN cases and approximately 25 % of CN cases. In CN and in CyN, a median of 2.8 % of CD34+ cells were early CD49f+ hematopoietic stem cells (eHSC) that did not express ELANE and thus escape from the unfolded protein response (UPR) caused by mutated NE. In CyN, the CD49f+ cells respond to G-CSF with a significant upregulation of the hematopoietic stem-cell-specific transcription factors, C/EBP/, MLL1, HOXA9, MEIS1, and HLF during the ascending arm of the cycle, resulting in the differentiation of myeloid cells to mature neutrophils at the cycle peak. However, NE protein released by neutrophils at the cycle's peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF. In contrast, in CN patients, CD49f+ cells failed to express mRNA levels of HSC-specific transcription factors mentioned above. Rescue of C/EBP//expression in CN restored granulopoiesis.

Regenerative medicine meets translational oncology: Modeling leukemic bone marrow niche.

Most studies on leukemia focus on leukemia cells as isolated objects without considering the bone marrow niche. Pal et al. have recreated the bone marrow niche using induced pluripotent stem cells (iPSCs), identifying CDH2 as a therapeutically druggable leukemia-promoting factor.1.

A topological refactoring design strategy yields highly stable granulopoietic proteins.

Protein therapeutics frequently face major challenges, including complicated production, instability, poor solubility, and aggregation. De novo protein design can readily address these challenges. Here, we demonstrate the utility of a topological refactoring strategy to design novel granulopoietic proteins starting from the granulocyte-colony stimulating factor (G-CSF) structure. We change a protein fold by rearranging the sequence and optimising it towards the new fold. Testing four designs, we obtain two that possess nanomolar activity, the most active of which is highly thermostable and protease-resistant, and matches its designed structure to atomic accuracy. While the designs possess starkly different sequence and structure from the native G-CSF, they show specific activity in differentiating primary human haematopoietic stem cells into mature neutrophils. The designs also show significant and specific activity in vivo. Our topological refactoring approach is largely independent of sequence or structural context, and is therefore applicable to a wide range of protein targets.

iPSC modeling of stage-specific leukemogenesis reveals BAALC as a key oncogene in severe congenital neutropenia.

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3R and RUNX1 are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1 mutant BAALC(hi) de novo AML.

Cooperating, congenital neutropenia-associated Csf3r and Runx1 mutations activate pro-inflammatory signaling and inhibit myeloid differentiation of mouse HSPCs.

Patients with the pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have an approximately 15% risk of developing acute myeloid leukemia (AML; called here CN/AML). Most CN/AML patients co-acquire CSF3R and RUNX1 mutations, which play cooperative roles in the development of AML. To establish an in vitro model of leukemogenesis, we utilized bone marrow lin- cells from transgenic C57BL/6-d715 Csf3r mice expressing a CN patient-mimicking truncated CSF3R mutation. We transduced these cells with vectors encoding RUNX1 wild type (WT) or RUNX1 mutant proteins carrying the R139G or R174L mutations. Cells transduced with these RUNX1 mutants showed diminished in vitro myeloid differentiation and elevated replating capacity, compared with those expressing WT RUNX1. mRNA expression analysis showed that cells transduced with the RUNX1 mutants exhibited hyperactivation of inflammatory signaling and innate immunity pathways, including IL-6, TLR, NF-kappaB, IFN, and TREM1 signaling. These data suggest that the expression of mutated RUNX1 in a CSF3R-mutated background may activate the pro-inflammatory cell state and inhibit myeloid differentiation.

Gene Knockout in Hematopoietic Stem and Progenitor Cells Followed by Granulocytic Differentiation.

In this chapter, we present an optimized CRISPR/Cas9 RNP nucleofection approach for gene knockout (KO) in hematopoietic stem and progenitor cells (HSPCs). With experimentally proved active locus-specific sgRNAs, we routinely reach over 80% gene KO in HSPCs, thus avoiding the need for cell sorting or enrichment of targeted cell population. Additionally, we provide a protocol for in vitro granulocytic differentiation of HSPCs after gene KO and detailed description of granulocyte function tests which can be applied to study the effects of a particular gene KO.

CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients.

A Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 µg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. "Maturation arrest," the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and "maturation arrest" corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients' primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.

LMO2 activation by deacetylation is indispensable for hematopoiesis and T-ALL leukemogenesis.

Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.

Cardiac and electrophysiological effects of primary and refined extracts from Leonurus cardiaca L. (Ph.Eur.).

Although several antiarrhythmic drugs of chemical origin are in clinical use since decades, their application is often limited by their adverse effects and especially by their inherited proarrhythmic risk, which can lead to a significantly increased mortality in patients receiving these compounds. On the other hand, aqueous extracts from the aerial parts of the European Lamiaceae Leonurus cardiaca (Ph.Eur.) have been used for centuries as a remedy against tachyarrhythmia and other cardiac disorders. Nevertheless, a scientific basis for the claim of direct cardiac electrophysiological, antiarrhythmic, or functional effects of Leonurus cardiacae herba (LCH) preparations has not been established until now. In order to enrich the active constituents from the primary extract which was tested as the most cardioactive, namely the aqueous Soxhlet extract, and to eliminate undesired substances such as the dichloromethanic fraction or potassium, a bioassay guided fractionation procedure was applied, resulting in the development of a Leonurus cardiaca refined extract (LCRE) which was characterised together with Leonurus crude extracts by a newly developed gradient elution HPLC fingerprint analysis for separation and quantification of six major phenolics as well as by qNMR for determining the stachydrine content. This refined extract was applied intracoronarily in isolated rabbit hearts perfused according to the Langendorff technique. Mapping experiments with 256 electrodes on the heart surface showed a reduction of left ventricular pressure and an increase of relative coronary flow at concentrations of 1.0 and 2.0 mg/mL LCRE. Furthermore, the PQ-interval was prolonged and both the basic cycle length and the activation recovery interval increased. In addition, voltage-clamp measurements were performed on the following cell models in order to characterise the electrophysiological profile of LCRE: neonatal rat ventricular cardiomyocytes to investigate the effect on I(Na) and I(Ca.L), sinoatrial node cells and ventricular myocytes isolated from adult guinea pigs to test effects on I(f) and action potential (AP) duration, as well as HERG-transfected HEK 293 cells to analyse the influence on the I (K.r). In these voltage clamp experiments LCRE exerted a calcium-antagonistic activity by I(Ca.L) blockade, reduced the repolarising current I(K.r), and prolonged the AP-duration, while I(Na) was not affected. Although LCRE displayed only weak effects on the I(f) amplitude and voltage dependence, it significantly prolonged the activation time constant of I(f). Thus, LCRE acts on multiple electrophysiological targets, specifically I(Ca.L), I(K.r), and I(f), observed both at whole organ and single cell level.