The Polycomb Group Protein L3MBTL1 Represses a SMAD5-Mediated Hematopoietic Transcriptional Program in Human Pluripotent Stem Cells

SummaryEpigenetic regulation of key transcriptional programs is a critical mechanism that controls hematopoietic development, and, thus, aberrant expression patterns or mutations in epigenetic regulators occur frequently in hematologic malignancies. We demonstrate that the Polycomb protein L3MBTL1, which is monoallelically deleted in 20q- myeloid malignancies, represses the ability of stem cells to drive hematopoietic-specific transcriptional programs by regulating the expression of SMAD5 and impairing its recruitment to target regulatory regions. Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells. We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation. Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia

Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation

Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35) vectors for zinc-finger nuclease (ZFN)- or transcription activator-like effector nuclease (TALEN)-mediated genome editing in human CD34+ hematopoietic stem cells (HSCs) from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA)-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene -expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35. ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells). The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2) within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.

A method to sequence and quantify DNA integration for monitoring outcome in gene therapy

Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events

The AIM2 inflammasome exacerbates atherosclerosis in clonal haematopoiesis

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors(1). Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2(V617F) (JAK2(VF)) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease(1,2). Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2(VF) selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2(VF) lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2(VF) lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1 beta reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2(VF) macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1 beta or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk