The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity

Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the “core”, which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5′ core with a novel 3′ 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors

SMTP-44D Inhibits Atherosclerotic Plaque Formation in Apolipoprotein-E Null Mice Partly by Suppressing the AGEs-RAGE Axis

SMTP-44D has been reported to have anti-oxidative and anti-inflammatory reactions, including reduced expression of receptor for advanced glycation end products (RAGE) in experimental diabetic neuropathy. Although activation of RAGE with its ligands, and advanced glycation end products (AGEs), play a crucial role in atherosclerotic cardiovascular disease, a leading cause of death in diabetic patients, it remains unclear whether SMTP-44D could inhibit experimental atherosclerosis by suppressing the AGEs–RAGE axis. In this study, we investigated the effects of SMTP-44D on atherosclerotic plaque formation and expression of AGEs in apolipoprotein-E null (Apoe−/−) mice. We further studied here whether and how SMTP-44D inhibited foam cell formation of macrophages isolated from Apoe−/− mice ex vivo. Although administration of SMTP-44D to Apoe−/− mice did not affect clinical or biochemical parameters, it significantly decreased the surface area of atherosclerotic lesions and reduced the atheromatous plaque size, macrophage infiltration, and AGEs accumulation in the aortic roots. SMTP-44D bound to immobilized RAGE and subsequently attenuated the interaction of AGEs with RAGE in vitro. Furthermore, foam cell formation evaluated by Dil-oxidized low-density lipoprotein (ox-LDL) uptake, and gene expression of RAGE, cyclin-dependent kinase 5 (Cdk5) and CD36 in macrophages isolated from SMTP-44D-treated Apoe−/− mice were significantly decreased compared with those from saline-treated mice. Gene expression levels of RAGE and Cdk5 were highly correlated with each other, the latter of which was also positively associated with that of CD36. The present study suggests that SMTP-44D may inhibit atherosclerotic plaque formation in Apoe−/− mice partly by blocking the AGEs-RAGE-induced ox-LDL uptake into macrophages via the suppression of Cdk5-CD36 pathway

Oncogenic N-Ras and Tet2 haploinsufficiency collaborate to dysregulate hematopoietic stem and progenitor cells

N-Ras G12D and haploinsufficient Tet2 collaborate to induce lethal and highly penetrant CMML in mice with shortened overall survival. N-Ras G12D and haploinsufficient Tet2 together promote balanced proliferation and enhanced competitiveness and self-renewal in HSPCs. Concurrent genetic lesions exist in a majority of patients with hematologic malignancies. Among these, somatic mutations that activate RAS oncogenes and inactivate the epigenetic modifier ten-eleven translocation 2 ( TET2 ) frequently co-occur in human chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias, suggesting a cooperativity in malignant transformation. To test this, we applied a conditional murine model that endogenously expressed oncogenic Nras G12D and monoallelic loss of Tet2 and explored the collaborative role specifically within hematopoietic stem and progenitor cells (HSPCs) at disease initiation. We demonstrate that the 2 mutations collaborated to accelerate a transplantable CMML-like disease in vivo, with an overall shortened survival and increased disease penetrance compared with single mutants. At preleukemic stage, N-Ras G12D and Tet2 haploinsufficiency together induced balanced hematopoietic stem cell (HSC) proliferation and enhanced competitiveness. Nras G12D/+ / Tet2 +/− HSCs displayed increased self-renewal in primary and secondary transplantations, with significantly higher reconstitution than single mutants. Strikingly, the 2 mutations together conferred long-term reconstitution and self-renewal potential to multipotent progenitors, a pool of cells that usually have limited self-renewal compared with HSCs. Moreover, HSPCs from Nras G12D/+ / Tet2 +/− mice displayed increased cytokine sensitivity in response to thrombopoietin. Therefore, our studies establish a novel tractable CMML model and provide insights into how dysregulated signaling pathways and epigenetic modifiers collaborate to modulate HSPC function and promote leukemogenesis

Mechanism of Reduction in Titers From Lentivirus Vectors Carrying Large Inserts in the 3′LTR

Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3′-long terminal repeat (LTR), infectious titers decreased proportional to the length of the insert. The stage of vector life cycle affected by vectors carrying the large cHS4 3′LTR insert was compared to a control vector: there was no increase in read-through transcription with insertion of the 1.2-kb cHS4 in the 3′LTR. Equal amount of full-length viral mRNA was produced in packaging cells and viral assembly/packaging was unaffected, resulting in comparable amounts of intact vector particles produced by either vectors. However, LV vectors carrying cHS4 in the 3′LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency, and hence lower transduction titers. Therefore, vectors with large insertions in the 3′LTR are transcribed and packaged efficiently, but the LTR insert hinders viral-RNA (vRNA) processing and transduction of target cells. These studies have important implications in design of integrating vectors

A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction

We show that lentiviral delivery of human γ-globin gene under β-globin regulatory control elements in hematopoietic stem cells (HSCs) results in sufficient postnatal fetal hemoglobin (HbF) expression to correct sickle cell anemia (SCA) in the Berkeley “humanized” sickle mouse. Upon de-escalating the amount of transduced HSCs in transplant recipients, using reduced-intensity conditioning and varying gene transfer efficiency and vector copy number, we assessed critical parameters needed for correction. A systematic quantification of functional and hematologic red blood cell (RBC) indices, organ pathology, and life span was used to determine the minimal amount of HbF, F cells, HbF/F-cell, and gene-modified HSCs required for correcting the sickle phenotype. We show that long-term amelioration of disease occurred (1) when HbF exceeded 10%, F cells constituted two-thirds of the circulating RBCs, and HbF/F cell was one-third of the total hemoglobin in sickle RBCs; and (2) when approximately 20% gene-modified HSCs repopulated the marrow. Moreover, we show a novel model using reduced-intensity conditioning to determine genetically corrected HSC threshold that corrects a hematopoietic disease. These studies provide a strong preclinical model for what it would take to genetically correct SCA and are a foundation for the use of this vector in a human clinical trial