Rationally Designed Base Editors for Precise Editing of the Sickle Cell Disease Mutation.

Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment of transition mutations without double-strand DNA break intermediates. The breadth of potential base editing targets is frequently limited by the requirement of a suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures of Cas9 and TadA to design a set of inlaid base editors (IBEs), in which deaminase domains are internal to Cas9. Several of these IBEs exhibit shifted editing windows and greater editing efficiency, enabling editing of targets outside the canonical editing window with reduced DNA and RNA off-target editing frequency. Finally, we show that IBEs enable conversion of the pathogenic sickle cell hemoglobin allele to the naturally occurring HbG-Makassar variant in patient-derived hematopoietic stem cells.

Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy.

The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-ß superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.

Interleukin-33 contributes to both M1 and M2 chemokine marker expression in human macrophages.

BACKGROUND: Interleukin-33 is a member of the IL-1 cytokine family whose functions are mediated and modulated by the ST2 receptor. IL-33-ST2 expression and interactions have been explored in mouse macrophages but little is known about the effect of IL-33 on human macrophages. The expression of ST2 transcript and protein levels, and IL-33-mediated effects on M1 (i.e. classical activation) and M2 (i.e. alternative activation) chemokine marker expression in human bone marrow-derived macrophages were examined. RESULTS: Human macrophages constitutively expressed the membrane-associated (i.e. ST2L) and the soluble (i.e. sST2) ST2 receptors. M2 (IL-4 + IL-13) skewing stimuli markedly increased the expression of ST2L, but neither polarizing cytokine treatment promoted the release of sST2 from these cells. When added to naïve macrophages alone, IL-33 directly enhanced the expression of CCL3. In combination with LPS, IL-33 blocked the expression of the M2 chemokine marker CCL18, but did not alter CCL3 expression in these naive cells. The addition of IL-33 to M1 macrophages markedly increased the expression of CCL18 above that detected in untreated M1 macrophages. Similarly, alternatively activated human macrophages treated with IL-33 exhibited enhanced expression of CCL18 and the M2 marker mannose receptor above that detected in M2 macrophages alone. CONCLUSIONS: Together, these data suggest that primary responses to IL-33 in bone marrow derived human macrophages favors M1 chemokine generation while its addition to polarized human macrophages promotes or amplifies M2 chemokine expression.

T regulatory cells and attenuated bleomycin-induced fibrosis in lungs of CCR7-/- mice.

BACKGROUND: C-C chemokine receptor (CCR)7 is a regulator of dendritic cell and T cell migration, and its role in tissue wound healing has been investigated in various disease models. We have previously demonstrated that CCR7 and its ligand, chemokine (C-C motif) ligand (CCL)21, modulates wound repair in pulmonary fibrosis (PF) but the mechanism of this is unknown. The objective of this study was to investigate whether the absence of CCR7 protects against bleomycin (BLM)-induced PF. CCR7-/- mice failed to mount a fibrotic pulmonary response as assessed by histologic collagen staining and quantification by hydroxyproline. We hypothesized that the prominent characteristics of CCR7-/- mice, including elevated levels of cytokine and chemokine mediators and the presence of bronchus-associated lymphoid tissue (BALT) might be relevant to the protective phenotype. RESULTS: Pulmonary fibrosis was induced in CCR7+/+ and CCR7-/- mice via a single intratracheal injection of BLM. We found that the lung cytokine/chemokine milieu associated with the absence of CCR7 correlated with an increase in BALT, and might be attributable to regulatory T cell (Treg) homeostasis and trafficking within the lungs and lymph nodes. In response to BLM challenge, CCR7-/- mice exhibited an early, steady increase in lung CD4+ T cells and increased CD4+ CD25+ FoxP3+ Tregs in the lungs 21 days after challenge. These findings are consistent with increased lung expression of interleukin-2 and indoleamine 2,3-dioxygenase in CCR7-/- mice, which promote Treg expansion. CONCLUSIONS: Our study demonstrates that the protective phenotype associated with BLM-treated CCR7-/- mice correlates with the presence of BALT and the anchoring of Tregs in the lungs of CCR7-/- mice. These data provide novel evidence to support the further investigation of CCR7-mediated Treg trafficking in the modulation of BLM-induced PF.

Inefficient lymph node sensitization during respiratory viral infection promotes IL-17-mediated lung pathology.

Development of bronchus-associated lymphoid tissue has been suggested to enhance local antiviral immune responses; however, ectopic lymph node formation often corresponds to chronic inflammatory diseases. These studies investigated the role of ectopic pulmonary lymph nodes upon respiratory syncytial virus (RSV) infection using CCR7-deficient mice, which develop bronchus-associated lymphoid tissue early in life. CCR7(-/-) mice exhibited impaired secondary lymph node formation, enhanced effector T cell responses and pathogenic mucus production in the lung after RSV infection. IL-17 production from CD4 T cells in CCR7(-/-) mice was most remarkably enhanced. Wild-type animals reconstituted with CCR7(-/-) bone marrow recapitulated the pathogenic lung phenotype in CCR7(-/-) mice, whereas CCR7(-/-) animals reconstituted with wild-type bone marrow had normal lymph node development, diminished IL-17 production and reduced lung pathology. Mixed bone marrow chimeras revealed an alteration of immune responses only in CCR7(-/-) T cells, suggesting that impaired trafficking promotes local effector cell generation. Lymphotoxin-α-deficient mice infected with RSV were used to further examine locally induced immune responses and demonstrated increased mucus production and amplified cytokine responses in the lung, especially IL-17. Neutralization of IL-17 in CCR7(-/-) or in lymphotoxin-α-deficient animals specifically inhibited mucus hypersecretion and reduced IL-13. Thus, immune cell trafficking to secondary lymph nodes is necessary for appropriate cytokine responses to RSV as well as modulation of the local environment.

CCR7 impairs hematopoiesis after hematopoietic stem cell transplantation increasing susceptibility to invasive aspergillosis.

Hematopoietic stem cell transplantation (HSCT) is limited by patient susceptibility to opportunistic infections. One of the most devastating infections after HSCT is invasive aspergillosis (IA), a life-threatening disease caused by Aspergillus fumigatus. Transplantation of hematopoietic stem cells (HSCs) and myeloid progenitor cells (MPCs) has been shown to mediate protection against IA, but little is known about the factors that regulate HSC and MPC cell expansion after transplantation. Herein, we investigated the role of CCR7 in a murine model of IA after combined HSC and MPC transplantation into lethally irradiated wild-type (WT) mice. Nonirradiated CCR7(-/-) mice had expanded populations of HSCs in the bone marrow and spleen, compared with WT mice. Irradiated WT mice reconstituted with CCR7(-/-) HSCs and MPCs had increased survival, decreased fungal burden, and enhanced myeloid leukocyte numbers during IA, compared with WT controls. In addition, WT mice reconstituted with WT HSCs and MPCs and treated with anti-CCR7 exhibited accelerated myeloid cell expansion similar to that observed in CCR7(-/-)→WT chimeras. Thus, removal of the inhibitory effects of CCR7 through genetic alteration or ligand immunoneutralization enhanced myeloid reconstitution, thereby accelerating fungal clearance in a murine model of IA.

CCR7 deficiency on dendritic cells enhances fungal clearance in a murine model of pulmonary invasive aspergillosis.

Aspergillus fumigatus is a sporulating fungus found ubiquitously in the environment and is easily cleared from immunocompetent hosts. Invasive aspergillosis develops in immunocompromised patients, and is a leading cause of mortality in hematopoietic stem cell transplant recipients. CCR7 and its ligands, CCL19 and CCL21, are responsible for the migration of dendritic cells from sites of infection and inflammation to secondary lymphoid organs. To investigate the role of CCR7 during invasive aspergillosis, we used a well-characterized neutropenic murine model. During invasive aspergillosis, mice with a CCR7 deficiency in the hematopoietic compartment exhibited increased survival and less pulmonary injury compared with the appropriate wild-type control. Flow cytometric analysis of the chimeric mice revealed an increase in the number of dendritic cells present in the lungs of CCR7-deficient chimeras following infection with Aspergillus conidia. An adoptive transfer of dendritic cells into neutropenic mice provided a protective effect during invasive aspergillosis, which was further enhanced with the adoptive transfer of CCR7-deficient dendritic cells. Additionally, CCR7-deficient dendritic cells activated in vitro with Aspergillus conidia expressed higher TNF-alpha, CXCL10, and CXCL2 levels, indicating a more activated cellular response to the fungus. Our results suggest that the absence of CCR7 is protective during invasive aspergillosis in neutropenic mice. Collectively, these data demonstrate a potential deleterious role for CCR7 during primary immune responses directed against A. fumigatus.

Differential susceptibility of C57BL/6NCr and B6.Cg-Ptprca mice to commensal bacteria after whole body irradiation in translational bone marrow transplant studies.

BACKGROUND: The mouse is an important and widely utilized animal model for bone marrow transplant (BMT) translational studies. Here, we document the course of an unexpected increase in mortality of congenic mice that underwent BMT. METHODS: Thirty five BMTs were analyzed for survival differences utilizing the Log Rank test. Affected animals were evaluated by physical examination, necropsy, histopathology, serology for antibodies to infectious disease, and bacterial cultures. RESULTS: Severe bacteremia was identified as the main cause of death. Gastrointestinal (GI) damage was observed in histopathology. The bacteremia was most likely caused by the translocation of bacteria from the GI tract and immunosuppression caused by the myeloablative irradiation. Variability in groups of animals affected was caused by increased levels of gamma and X-ray radiation and the differing sensitivity of the two nearly genetically identical mouse strains used in the studies. CONCLUSION: Our retrospective analysis of thirty five murine BMTs performed in three different laboratories, identified C57BL/6NCr (Ly5.1) as being more radiation sensitive than B6.Cg-Ptprca/NCr (Ly5.2). This is the first report documenting a measurable difference in radiation sensitivity and its effects between an inbred strain of mice and its congenic counterpart eventually succumbing to sepsis after BMT.