Directed gene transfer into specific cell lineages in vivo is an attractive approach for both modulating gene expression and correcting inherited mutations such as emphysema caused by human alpha1 ...antitrypsin (hAAT) deficiency. However, somatic tissues are mainly comprised of heterogeneous, differentiated cell lineages that can be short lived and difficult to specifically transfect. Here, we describe an intratracheally instilled lentiviral system able to deliver genes selectively to as many as 70% of alveolar macrophages (AMs) in the mouse lung. Following a single in vivo lentiviral transduction, genetically tagged AMs persisted in lung alveoli and expressed transferred genes for the lifetime of the adult mouse. A prolonged macrophage lifespan, rather than precursor cell proliferation, accounted for the surprisingly sustained presence of transduced AMs. We utilized this long-lived population to achieve localized secretion of therapeutic levels of hAAT protein in lung epithelial lining fluid. In an established mouse model of emphysema, lentivirally delivered hAAT ameliorated the progression of emphysema, as evidenced by attenuation of increased lung compliance and alveolar size. After 24 weeks of sustained gene expression, no humoral or cellular immune responses to hAAT protein were detected. Our results challenge the dogma that AMs are short lived and suggest that these differentiated cells may be a possible target cell population for in vivo gene therapy applications, including the sustained correction of hAAT deficiency.
The retinoblastoma protein, pRB, and the closely related proteins p107 and p130 are important regulators of the mammalian cell cycle. Biochemical and genetic studies have demonstrated overlapping as ...well as distinct functions for the three proteins in cell cycle control and mouse development. However, the role of the pRB family as a whole in the regulation of cell proliferation, cell death, or cell differentiation is not known. We generated embryonic stem (ES) cells and other cell types mutant for all three genes. Triple knock-out mouse embryonic fibroblasts (TKO MEFs) had a shorter cell cycle than wild-type, single, or double knock-out control cells. TKO cells were resistant to G
1
arrest following DNA damage, despite retaining functional p53 activity. They were also insensitive to G
1
arrest signals following contact inhibition or serum starvation. Finally, TKO MEFs did not undergo senescence in culture and do possess some characteristics of transformed cells. Our results confirm the essential role of the Rb family in the control of the G
1
/S transition, place the three Rb family members downstream of multiple cell cycle control pathways, and further the link between loss of cell cycle control and tumorigenesis.
Defects in neural tube formation are among the most common malformations leading to infant mortality. Although numerous genetic loci appear to contribute to the defects observed in humans and in ...animal model systems, few of the genes involved have been characterized at the molecular level. Mice lacking the p53 tumour suppressor gene are predisposed to tumours, but the viability of these animals indicates that p53 function is not essential for embryonic development. Here, we demonstrate that a fraction of p53-deficient embryos in fact do not develop normally. These animals display defects in neural tube closure resulting in an overgrowth of neural tissue in the region of the mid-brain, a condition known as exencephaly.
Here, we describe a system for the exogenous control of gene expression in mammalian cells that relies on the control of translational termination. To achieve gene regulation, we modified ...protein-coding sequences by introduction of a translational termination codon just downstream from the initiator AUG codon. Translation of the resulting mRNA leads to potent reduction in expression of the desired gene product. Expression of the gene product can be controlled by treating cells that express the mRNA with either aminoglycoside antibiotics or several nonantibiotic compounds. We show that the extent of regulation of gene expression can be substantial (60-fold) and that regulation can be achieved in the case of a variety of different genes, in different cultured cell lines and in primary cells in vivo. This gene regulation strategy offers significant advantages over existing methods for controlling gene expression and should have both immediate experimental application and possible clinical application.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of ...terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107-deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb-deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis.
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BFBNIB, NUK, PNG, UL, UM, UPUK
Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 ...independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Play is essential to development because it contributes to the cognitive, physical, social, and emotional well-being of children and youth. Play also offers an ideal opportunity for parents to engage ...fully with their children. Despite the benefits derived from play for both children and parents, time for free play has been markedly reduced for some children. This report addresses a variety of factors that have reduced play, including a hurried lifestyle, changes in family structure, and increased attention to academics and enrichment activities at the expense of recess or free child-centered play. This report offers guidelines on how pediatricians can advocate for children by helping families, school systems, and communities consider how best to ensure that play is protected as they seek the balance in children's lives to create the optimal developmental milieu.
Abstract 2801
MLN4924 is an investigational inhibitor of Nedd8-activating enzyme (NAE), which plays an essential role in regulating the activity of the cullin-RING E3 ligases (CRLs). NAE controls the ...neddylation cascade that results in Nedd8 conjugation to the CRLs, which is required for ligase activity. NAE inhibition thus inhibits ubiquitination and proteasomal degradation of CRL substrates, which include proteins involved in cell-cycle regulation (p27), signal transduction (pIκBα), DNA replication (Cdt-1), stress response (Nrf-2), and other processes important to tumor cell growth and survival. In lymphoma cells, NAE inhibition with MLN4924 has been shown to result in apoptosis either through increased Cdt-1 levels, S-phase accumulation, and DNA re-replication, or via pIκBα stabilization and consequent NF-κB pathway inhibition. In vivo, MLN4924 treatment resulted in tumor growth inhibition and regressions in lymphoma xenograft models. This phase 1 dose-escalation study is the first investigation of MLN4924 in multiple myeloma (MM) and lymphoma patients. We have previously reported (Shah et al, ASH 2009) that the maximum tolerated dose (MTD) of MLN4924 on Schedule A of this study (days 1, 2, 8, and 9 of 21-day cycles) was 110 mg/m2, with dose-limiting toxicities (DLTs) including muscle cramps and febrile neutropenia. Analyses of peripheral blood mononuclear cells and skin biopsies indicated MLN4924 exerted the predicted pharmacodynamic (PD) effects in peripheral blood and skin, including inhibition of neddylated cullins and induction of pIκBα, Cdt-1, and Nrf-2. Two additional schedules of MLN4924 administration are now being investigated with the aim of increasing tolerability and the deliverable dose.
Patients aged ≥18 years with ECOG performance status 0–2 and relapsed and/or refractory MM or lymphoma, including any B- or T-cell non-Hodgkin's lymphoma (NHL) and Hodgkin lymphoma (HL), following ≥2 prior lines of therapy were eligible. Primary objectives were to determine the MTD and safety profile of MLN4924 on the different dosing schedules, describe the pharmacokinetics (PK) and PD of MLN4924 in blood, and investigate PD effects in skin and tumor. Secondary objectives included evaluation of disease response. Patients received MLN4924 via a 60-minute intravenous infusion on either days 1, 4, 8, and 11 (Schedule B) or days 1 and 8 (Schedule C) of 21-day cycles for up to 12 months. Doses of 25–147 mg/m2 were investigated on Schedule A; for Schedules B and C, dose escalation started at the MTD of Schedule A, 110 mg/m2, and proceeded in 1.33-fold increments using a Bayesian continual reassessment method based on the occurrence of DLTs in cycle 1. The MTD was defined as the dose level closest to that predicted to result in a DLT rate of 25%. Adverse events (AEs) were graded using National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0.
To date, 7 patients (5 male, median age 60 years range 48–68) have been enrolled to Schedule B, 2 each at 110, 147, and 196, and 1 at 261 mg/m2; 2 have MM and 5 lymphoma (2 diffuse large B-cell lymphoma DLBCL, 1 small lymphocytic lymphoma, 1 mantle cell lymphoma MCL, 1 nodular sclerosis HL). Patients have received a median of 3 cycles (range 2–5) to date. A total of 7 patients (all male, median age 49 years range 45–66) have been enrolled to Schedule C, 2 each at 110 and 147, and 3 at 196 mg/m2; 4 have MM and 3 lymphoma (1 follicular lymphoma FL, 1 MCL, 1 nodular sclerosis HL). Median number of cycles received is 4 (range 1–7). No DLTs and no grade ≥3 AEs have been reported on either schedule to date. On Schedule B, only grade 1 dyspnea and myalgia (both n=2) have been reported in >1 patient, with grade 1 diarrhea (n=3), constipation, fatigue, and nausea (each n=2) reported on Schedule C. PK data for the 110 mg/m2 cohorts of Schedules B and C are consistent with the lack of significant accumulation of MLN4924 in plasma shown on Schedule A. One patient with HL on Schedule A achieved a partial response; no responses have been reported to date on Schedules B and C though some heavily treated patients have demonstrated stable disease for 5 or more cycles (1 FL, 7 cycles; 1 DLBCL, 5 cycles; 1 MM, 5 cycles).
Enrollment and dose escalation are proceeding on Schedules B and C (at doses above Schedule A MTD) to determine the MTD on each schedule; updated clinical data will be presented, together with data on the PK and PD of MLN4924, on these dosing schedules.
Shah:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding. Off Label Use: Investigational agent in clinical development for the treatment of multiple myeloma and lymphoma. O'Connor:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Centocor Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Smith:Millennium Pharmaceuticals, Inc.: Honoraria, Research Funding, Speakers Bureau. Orlowski:Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding. Mulligan:Millennium Pharmaceuticals: Employment. Smith:Millennium Pharmaceuticals, Inc.: Employment. Pickard:Millennium Pharmaceuticals, Inc.: Employment. Dezube:Millennium Pharmaceuticals: Employment, Equity Ownership. Lonial:Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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