An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance—i.e., 100% of treated animals showing sustained expression after 4 months—was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.

Pro-apoptotic gene expression mediated by the p38 mitogen-activated protein kinase signal transduction pathway

Neurotrophic factor deprivation causes apoptosis by a mechanism that requires macromolecular synthesis. This fact suggests that gene expression is necessary to achieve cell death. To identify mRNA that is expressed in apoptotic cells we used subtractive hybridization with cDNA prepared from neuronal pheochromocytoma cells. Monoamine oxidase (MAO) expression was increased in cells during nerve growth factor withdrawal-induced apoptosis. The increased apoptosis and induction of MAO was prevented by inhibition of the p38 mitogen-activated protein (MAP) kinase pathway. MAO may contribute to the apoptotic process because inhibition of MAO activity suppressed cell death. Together, these data indicate that MAO may be a target of pro-apoptotic signal transduction by the p38 MAP kinase pathway.

Analysis of gene expression during myc oncogene-induced lymphomagenesis in the bursa of Fabricius

The transcriptional effects of deregulated myc gene overexpression are implicated in tumorigenesis in a spectrum of experimental and naturally occurring neoplasms. In follicles of the chicken bursa of Fabricius, myc induction of B-cell neoplasia requires a target cell population present during early bursal development and progresses through preneoplastic transformed follicles to metastatic lymphomas. We developed a chicken immune system cDNA microarray to analyze broad changes in gene expression that occur during normal embryonic B-cell development and during myc-induced neoplastic transformation in the bursa. The number of mRNAs showing at least 3-fold change was greater during myc-induced lymphomagenesis than during normal development, and hierarchical cluster analysis of expression patterns revealed that levels of several hundred mRNAs varied in concert with levels of myc overexpression. A set of 41 mRNAs were most consistently elevated in myc-overexpressing preneoplastic and neoplastic cells, most involved in processes thought to be subject to regulation by Myc. The mRNAs for another cluster of genes were overexpressed in neoplasia independent of myc expression level, including a small subset with the expression signature of embryonic bursal lymphocytes. Overexpression of myc, and some of the genes overexpressed with myc, may be important for generation of preneoplastic transformed follicles. However, expression profiles of late metastatic tumors showed a large variation in concert with myc expression levels, and some showed minimal myc overexpression. Therefore, high-level myc overexpression may be more important in the early induction of these lymphomas than in maintenance of late-stage metastases.

Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells

We describe a mouse model in which p27Kip1 transgene expression is spatially restricted to the central nervous system neuroepithelium and temporally controlled with doxycycline. Transgene-specific transcripts are detectable within 6 h of doxycycline administration, and maximum nonlethal expression is approached within 12 h. After 18–26 h of transgene expression, the G1 phase of the cell cycle is estimated to increase from 9 to 13 h in the neocortical neuroepithelium, the maximum G1 phase length attainable in this proliferative population in normal mice. Thus our data establish a direct link between p27Kip1 and control of G1 phase length in the mammalian central nervous system and unveil intrinsic mechanisms that constrain the G1 phase length to a putative physiological maximum despite ongoing p27Kip1 transgene expression.