AAV5-mediated gene transfer to the parotid glands of non-human primates

Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n3 per group; 1010 or 3 × 1011 particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by & ∼80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (& ∼1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands. © 2010 Macmillan Publishers Limited All rights reserved

Contributions of Gene Marking to Cell and Gene Therapies

The first human genetic modification studies used replication-incompetent integrating vector vectors to introduce marker genes into T lymphocytes and subsequently into hematopoietic stem cells. Such studies have provided numerous insights into the biology of hematopoiesis and immune reconstitution and contributed to clinical development of gene and cell therapies. Tracking of hematopoietic reconstitution and analysis of the origin of residual malignant disease after hematopoietic transplantation has been possible via gene marking. Introduction of selectable marker genes has enabled preselection of specific T-cell populations for tumor and viral immunotherapy and reduced the threat of graft-versus-host disease, improving the survival of patients after allogeneic marrow transplantation. Marking studies in humans, murine xenografts, and large animals have helped optimize conditions for gene transfer into CD34+ hematopoietic progenitors, contributing to the achievement of gene transfer efficiencies sufficient for clinical benefit in several serious genetic diseases such as X-linked severe combined immunodeficiency and adrenoleukodystropy. When adverse events linked to insertional mutagenesis arose in clinical gene therapy trials for inherited immunodeficiencies, additional animal studies using gene-marking vectors have greatly increased our understanding of genotoxicity. The knowledge gained from these studies is being translated into new vector designs and clinical protocols, which we hope will continue to improve the efficiency, effectiveness and safety of these promising therapeutic approaches

Interactive effects of iron, irradiance and CO₂ on Ross Sea phytoplankton

We conducted a factorial shipboard continuous culture experiment to examine the interactive effects of altered iron, irradiance and CO2 on the summer phytoplankton community of the Ross Sea, Antarctica. After 18 days of continuous incubation, iron enrichment increased phytoplankton biomass, nutrient drawdown, diatom and Phaeocystis abundance, and some photosynthetic parameters. High irradiance significantly increased the number of Phaeocystis antarctica colonies, as well as P. antarctica abundance relative to diatoms. Iron and light had significant interactive effects on diatom and P. antarctica pigment concentrations. P. antarctica colony abundance, and Si:N, Si:C, and N:P ratios. The major influence of high CO2 was on diatom community structure, by favoring the large centric diatom Chaetoceros lineola over the small pennate species Cylindrotheca closterium. The ratio of centric to pennate diatoms was significantly responsive to changes in all three variables individually, and to all of their possible two- and three-way combinations. These results suggest that shifts in light, iron, and CO2 and their mutual interactions all play a role in controlling present day Ross Sea plankton community structure, and need to be considered when predicting the possible future responses of biology and biogeochemistry in this region