Substantial effort has been invested in developing methodologies for efficient gene transfer into human, repopulating, hematopoietic stem cells. Oncoretroviral vectors are limited by the lack of nuclear mitosis in quiescent stem cells during ex vivo transduction, whereas the preintegration complex of lentiviral vectors contains nuclear-localizing signals that permit genome integration without mitosis. We have developed a flexible and versatile system for generating lentiviral vector particles and have pseudotyped such particles with amphotropic, ecotropic, feline endogenous virus (RD114) or vesicular stomatitis virus (VSV-G) envelope proteins. Particles of all four types could be concentrated approximately 100-fold by ultracentrifugation or ultrafiltration. RD114 or amphotropic particles were more efficient than VSV-G-pseudotyped particles at transducing human cord blood CD34(+) cells and clonogenic progenitors within that population. Amphotropic particles transduced cytokine-mobilized, human peripheral blood CD34(+) cells capable of establishing hematopoiesis in immunodeficient mice more efficiently than the other two types of particles. We conclude that the use of amphotropic pseudotyped lentiviral vector particles rather than the commonly used VSV-G-pseudotyped particles should be considered in potential applications of lentiviral vectors for gene transfer into this therapeutically relevant target cell population.

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Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector–mediated gene transfer. Human hematopoietic cell lines and cord blood–derived CD34+ and CD34+, CD38− cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34+ cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive for enhanced green fluorescent protein represented as much as 90% of the graft. The use of RD114-pseudotyped vectors may be advantageous for therapeutic gene transfer into hematopoietic stem cells.

The functional classification of genes on a genome-wide scale is now in its infancy, and we make a first attempt to assess existing methods and identify sources of error. To this end, we compared two independent efforts for associating proteins with functions, one implemented by FlyBase and the other by PANTHER at Celera Genomics. Both methods make inferences based on sequence similarity and the available experimental evidence. However, they differ considerably in methodology and process. Overall, assuming that the systematic error across the two methods is relatively small, we find the protein-to-function association error rate of both the FlyBase and PANTHER methods to be <2%. The primary source of error for both methods appears to be simple human error. Although homology-based inference can certainly cause errors in annotation, our analysis indicates that the frequency of such errors is relatively small compared with the number of correct inferences. Moreover, these homology errors can be minimized by careful tree-based inference, such as that implemented in PANTHER. Often, functional associations are made by one method and not the other, indicating that one of the greatest challenges lies in improving the completeness of available ontology associations.

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