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Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and longterm gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/10[sup 7] particles) and adenovirus. Transduction of CD34[sup +] cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated, quiescent CD34[sup +] cells were resistant to transduction (less than 4%), but transduction increased to 23 ± 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor α (51 ± 4 %) at a multiplicity of infection of 10[sup 6]. rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34[sup +] and CD34[sup +], CD38[sup -] subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 ± 7% of CD34[sup +] progenitors and 21 ± 6% of CD34[sup +], CD38[sup -] progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient. [ABSTRACT FROM AUTHOR]

One factor limiting the ability to modify human repopulating hematopoietic cells genetically with retroviral vectors is the relatively low expression of the cognate viral receptor. We have tested sequential transduction of human hematopoietic cells with an adenoviral vector encoding the ecotropic retroviral receptor followed by transduction with an ecotropic retroviral vector. Adenoviral transduction of K562 erythroleukemia cells was highly efficiently with >95% of cells expressing the ecotropic receptor at a multiplicity of infection (MOI) of 103with a correspondingly high transduction with a retroviral vector. Ecotropic receptor expression in CD34+ cells following transduction with adenoviral vectors was increased by at least two-fold (from 20 to 48%) by replacing the RSV promoter with the CMV E1a promoter, resulting in a parallel increase in retroviral transduction efficiency. Replacing the head portion of the fiber protein in conventional adenoviral vectors (serotype 5) with the corresponding portion from an adenoviral 3 serotype resulted in ecotropic receptor expression in 60% of CD34+ cells at an MOI of 104 and a retroviral transduction of 60% of hematopoietic clonogenic progenitors. The sequential transduction strategy also resulted in efficient transduction of the primitive CD34+CD38- subset suggesting that it may hold promise for genetic modification of human hematopoietic stem cells. [ABSTRACT FROM AUTHOR]

Presents a case report on palmoplantar pustulosis and bone lesions. Discussion on fracture detection; Utilization of magnetic resonance imaging in checking tissue inflammation; Treatment of multifocal osteomyelitis.