Biomedical subjects

J Kraunus

Publications and source records attributed to J Kraunus.

3 recordsLinked to original sources

PubMed2004

B-cell-specific transgene expression using a self-inactivating retroviral vector with human CD19 promoter and viral post-transcriptional regulatory element.

Retroviral gene transfer resulting in transgene expression selectively restricted to specific cell lineages would be desirable for many gene therapeutic applications. Such transcriptional targeting of retroviruses can be accomplished by employing eukaryotic control elements in self-inactivating (SIN) retroviral vectors, but use of these vectors is complicated by an accompanying reduction in viral titers. To overcome this restriction and address the influence of the post-transcriptional regulatory element of the Woodchuck hepatitis virus (WPRE) on viral titers and transgene expression, we developed SIN-vectors with and without WPRE. Using the enhancer-promoter of the Spleen Focus Forming virus (SFFV) to direct eGFP expression to multiple hematopoietic lineages, we show that WPRE significantly (>10 x) increased viral titers (>10(6) per ml of unconcentrated supernatant) and transgene expression in NIH3T3 cells in vitro. Gene expression in vivo was significantly lowered in lymphoid cells, but not in myeloid cells when WPRE was present. Furthermore, the use of WPRE in combination with the B-cell lineage-specific CD19 promoter significantly increased viral titers and allowed targeting of transgene expression by SIN-vectors specifically to B cells throughout their development in primary and secondary lymphoid organs.

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PubMed2004

Self-inactivating retroviral vectors with improved RNA processing.

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5' untranslated region (5'UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3'UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3' long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5'UTR, taking advantage of retroviral splice sites surrounding Psi. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10(6) infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

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PubMed1997

Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice.

TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.

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