Self-inactivating retroviral vectors with improved RNA processing

TY - JOUR

T1 - Self-inactivating retroviral vectors with improved RNA processing

AU - Kraunus, J

AU - Schaumann, D H S

AU - Meyer, J

AU - Modlich, U

AU - Fehse, B

AU - Brandenburg, G

AU - von Laer, D

AU - Klump, H

AU - Schambach, A

AU - Bohne, J

AU - Baum, C

PY - 2004/11

Y1 - 2004/11

N2 - 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).

AB - 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).

KW - Animals

KW - Cell Line, Tumor

KW - Gene Expression

KW - Genetic Engineering

KW - Genetic Therapy

KW - Genetic Vectors/genetics

KW - Hematopoietic Stem Cells/virology

KW - Hepatitis B Virus, Woodchuck/genetics

KW - Humans

KW - Lymphocytes/virology

KW - Mice

KW - Mice, Inbred C57BL

KW - RNA Processing, Post-Transcriptional

KW - Safety

KW - Transfection/methods

KW - Transgenes

KW - Virus Inactivation

U2 - 10.1038/sj.gt.3302309

DO - 10.1038/sj.gt.3302309

M3 - SCORING: Journal article

C2 - 15372067

VL - 11

SP - 1568

EP - 1578

JO - GENE THER

JF - GENE THER

SN - 0969-7128

IS - 21

ER -