Recombinant AAV-2 harboring gfp-antisense/ribozyme fusion sequences monitor transduction, gene expression, and show anti-HIV-1 efficacy.
Vector-mediated delivery of potentially antivirally active genes is a key step in somatic gene therapy including therapeutic approaches against AIDS. A crucial technical prerequisite is to monitor DNA transfer into target cells. Here, we describe recombinant infectious particles derived from the adeno-associated virus type 2 (AAV-2) that are suitable to deliver effective HIV-1-directed antisense and ribozyme genes into target cells. To monitor transduction, we designed and tested a number of fusions between indicator-coding sequences of luciferase or gfp with effective HIV-1-directed antisense or ribozyme sequences. The combination of an indicator function and an antiviral func- tion in cis allows successful identification of transduced cells and measurement of effects on the replication of HIV-1 in antisense/ribozyme-expressing cells only. The fusion genes were shown to express the indicator genes. Inhibition of HIV-1 replication mediated by the antisense/ribozyme portion of the fusion transcripts was similar to parental constructs and neither acute nor long-term toxicity of fusion genes and their gene products was observed. These results suggest the use of rAAV constructs described here as tools to study the transducibility of target cells, gene expression and efficacy of HIV-1-directed antisense and ribozyme genes.
Hörster
A
A
Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany.
Teichmann
B
B
Hormes
R
R
Grimm
D
D
Kleinschmidt
J
J
Sczakiel
G
G
D000163:Acquired Immunodeficiency Syndrome / Q000628:therapy*; D015496:CD4-Positive T-Lymphocytes; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics*; D016372:RNA, Antisense; D016337:RNA, Catalytic / Q000235:genetics; D014162:Transfection; D014779:Virus Replication
D008164:Luminescent Proteins; D016372:RNA, Antisense; D016337:RNA, Catalytic; D049452:Green Fluorescent Proteins; D008156:Luciferases