Retrovirally expressed anti-HIV ribozymes confer a selective survival advantage on CD4+ T cells in vitro.
To date, a selective advantage of cells expressing anti-HIV ribozymes has not been shown. This study was undertaken to determine whether such a selective advantage can be demonstrated in vitro. A retroviral vector coding for a hairpin ribozyme targeting the HIV 5'LTR and for the low affinity nerve growth factor receptor (LNGF-RDelta) was designed. Since we demonstrated by RT-PCR that the amount of ribozyme transcripts was highly correlated with the level of surface LNGF-RDelta expression, the vector was utilized to assess ribozyme expression by flow cytometry. Transduced Hut78 and primary CD4+ T cells were purified and subsequently mixed with unmodified cells. After HIV challenge the percentage of ribozyme expressing cells in the cell mixture was monitored by flow cytometry. Twenty-one days after HIV infection the proportion of ribozyme expressing CD4+ T cells was 2.6 times higher in comparison to cells with the control vector. CD4+ T cells with a strong ribozyme expression conferred a 7.4-fold selective advantage at day 21 and a 11.7-fold at day 28. For Hut78 cells a selective advantage was detected exclusively for strongly ribozyme expressing cells. As a mechanism underlying the selective advantage an inhibition of HIV induced apoptosis was shown. These results demonstrate that anti-HIV ribozymes are able to confer a selective survival advantage and indicate that the protective effect is dependent on the amount of ribozyme expression. Gene Therapy (2000) 7, 408-416.
Klebba
C
C
Medizinische Klinik III der Johann Wolfgang Goethe Universität, Frankfurt, Germany.
Ottmann
O G
OG
Scherr
M
M
Pape
M
M
Engels
J W
JW
Grez
M
M
Hoelzer
D
D
Klein
S A
SA
D017209:Apoptosis; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D005822:Genetic Vectors / Q000235:genetics*; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D020932:Nerve Growth Factor / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D012641:Selection, Genetic; D014779:Virus Replication / Q000235:genetics
D016337:RNA, Catalytic; D012333:RNA, Messenger; D020932:Nerve Growth Factor