Interference between effector RNAs expressed from conventional dual-function anti-HIV retroviral vectors can be circumvented using dual-effector-cassette retroviral vectors.
Coexpression of different effector molecules from a single vector (a dual-function vector) may provide enhanced efficacy. Thus far most of the reported anti-HIV dual-function vectors express different effector RNAs as a chimeric molecule. In our study involving retroviral vectors coexpressing a U5 ribozyme and either an anti-tat or anti-rev antisense RNA, chimeric vectors exhibit poor potency in several important functional aspects, including inhibition of HIV replication, protection against cytopathic effects, and suppression of target gene function. Surprisingly, such a poor efficacy of chimeric vector function was not associated with a lower level of effector RNA expression. These results indicate that expression of two effector RNAs as a chimeric molecule can lead to interference, reducing their global biological effects. More importantly, we have demonstrated that such interference can be avoided by coexpressing these effector RNAs as separate molecules through a new dual-function vector, called a dual-effector cassette (Dec) vector, developed in this study. We also define some of the design alterations that might affect the efficacy of the Dec vector and demonstrate that forward-designed Dec vectors are more efficacious than reverse-designed Dec vectors, which express a lower level of effector RNA owing to the instability of the 5' effector cassettes in the provirus. We believe that the principle of Dec vector design may also be applicable for the coexpression of other therapeutic RNA effectors in many gene therapy applications.
National Centre for HIV Virology Research, Infectious Diseases Laboratories Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia. Pengh@ll.visn22.med.va.gov
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D002460:Cell Line; D015696:Gene Products, tat / Q000032:analysis; D015316:Genetic Therapy; D005822:Genetic Vectors; D006678:HIV / Q000276:immunology; D016655:HIV Core Protein p24 / Q000032:analysis; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D008954:Models, Biological; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D016655:HIV Core Protein p24; D016337:RNA, Catalytic; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA