Interference between Effector RNAs Expressed from Conventional Dual-Function Anti-HIV Retroviral Vectors Can Be Circumvented Using Dual-Effector-Cassette Retroviral Vectors

Abstract

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.

Assessment of Transduction Rates of Porcine Bone Marrow

“…3). These results are consistent with recent reports on the instability of some recombinant proviral genomes, and more specifically of genes inserted in the LTRs of the N2-based double copy vector used in our experiments (22)(23)(24)(25)(26).…”

Current develoments and future prospects for HIV gene therapy using interfering RNA-based strategies

“…Double copy retroviral vectors were designed to express tRNAi Met -hammerhead ribozyme targeted against the U5 region or tRNAi Met -antisense (tat or rev) RNA (209). Vectors were also designed to co-express these RNAs as part of a single transcript (tRNAi Met -U5 ribozyme-tat or rev antisense RNA) or as two separate transcripts (tRNAi Met -U5 ribozyme and tRNAi Met -tat or rev antisense RNA).…”