Decreased phenotypic susceptibility to etravirine in patients with predicted genotypic sensitivity.

BACKGROUND: A sensitive, phenotypic reverse transcriptase (RT)-based drug susceptibility assay for the detection of etravirine (ETR) resistance in patient isolates was developed and compared with the results from direct sequencing and ultra-deep pyrosequencing (UDPS). METHODS: Samples were obtained from 15 patients with antiretroviral therapy (ART) failure and from five non-nucleoside reverse transcriptase inhibitor (NNRTI)-naïve patients of whom four were infected by an NNRTI-resistant strain (transmitted drug resistance, TDR). In five patients, two consecutive samples (a and b) were taken for follow up of the virological response. HIV-1 RT was purified and drug susceptibility (IC50) to ETR was estimated. Direct sequencing was performed in all samples and UDPS in samples from nine patients. RESULTS: Increased IC50 to ETR was found in samples from 13 patients where direct sequencing predicted resistance in only four. UDPS identified additional (N = 11) NNRTI resistance associated mutations (RAMs) in six of nine tested patients. During early failure, IC50 increases were observed in three of six patients without any ETR-RAMs detected by direct sequencing. In further two patients, who stopped NNRTI before sampling, increased IC50 values were found shortly after, despite absence of ETR-RAMs. In two patients who had stopped NNRTI for >1 year, a concordance between phenotype and genotypes was found. Two patients with TDR had increased IC50 despite no ETR-RAMs were detected by direct sequencing. UDPS revealed additional ETR-RAMs in four patients with a discrepancy between phenotype and direct sequencing. CONCLUSIONS: The RT-based phenotypic assay showed decreased ETR susceptibility in patients where direct sequencing predicted ETR-sensitive virus. This increased phenotypic sensitivity was to a large extent supported by UDPS and treatment history. Our method could be valuable for further studies on the phenotypic kinetics of NNRTI resistance. The clinical relevance remains to be studied in larger patient-populations.

Efficacy and mode of action of hammerhead and hairpin ribozymes against various HIV-1 target sites.

Ribozymes have been proposed as gene therapy agents against HIV-1, although many fundamental questions about their mechanism of action remain unclear. Few studies have compared directly the potential of different modified ribozyme species against a particular target. Here we compare the relative abilities of hammerhead (HhU5) and hairpin (HpU5) ribozymes directed against a well-studied target RNA that has therapeutic potential, located in the untranslated 5' region (U5), to inhibit HIV-1 replication. The two types of ribozymes showed similar antiviral efficacy after being stably transfected into HUT78 cells and subsequently challenged with HIV-1(SF2), but the HhU5 ribozyme showed faster cleavage kinetics when tested in a cell-free system. In the second part of this study, we examined whether different ribozymes were able to inhibit the integration of proviral DNA in infected HUT78 cells. We found that cell pools stably expressing HpU5 could limit the appearance of integrated provirus, indicating that they could inhibit the infecting viral RNA before reverse transcription. A preintegration effect was also found for cell pools expressing a ribozyme targeting the nef gene (HhNef) or a ribozyme targeting the LTR (HhLTR). However, no discernible preintegration effects were seen for the HhU5 ribozyme or an active ribozyme directed against an RNA target site in the pol gene (HhPol). Thus, the results suggest that the mode of ribozyme action varied between sites and is not dependent solely on inhibiting the infecting viral RNA. Evidence for a preintegration effect is extremely encouraging and indicates that "resistant" cells have some chance to repopulate the immune system through such a selective advantage. We also studied the ability of the different ribozymes to down regulate viral RNA postintegration.

Cis-cleavage affects hammerhead and hairpin ribozyme steady-state levels differently and has strong impact on trans-targeting efficiency.

Trans-cleaving hammerhead or hairpin ribozymes were expressed in transgenic mice and in cell lines, using a cassette containing a second cis-cleaving hammerhead ribozyme positioned 3' of the trans-cleaving hammerhead or hairpin ribozyme. Cis-cleavage could be detected readily in transgenic mice, demonstrating in vivo release of the desired short trans-cleaving ribozyme transcript with a defined 3'-end. In transgenic organs, all cis-cleavage products containing a hairpin ribozyme were found at significantly higher steady-state levels than products containing a hammerhead ribozyme. Furthermore, an organ difference - kidney > liver > lung > spleen - regarding steady-state levels of both 5' and 3' cleavage products was found. In pools of stably transfected human T cells (HUT78), the efficacy of the 3' cis-cleavage was found to affect both the steady-state level and the antiviral efficiency of a trans-cleaving hairpin ribozyme targeting HIV-1. Insertion of a point mutation, efficiently inhibiting the cis-cleavage mechanism, led to higher overall steady-state levels of the noncleaved full-length transcript but, at the same time, also abolished the hairpin ribozyme protection against HIV-1 infection. We conclude that the cis-cleavage affects hammerhead and hairpin ribozyme steady-state levels differently and that it has a strong impact on trans-targeting efficiency.