An RNAi in silico approach to find an optimal shRNA cocktail against HIV-1

Abstract

BackgroundHIV-1 can be inhibited by RNA interference in vitro through the expression of short hairpin RNAs (shRNAs) that target conserved genome sequences. In silico shRNA design for HIV has lacked a detailed study of virus variability constituting a possible breaking point in a clinical setting. We designed shRNAs against HIV-1 considering the variability observed in naïve and drug-resistant isolates available at public databases.MethodsA Bioperl-based algorithm was developed to automatically scan multiple sequence alignments of HIV, while evaluating the possibility of identifying dominant and subdominant viral variants that could be used as efficient silencing molecules. Student t-test and Bonferroni Dunn correction test were used to assess statistical significance of our findings.ResultsOur in silico approach identified the most common viral variants within highly conserved genome regions, with a calculated free energy of ≥ -6.6 kcal/mol. This is crucial for strand loading to RISC complex and for a predicted silencing efficiency score, which could be used in combination for achieving over 90% silencing. Resistant and naïve isolate variability revealed that the most frequent shRNA per region targets a maximum of 85% of viral sequences. Adding more divergent sequences maintained this percentage. Specific sequence features that have been found to be related with higher silencing efficiency were hardly accomplished in conserved regions, even when lower entropy values correlated with better scores. We identified a conserved region among most HIV-1 genomes, which meets as many sequence features for efficient silencing.ConclusionsHIV-1 variability is an obstacle to achieving absolute silencing using shRNAs designed against a consensus sequence, mainly because there are many functional viral variants. Our shRNA cocktail could be truly effective at silencing dominant and subdominant naïve viral variants. Additionally, resistant isolates might be targeted under specific antiretroviral selective pressure, but in both cases these should be tested exhaustively prior to clinical use.

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…However, this approach was not completely robust as escape was observed from combinatorial shRNAs despite these being specifically designed to target previously characterized resistant viral variants [197] . Since then, multiple design approaches have been developed using a variety of strategies in search of the best combination of siRNA/ shRNAs molecules that might prevent viral escape [198,199] . Following these findings, shRNAs targeting both conserved viral genes and host cellular genes required for viral replication became the preferred way to overcome this problem.…”

A comparison of multiple shRNA expression methods for combinatorial RNAi

“…This idea is supported by several wet studies showing that in laboratory conditions HIV-1 escape can be delayed by using more than one shRNA [ 11 , 23 - 26 ]. In a clever variation on this idea, some have designed anticipatory shRNAs specifically to block known escape routes, though when tested it was found that the virus still evolved around these [ 27 , 28 ]. There is now clearly a need for an evaluation of multiple shRNA expression strategies to identify those that can be readily integrated into current anti-HIV gene therapy research programs.…”

Microscale Symmetrical Electroporator Array as a Versatile Molecular Delivery System

“…The delivery of macromolecules such as protein 1 2 , DNA 3 4 and RNA 5 6 into cells is a crucial but challenging task for biological and clinical research to elucidate the role of these molecules in regulating cellular functions. Particularly, intracellular co-delivery of multiple molecules would enable identification of optimum combinatorial molecular ratios for various applications, including drug screening for combination therapy 7 8 9 and cellular reprogramming, utilizing multiple transduction factors 10 11 12 . The main hurdle of intracellular macromolecule delivery lies with the difficulty in simultaneously achieving high transfection efficiency, prolonged efficacy, and low cytotoxicity for a wide range of molecules.…”