Potente HIV-gerichtete RNA-Interferenz mittels Selektion einer randomisierten lentiviralen shRNA-Bibliothek

Um neue potente shRNA-Sequenzen gegen HIV-1 zu identifizieren, wurde zum ersten Mal eine randomisierte lentivirale shRNA-Bibliothek konstruiert. Die Expression der shRNAs wurde dabei von einem RNA Polymerase III Promoter (Pol III) gesteuert. Die Selektion potenter shRNA-Sequenzen erfolgte über zu diesem Zweck etablierten Zelllinien. Sowohl die Konstruktion als auch das Selektionssystem basierten auf etablierten Technologien, die jedoch in einigen Punkten zwecks erhöhter Diversität der Bibiothek und optimierter Selektionsbedingungen modifiziert wurden. Die Validierung der selektierten shRNA-Sequenzen zeigte, dass mehr als die Hälfte der shRNAs ein inhibitorisches Potential von über 70% aufwiesen. Sequenzanalysen subklonierter shRNA-Expressionskassetten demonstrierten ihre HIV-1 Spezifität. Außerdem zeigten Vergleichsanalysen mit öffentlichen siRNA-Algorithmen, dass die meisten der hier selektierten shRNAs die Parameter der Algorithmen nicht erfüllen konnten. Daraus resultiert, dass die selektierten shRNA-Sequenzen zusammen mit anderen selektierten Bibliotheken einen wichtigen Beitrag zur Verbesserung der entscheidenden Parameter der siRNA-Algorithmen leisten könnten, weil sie aufgrund ihrer Effizienz und möglicher Toxizität und nicht ihrer Sequenz selektiert wurden. Um die Konvertibilität der inhibitorischen Sequenzen zu überprüfen, wurde das Pol III System mit synthetischen siRNAs und RNA Polymerase II gesteuerten miRNAs verglichen. Dabei stellte sich das Pol III System als das effizienteste heraus. Synthetische siRNA-Derivate ausgewählter hochpotenter shRNA-Sequenzen wiesen zwar ein vergleichbar starkes inhibitorisches Potential auf, die transiente Wirkdauer und die geringe Stabilität einiger siRNA-Sequenzen in Serum limitiert jedoch ihren Einsatz in klinischen Studien. Die Expression der shRNAs in einer miRNA-Umgebung offenbarte wiederum nur eine geringe inhibitorische Wirkung und erfordert weitere Optimierungen dieses Expressionssystems. Die stabile lentivirale Expression von shRNAs in HIV permissiven HeLa-P4-Zellen zeigte eine effiziente Inhibition der Virusreplikation nach Infektion mit HIV-1. Da die inserierten shRNAs in Zelllinien selektiert und reevaluiert wurden, kann außerdem von einer geringen Zytotoxizität ausgegangen werden. Dies macht sie für einen etwaigen Einsatz in gentherapeutischen Ansätzen interessant

Selection of Potent Non-Toxic Inhibitory Sequences from a Randomized HIV-1 Specific Lentiviral Short Hairpin RNA Library

RNA interference (RNAi) has been considered as an efficient therapeutic approach against the human immunodeficiency virus type 1 (HIV-1). However, to establish a durable inhibition of HIV-1, multiple effective short hairpin RNAs (shRNAs) need to be stably expressed to prevent the emergence of viral escape variants. In this study, we engineered a randomized lentiviral H1-promoter driven shRNA-library against the viral genome. Potent HIV-1 specific shRNAs were selected by ganciclovir treatment of cell lines stably expressing the cDNA of Herpes Simplex Virus thymidine kinase (HSV-TK) fused to HIV-1 nucleotide sequences. More than 50% of 200 selected shRNAs inhibited an HIV-1 based luciferase reporter assay by more than 70%. Stable expression of some of those shRNAs in an HIV-1 permissive HeLa cell line inhibited infection of wild-type HIV-1 by more than 90%. The combination of a randomized shRNA-library directed against HIV-1 with a live cell selection procedure yielded non-toxic and highly efficient HIV-1 specific inhibitory sequences that could serve as valuable candidates for gene therapy studies

Clone size of randomized shRNA-libraries.

<p>To indicate the complexity of the shRNA library, the number of bacterial colonies of each transformation step (primary library, secondary library and final library) was documented as colony forming units (cfu).</p

Construction of the shRNA-library.

<p><b>a</b>) The 3 HIV segments of pNL4.3 were fragmented using DNaseI and blunt-ended. <b>b</b>) Fragments of 150–300 bp were eluted and ligated to the 3′ loop. To limit the size of the HIV-1 inverted repeats, a recognition site for MmeI, which cleaves exactly 20nt from its recognition site and leaves 2 nt 5′ overhangs, was engineered into the 3′ loop. <b>c</b>) Ligation of the 5′ loop to the MmeI-digested fragments generated a quasi-circular single-stranded structure. <b>d</b>) Rolling circle amplification (RCA) reactions using Φ 29 DNA polymerase and the primers RCA1 and RCA2 were performed to amplify the single-stranded circular DNA and to generate the complementary strand yielding a DNA concatemere of palindromic, inverted repeats encoding siRNA molecules. Digestion with BglII and MlyI liberated shRNA sequences which were inserted into the expression vector pENTR/siLib. <b>d</b>) The shRNA sequences were cloned into the linearized pENTR/Lib generating the primary library which was religated after BamHI digestion yielding the secondary library. The final lentiviral shRNA-library was generated by LR recombination of the secondary library into pL/EGP/siLib.</p

Specificity of shRNAs against HIV-1.

<p><b>a)</b> Designation of the identified shRNAs <b>b)</b> shRNA-target sequence of the HIV-1 proviral clone pNL4.3 <b>c)</b> starting nucleotide of the shRNA based on +1 being the transcriptional start site. <b>d)</b> Number of known isolates which include the target sequence.</p

Reconfirmation of the inhibitory potential of selected shRNA-sequences.

<p><b>a</b>) ShRNAs were PCR-amplified from genomic DNA of selected cell clones as shRNA expression cassettes consisting of H1 promoter, shRNA-sequence and polyT. Each of the 200 individual cassettes were co-transfected with the HIV-1 specific Luciferase reporter construct pNL4.3Luc.R-E- into HEK 293 FT cells. Luciferase expression was measured 48 h p.t. Cells transfected with pNL4.3Luc.R-E- and a scrambled shRNA (sh scr) expressing cassette were used as control. <b>b</b>) Map of the identified shRNAs and re-evaluation of their inhibitory potential upon co-transfection as described in <b>a</b>). Cells which were transfected with pNL4.3LucR-E- alone or co-transfected with a scrambled siRNA control served as controls. Error bars indicate +/− SD of mean of three independent experiments.</p

Expression of the shRNAs from different polymerase III promoters.

<p><b>a</b>) 6 potent shRNA sequences were subcloned to allow for U6 promoter driven shRNA expression. U6 or H1 promoter expression vectors and pNL4.3LucR-E- were co-transfected into HEK 293 FT cells. Western blot analysis with a HIV-1 Integrase specific antibody 48 h p.t. indicated that the shRNAs can be efficiently expressed from both polymerase III promoters. Actin was used as loading control. <b>b</b>) <b>c</b>) Published shRNA-sequences against pol, nef, rev/env, gag and vpu/env were cloned to be expressed by the U6 promoter. These constructs as well as 14 library shRNAs and pNL4.3LucR-E- were co-transfected into HEK 293 FT cells and analysed 48 h post transfection. Western blot analysis <b>b</b>) with a HIV-1 Integrase specific antibody or <b>c</b>) luciferase assays demonstrated that the newly identified shRNAs are as potent as the published shRNAs. A scrambled shRNA (sh scr) and non-transfected cells were used as controls.</p

A tissue-specific screen of ceramide expression in aged mice identifies ceramide synthase-1 and ceramide synthase-5 as potential regulators of fiber size and strength in skeletal muscle

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi-organ investigation of age-associated changes of ceramide species, a unique and distinctive change pattern of C-16:0 and C-18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C-16:0 and C-18:0 acyl chains, respectively, was down-regulated in skeletal muscle of aged mice. Similarly, an age-dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1(Delta/Delta) and CerS5(Delta/Delta) knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1- and CerS5-deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age-related muscle wasting disorders in humans