Analysis of Adenovirus-Host Interactions to Improve Recombinant Adenoviral Vectors for Gene Therapy

Recombinant adenoviral vectors are among the most commonly used vehicles in gene therapy. Replication-deficient adenoviruses include early generation adenoviruses, which are deleted in less than three adenoviral genes, and the high-capacity adenoviruses (HC-AdV) as the most advanced form. HC-AdVs are deleted for all viral sequences leaving only the two inverted terminal repeat sequences (ITRs) and the packaging signal from the original viral genome. Therefore, up to 36 kilobases of foreign DNA can be packaged by HC-AdV particles and transduced into the desired target cell. Efficacy of these vectors was shown in several animal models, in which a single injected virus dose resulted in up to 3 years of transgene expression. However, also for recombinant adenoviruses including HC-AdVs limiting factors remain, which were investigated and improved in the course of this work. The production of HC-AdV represents one limiting factor because it is a labour intensive and sophisticated process that requires some experience. Therefore, in this study, a protocol was developed that simplifies the generation of these viruses starting with an improved cloning procedure and ending with precise titration of the purified particles. In addition, this improved virus production procedure was used to demonstrate the feasibility of HC-AdV to delivery short-hairpin RNAs, thus reducing hepatitis B RNA molecules in vitro and in vivo. The majority of HC-AdVs are currently based on the adenovirus serotype 5 (Ad5). However, DNA sequences inserted into the HC-AdV genome and remaining viral sequences were shown to influence duration and stability of transgene expression, which can negatively influence the outcome of a therapeutic approach. By analyzing viral ITR sequences derived from different adenoviral serotypes, this work demonstrated that ITR-driven transcriptional activity from several serotypes but also inhibiting functions occur leading to reduced transgene expression. Furthermore, a negative impact of ITR sequences on nearby promoters could be observed. The data obtained in this work suggest that it could be beneficial to introduce shielding sequences into the HC-AdV genome, which flank the transgene expression cassettes and therefore, prevent undesired side effects. Moreover, the results indicated, that pursuing ITRs from adenovirus serotype 7 in the context of an adenoviral vector could be advantageous, as it demonstrated most suitable features regarding transcriptional activation and influence on promoter performance. The efficiency of HC-AdV in terms of long-term expression of foreign DNA sequences is mainly based on the stability of vector genomes in quiescent cells. In dividing cells, however, a continuous reduction of the viral DNA reduces the therapeutic effect. Thus, integration systems on the basis of viral hybrid vectors were developed, which result into recombinase-mediated somatic integration of the therapeutic DNA from the HC-AdV genome into the chromosomal DNA. The most prominent representative of non-viral integration systems is the Sleeping Beauty (SB) transposase. Although function and efficacy of this transposase was shown in the context of an HC-AdV, it turned out, that transgene expression is decreased after Sleeping Beauty mediated transposition. Herein, analysis of transposition in cells with suppressed RNA interference pathway, showed a higher transposition rate in RNA interference knockdown cells compared to control cells, which was mainly based on an increased transgene expression. Therefore, this work shows for the first time that due to convergent transcription, originated from the two SB recognition sequences (IRs) flanking the transposon, formation of double-stranded RNAs (dsRNAs) can occur. These dsRNAs can be substrates for the RNAi mechanism and contribute to the silencing of gene expression. In the future this finding can be used to significantly improve the SB transposon technology. Moreover the influence of the RNAi mechanism on the adenovirus life cycle could be demonstrated within this project. By the suppression of the RNAi pathway using an RNAi suppressor protein we could improve recombinant adenovirus replication and viral particle production, up to 100-fold. In addition, this RNAi suppressor protein increased production of HC-AdV up to 6-fold. This upregulation was mainly based on the increased expression of viral regulatory proteins as well as the suppression of small adenoviral RNAs. In conclusion, this work provides different strategies to improve HC-AdVs for gene therapeutic purposes. Furthermore, it investigated mechanisms that negatively interfere with the therapeutic outcome, which need to be considered in future work. In particular, the influence of the RNA interference pathway on the replication profile of recombinant adenoviruses could be demonstrated for the first time essentially broadening the potential of these vectors with respect to viral production and design of oncolytic adenoviruses. In summary, this study emphasizes the importance of understanding the biology of viral vectors systems, which then can be translated into the development of optimized vectors for gene therapeutic applications

Hyperactive Sleeping Beauty Transposase Enables Persistent Phenotypic Correction in Mice and a Canine Model for Hemophilia B

Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases

RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration

Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system

Generation and characterization of the RNAi knockdown cell lines.

<p>(A) DNA sequences used to generate stable <i>p19</i> expressing cell lines. Kp19 was used for stable plasmid transfection of HEK293 cells. The plasmid p19-MIE was used to produce a P19 expressing recombinant retrovirus for stable infection of HEK293 cells. K: Kozak sequence; pCMV: promoter of the cytomegalovirus; p19: p19 expression cassette; pRSV: promoter of the rous sarcoma virus; RGS-His: 6 histidin residues connected to the P19 protein by an arginin-glycin-serin motive; Neo: neomycin resistance cassette that mediates G418 resistance; poly A: polyadenylation signal derived from the simian virus; GFP: green fluorescent protein expression cassette; LTR: long terminal repeats; IRES: internal ribosome entry site. (B) Flow cytometric analysis of cell clones generated by retroviral transduction. Single cell clones from cell sorting were amplified and analysed by flow cytometry. Cells appearing in quadrant Q2 refer to GFP+cells. X-axis: GFP amount; Y-Axis: SSC: side scatter, to measure cell viability. <b>(</b>C) Quantitative analysis of GFP positive clones generated by cell sorting shown in <b>Fig. 1B</b>. (D) Expression of <i>p19</i> mRNA in the stable cell lines G3, G4, G5 and G16. The generated cDNA was used for PCR amplification with <i>p19</i> specific primers and a 519 bp band indicates positive cell clones. As positive control the p19 expression cassette from the plasmid Kp19 (+c) was amplified. +: sample with RT; −: sample without RT; 0: untreated HEK293 cells; M: marker. (E) Detection of P19 expression by Western Blot analysis in stable cell lines, which express the His-tagged version of the P19 protein. Monomeric and dimeric P19 molecules were detected using a peroxidase labeled anti-His antibody at 19 kDa and 38 kDa indicated by an arrow in the diagram. As positive control, HEK293 cells were transiently transfected with p19 expressing plasmids (left lane, +c) or mock transfected (-c). (F) Functionality of P19. RNA was isolated from HEK293, B6, G3, G4, G5, G16 cells and reverse transcribed. The cDNA was used for quantification of the HoxB8 mRNA amount by qRT-PCR. An increase in the HoxB8 level indicates a functional P19 protein because functional P19 inhibits miR169a- mediated downregulation of HoxB8. Normalization was performed by GAPDH measurement with GAPDH specific primers. The fold increase of the HoxB8 amount in the RNAi knockdown cell lines was determined in a semi-quantitative manner. *: p-value<0.05.</p

Analysis of the effect of insulator sequences on SB mediated transposition in HEK293 cells and the RNAi knockdown cell line G4.

<p>(A) DNA construct used for the study. pCMV: major immediate early promoter/enhancer; polyA: poly adenylation signal of the simian virus 40; SB: Sleeping Beauty (SB) transposase; mSB: non-functional version of the SB transposase; neo: neomycin re<i>sistance cassette; HS4: chicken insulator.</i> (B) The substrate plasmid T/neo-HS4 was transfected into HEK293 or G4 cells along with either the functional Sleeping Beauty transposase (SB) or the inactive version of SB (mSB). After 2 weeks of G418 selection, cells were stained and blue colonies were counted. The ratios of transposition events (SB to mSB) in G4 cells and HEK293 cells are displayed.</p

Quantification of transposon-derived transgene expression after Sleeping Beauty (SB) mediated transposition in HEK293 cells and the RNAi knockdown cell line G4 and B6.

<p>Transposon donor plasmids (pTnori for G4 cells and pTMCS-IP for B6 cells) and active or inactive SB transposase encoding plasmids (pCMC-SB and pCMV-mSB) were co-transfected into HEK293 cells and RNAi knockdown cell lines G4 and B6. Ten days (G4 cells) and 21 days (B6 cells) post-transfection, RNA was isolated from non-selected cells and reverse transcribed. The cDNA was then subjected to quantitative Real-Time PCR using neomycin or puromycin specific primers. Results were normalized to expression of 1000 human beta2 microglobulin RNA molecules. (A) Normalized neomycin (neo) expression in G4 cells compared to HEK293 cells. (B) Normalized puromycin (puro) expression in B6 and HEK293 cells. (C) The fold increase in neomycin expression is shown. Transgene expression in HEK293 cells was set to 1. (D) The fold increase in puromycin expression levels is is displayed. All data are statistically relevant (p-value<0.05).</p

Influence of the RNA interference (RNAi) pathway on integration events mediated by Sleeping Beauty (SB) transposase and Frog Prince (FP) transposase.

<p>(A) Shown are all DNA constructs used to analyze the influence of the RNAi pathway on SB mediated transposition in HEK293 cells and the RNAi knockdown cell lines G4 and B6. The vector pTnori with the neomycin resistance gene was used as a transposon donor vector in G4 cells and pTMCS-IP was used in B6 cells. pCMV: major immediate early promoter/enhancer; IRES: internal ribosome entry side of the encephalomyocarditis virus (ECMV); Puro: puromycin-N-acetyl-transferase gene mediates puromycin resistance; bpolyA: bovine growth hormone A polyA signal; polyA: SV40 polyA signal; Neo: neomycin resistance cassette mediating G418 resistance; SB: SB transposase; mSB: mutated version of the Sleeping Beauty transposase; IR: inverted repeats recognized by SB. (B) Analysis of transfection efficiencies in HEK293, G4 (left panel) and B6 cells (right panel) two days post-transfection of the DNA constructs used for SB-mediated transposition. Instead of 100 ng stuffer plasmid as used for integration assays, 100 ng of the renilla luciferase expressing plasmid pRL-TK were transfected into 6-well plates and relative light units were determined. For details please refer to the material and methods section. The amount of relative light units correlates with the transfection efficiency. (C) Total number of colony forming units (CFUs) after SB transposase mediated integration in HEK293 cells and the RNAi knockdown cell lines G4 (left panel) or B6 (right panel). The fold increase in transposition events compared to the inactive protein is shown in numbers above the respective bars. All experiments were performed in triplicates and were statistically relevant (p-value<0.05). *: p-value<0.05. (D) The fold increase in integration events directly comparing the parental cell line HEK293 and the respective RNAi knockdown cell line for the SB transposase and the Frog Prince (FP) transposase are shown. (E) Shown are all DNA constructs used to analyze the influence of the RNAi pathway on FP mediated transposition in HEK293 cells and the RNAi knockdown cell lines G4 and B6. The transposon donor vector pFP-IP with the puromycin resistance gene was used in B6 cells and pFP-neo2 was used in G4 cells. P beta actin: beta actin promoter; FP: Frog Prince transposase; 4a: empty vector without FP transposase; IR: inverted repeats recognized by FP. (F) Analysis of transfection efficiencies in HEK293, G4 and B6 cells for the FP transposase system two days post-transfection. The amount of relative light units correlates with the transfection efficiency. (G) Total number of CFUs after Frog Prince mediated transposition in HEK293 cells and the RNAi knockdown cell lines G4 (left panel) or B6 (right panel). The fold increase in transposition events is expressed in numbers above the respective bars. The data comparing the active transposase with its inactive version within one cell line are statistically relevant (p-value<0.05). All experiments were performed in triplicates. * p-value<0.05.</p