Viral Hybrid Vectors for Somatic Integration - Are They the Better Solution?

The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system

Ad 2.0: a novel recombineering platform for high-throughput generation of tailored adenoviruses

stranded DNA genome of 26-45 kb were broadly explored in basic virology, for vaccination purposes, for treatment of tumors based on oncolytic virotherapy, or simply as a tool for efficient gene transfer. However, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types and their genetic modification. Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here, we adopted a seamless recombineering technology for high-throughput and arbitrary genetic engineering of recombinant adenoviral DNA molecules. Our cloning platform which also includes a novel recombination pipeline is based on bacterial artificial chromosomes (BACs). It enables generation of novel recombinant adenoviruses from different sources and switching between commonly used early generation AdVs and the last generation high-capacity AdVs lacking all viral coding sequences making them attractive candidates for clinical use. In combination with a novel recombination pipeline allowing cloning of AdVs containing large and complex transgenes and the possibility to generate arbitrary chimeric capsid-modified adenoviruses, these techniques allow generation of tailored AdVs with distinct features. Our technologies will pave the way toward broader applications of AdVs in molecular medicine including gene therapy and vaccination studies

Oncolytic Adenoviruses Armed with Tumor Necrosis Factor Alpha and Interleukin-2 Enable Successful Adoptive Cell Therapy

Adoptive cell therapy holds much promise in the treatment of cancer but results in solid tumors have been modest. The notable exception is tumor-infiltrating lymphocyte (TIL) therapy of melanoma, but this approach only works with high-dose preconditioning chemotherapy and systemic interleukin (IL)-2 postconditioning, both of which are associated with toxicities. To improve and broaden the applicability of adoptive cell transfer, we constructed oncolytic adenoviruses coding for human IL-2 (hIL2), tumor necrosis factor alpha (TNF-alpha), or both. The viruses showed potent antitumor efficacy against human tumors in immunocompromised severe combined immunodeficiency (SCID) mice. In immunocompetent Syrian hamsters, we combined the viruses with TIL transfer and were able to cure 100% of the animals. Cured animals were protected against tumor re-challenge, indicating a memory response. Arming with IL-2 and TNF-alpha increased the frequency of both CD4(+) and CD8(+) TILs in vivo and augmented splenocyte proliferation ex vivo, suggesting that the cytokines were important for T cell persistence and proliferation. Cytokine expression was limited to tumors and treatment-related signs of systemic toxicity were absent, suggesting safety. To conclude, cytokine-armed oncolytic adenoviruses enhanced adoptive cell therapy by favorable alteration of the tumor microenvironment. A clinical trial is in progress to study the utility of Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (TILT-123) in human patients with cancer.Peer reviewe

Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation

Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35) vectors for zinc-finger nuclease (ZFN)- or transcription activator-like effector nuclease (TALEN)-mediated genome editing in human CD34+ hematopoietic stem cells (HSCs) from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA)-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene -expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35. ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells). The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2) within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.

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