Oliver Coutelle

Recent developments in adeno‐associated virus vector technology

Adeno‐associated virus (AAV), a single‐stranded DNA parvovirus, is emerging as one of the leading gene therapy vectors owing to its nonpathogenicity and low immunogenicity, stability and the potential to integrate site‐specifically without known side‐effects. A portfolio of recombinant AAV vector types has been developed with the aim of optimizing efficiency, specificity and thereby also the safety of in vitro and in vivo gene transfer. More and more information is now becoming available about the mechanism of AAV/host cell interaction improving the efficacy of recombinant AAV vector (rAAV) mediated gene delivery. This review summarizes the current knowledge of the infectious biology of AAV, provides an overview of the latest developments in the field of AAV vector technology and discusses remaining challenges. Copyright

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

Background In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. Methodology HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. Principal Findings The 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. Conclusions Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.

Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy.

Background:The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC.Methods:Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification.Results:Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31%; P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05).Conclusion:Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment.

The Proteasome Inhibitor Bortezomib Sensitizes Melanoma Cells toward Adoptive CTL Attack

Adoptive transfer of tumor-specific cytolytic T lymphocytes (CTL) results in target cell lysis by activating the intrinsic apoptotic cell death program. Not surprisingly, deregulation of the apoptotic machinery is one of the central mechanisms by which tumor cells escape immune destruction despite specific CTL recognition. Here we show that treatment with the proteasome inhibitor bortezomib sensitizes previously resistant tumor cells for cytolytic T-cell attack. Human T cells were redirected toward melanoma cells by engineered expression of an immunoreceptor with binding specificity for high molecular weight-melanoma-associated antigen. Established melanoma cell lines as well as primary melanoma cells from tumor biopsies, which are notoriously resistant toward T-cell lysis, became sensitive upon bortezomib treatment. Detailed analysis of the underlying molecular mechanism revealed that bortezomib treatment induced mitochondrial accumulation of NOXA, which potentiated the release of mitochondrial second mitochondria-derived activator of caspase (SMAC) in response to CTL effector functions, including caspase-8 and granzyme B. Our data indicate that proteasome inhibition increases the sensitivity of tumor cells toward cytolytic T-cell attack by NOXA-mediated enhancement of mitochondrial SMAC release.

Intrinsic phospholipase A2 activity of adeno-associated virus is involved in endosomal escape of incoming particles.

The unique region of the VP1 capsid protein of adeno-associated viruses (AAV) in common with autonomously replicating parvoviruses comprises a secreted phospholipase A2 (sPLA2) homology domain. While the sPLA2 domain of Minute Virus of Mice has recently been shown to mediate endosomal escape by lipolytic pore formation, experimental evidence for a similar function in AAV infection is still lacking. Here, we explored the function of the sPLA2 domain of AAV by making use of the serotype 2 mutant (76)HD/AN. The sPLA2 defect in (76)HD/AN, which severely impairs AAVs infectivity, could be complemented in trans by co-infection with wild-type AAV2. Furthermore, co-infection with endosomolytically active, but not with inactive adenoviral variants partially rescued (76)HD/AN, providing the first evidence for a function of this domain in endosomal escape of incoming AAV particles.

Ubiquitin C-Terminal Hydrolase-L1 Potentiates Cancer Chemosensitivity by Stabilizing NOXA

The BH3-only protein NOXA represents one of the critical mediators of DNA-damage-induced cell death. In particular, its involvement in cellular responses to cancer chemotherapy is increasingly evident. Here, we identify a strategy of cancer cells to escape genotoxic chemotherapy by increasing proteasomal degradation of NOXA. We show that the deubiquitylating enzyme UCH-L1 is a key regulator of NOXA turnover, which protects NOXA from proteasomal degradation by removing Lys(48)-linked polyubiquitin chains. In the majority of tumors from patients with melanoma or colorectal cancer suffering from high rates of chemoresistance, NOXA fails to accumulate because UCH-L1 expression is epigenetically silenced. Whereas UCH-L1/NOXA-positive tumor samples exhibit increased sensitivity to genotoxic chemotherapy, downregulation of UCH-L1 or inhibition of its deubiquitylase activity resulted in reduced NOXA stability and resistance to genotoxic chemotherapy in both human and C. elegans cells. Our data identify the UCH-L1/NOXA interaction as a therapeutic target for overcoming cancer chemoresistance.

Embelin inhibits endothelial mitochondrial respiration and impairs neoangiogenesis during tumor growth and wound healing

In the normal quiescent vasculature, only 0.01% of endothelial cells (ECs) are proliferating. However, this proportion increases dramatically following the angiogenic switch during tumor growth or wound healing. Recent evidence suggests that this angiogenic switch is accompanied by a metabolic switch. Here, we show that proliferating ECs increasingly depend on mitochondrial oxidative phosphorylation (OxPhos) for their increased energy demand. Under growth conditions, ECs consume three times more oxygen than quiescent ECs and work close to their respiratory limit. The increased utilization of the proton motif force leads to a reduced mitochondrial membrane potential in proliferating ECs and sensitizes to mitochondrial uncoupling. The benzoquinone embelin is a weak mitochondrial uncoupler that prevents neoangiogenesis during tumor growth and wound healing by exhausting the low respiratory reserve of proliferating ECs without adversely affecting quiescent ECs. We demonstrate that this can be exploited therapeutically by attenuating tumor growth in syngenic and xenograft mouse models. This novel metabolic targeting approach might be clinically valuable in controlling pathological neoangiogenesis while sparing normal vasculature and complementing cytostatic drugs in cancer treatment.

Combined Paracrine and Endocrine AAV9 mediated Expression of Hepatocyte Growth Factor for the Treatment of Renal Fibrosis

In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1alpha1 (Col1A1), platelet-derived growth factor receptor-beta (PDGFR-beta), and alpha-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella

The X‐linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti‐apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase‐mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP‐mediated immune response by inducing the BID‐dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain‐dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

Elevated XIAP expression alone does not confer chemoresistance

Background:In various tumour types, elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been observed and XIAP targeting in diverse tumour entities enhanced the susceptibility to chemotherapeutic agents. Therefore, XIAP has been described and reviewed repeatedly as a chemoresistance factor in different tumour entities. However, rather than being an adverse prognostic marker, recent data suggest that elevated XIAP expression may be associated with a favourable clinical outcome. These somewhat conflicting findings, and the fact that in early studies XIAP suppressed apoptosis only when expressed transiently at levels far in excess of its physiological concentration, argue that the function of XIAP as an anti-apoptotic factor in tumour cells is both more complex and diverse than previously appreciated.Methods:To better understand the impact of long-term elevated XIAP expression on resistance to chemotherapy, we generated cell lines stably overexpressing XIAP. The role of mitochondria was examined by stable expression of Bcl2 or stable knockdown of second mitochondria-derived activator of caspase (SMAC) in combination with up- or downregulation of XIAP expression.Results:Our data show that long-term expression of XIAP at concentrations comparable to that in tumour cells (two- to five-fold increase) resulted in little or no resistance towards chemotherapeutic drugs. The XIAP overexpression only in conjunction with stable knockdown of a single XIAP-antagonising factor such as SMAC resulted in severe resistance to cytostatic agents demonstrating XIAP as a potent chemoresistance factor only in cells lacking functional XIAP regulatory circuits.Conclusion:Our results demonstrated that elevated XIAP expression alone cannot serve as a predictive marker of chemoresistance. Our data suggest that in order to predict the impact of XIAP on chemosusceptibility for a given tumour entity, the expression levels and functional states of all XIAP modulators need to be taken into account.