Mathieu Nonnenmacher

Intracellular transport of recombinant adeno-associated virus vectors

Recombinant adeno-associated viral vectors (rAAVs) have been widely used for gene delivery in animal models, and are currently evaluated for human gene therapy after successful clinical trials in the treatment of inherited, degenerative or acquired diseases, such as Leber congenital amaurosis, Parkinson disease or heart failure. However, limitations in vector tropism, such as limited tissue specificity and insufficient transduction efficiencies of particular tissues and cell types, still preclude therapeutic applications in certain tissues. Wild-type adeno-associated viruses (AAVs) are defective viruses that require the presence of a helper virus to complete their life cycle. On the one hand, this unique property makes AAV vectors one of the safest available viral vectors for gene delivery. On the other, it also represents a potential obstacle because rAAV vectors have to overcome several biological barriers in the absence of a helper virus to transduce successfully a cell. Consequently, a better understanding of the cellular roadblocks that limit rAAV gene delivery is crucial and, during the last 15 years, numerous studies resulted in an expanding body of knowledge of the intracellular trafficking pathways of rAAV vectors. This review describes our current understanding of the mechanisms involved in rAAV attachment to target cells, endocytosis, intracellular trafficking, capsid processing, nuclear import and genome release with an emphasis on the most recent discoveries in the field and the emerging strategies used to improve the efficiency of AAV-derived vectors.

Adeno-Associated Virus 2 Infection Requires Endocytosis through the CLIC/GEEC Pathway

Adeno-associated viruses (AAVs) are nonpathogenic, nonenveloped, single-stranded DNA viruses in development as gene therapy vectors. AAV internalization was postulated to proceed via a dynamin-dependent endocytic mechanism. Revisiting this, we find that infectious endocytosis of the prototypical AAV, AAV2, is independent of clathrin, caveolin, and dynamin. AAV2 infection is sensitive to EIPA, a fluid-phase uptake inhibitor, but is unaffected by Rac1 mutants or other macropinocytosis inhibitors. In contrast, AAV2 infection requires actin cytoskeleton remodeling and membrane cholesterol and is sensitive to inhibition of Cdc42, Arf1, and GRAF1, factors known to be involved in the formation of clathrin-independent carriers (CLIC). AAV2 virions are internalized in the detergent-resistant GPI-anchored-protein-enriched endosomal compartment (GEEC) and translocated to the Golgi apparatus, similarly to the CLIC/GEEC marker cholera toxin B. Our results indicate that-unlike the viral entry mechanisms described so far-AAV2 uses the pleiomorphic CLIC/GEEC pathway as its major endocytic infection route.

Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy

A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca2+ handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.

Variation in the Nucleotide Sequence of Cottontail Rabbit Papillomavirus a and b Subtypes Affects Wart Regression and Malignant Transformation and Level of Viral Replication in Domestic Rabbits

ABSTRACT We previously reported the partial characterization of two cottontail rabbit papillomavirus (CRPV) subtypes with strikingly divergent E6 and E7 oncoproteins. We report now the complete nucleotide sequences of these subtypes, referred to as CRPVa4 (7,868 nucleotides) and CRPVb (7,867 nucleotides). The CRPVa4 and CRPVb genomes differed at 238 (3%) nucleotide positions, whereas CRPVa4 and the prototype CRPV differed by only 5 nucleotides. The most variable region (7% nucleotide divergence) included the long regulatory region (LRR) and the E6 and E7 genes. A mutation in the stop codon resulted in an 8-amino-acid-longer CRPVb E4 protein, and a nucleotide deletion reduced the coding capacity of the E5 gene from 101 to 25 amino acids. In domestic rabbits homozygous for a specific haplotype of the DRA and DQA genes of the major histocompatibility complex, warts induced by CRPVb DNA or a chimeric genome containing the CRPVb LRR/E6/E7 region showed an early regression, whereas warts induced by CRPVa4 or a chimeric genome containing the CRPVa4 LRR/E6/E7 region persisted and evolved into carcinomas. In contrast, most CRPVa, CRPVb, and chimeric CRPV DNA-induced warts showed no early regression in rabbits homozygous for another DRA-DQA haplotype. Little, if any, viral replication is usually observed in domestic rabbit warts. When warts induced by CRPVa and CRPVb virions and DNA were compared, the number of cells positive for viral DNA or capsid antigens was found to be greater by 1 order of magnitude for specimens induced by CRPVb. Thus, both sequence variation in the LRR/E6/E7 region and the genetic constitution of the host influence the expression of the oncogenic potential of CRPV. Furthermore, intratype variation may overcome to some extent the host restriction of CRPV replication in domestic rabbits.

Cottontail rabbit papillomavirus E8 protein is essential for wart formation and provides new insights into viral pathogenesis.

ABSTRACT The cottontail rabbit papillomavirus (CRPV) a and b subtypes display a conserved E8 open reading frame encoding a 50-amino-acid hydrophobic protein, with structural similarities to the E5 transmembrane oncoprotein of genital human PVs (HPVs). CRPV E8 has been reported to play a role in papilloma growth but not to be essential in papilloma formation. Here we report that the knockout of E8 start codon almost prevented wart induction upon biobalistic inoculation of viral DNA onto rabbit skin. The scarce warts induced showed very slow growth, despite sustained expression of E6 and E7 oncogenes. This points to an essential role of E8 in disturbing epidermal homeostasis. Using a yeast two-hybrid screen, we found that E8 interacted with the zinc transporter ZnT1, protocadherin 1 (PCDH1), and AHNAK/desmoyokin, three proteins as yet unrelated to viral pathogenesis or cell transformation. HPV16 E5 also interacted with these proteins in two-hybrid assay. CRPV E8 mainly localized to the Golgi apparatus and the early endosomes of transfected keratinocytes and colocalized with ZnT1, PCDH1, and AHNAK. We showed that ZnT1 and PCDH1 formed a complex and that E8 disrupted this complex. CRPV E8, like HPV16 E5, increased epidermal growth factor (EGF)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and both the EGF-dependent and the EGF-independent activity of activating protein-1 (AP-1). Competition experiments with a nonfunctional truncated ZnT1 protein showed that E8-ZnT1 interaction was required for AP-1 activation. Our data identify CRPV E8 as a key player in papilloma induction and unravel novel cellular targets for inducing the proliferation of keratinocytes.

Syntaxin 5-Dependent Retrograde Transport to the trans-Golgi Network Is Required for Adeno-Associated Virus Transduction

ABSTRACT Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment. IMPORTANCE Gene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne muscular dystrophy, hemophilia, heart failure, Parkinsons disease, and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to the trans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from that of known Golgi apparatus-targeted cargos, and we raise the possibility that this nonclassical pathway is shared by most AAV variants, regardless of their attachment receptors.

Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling

Background— Stromal interaction molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiomyocytes. STIM1 is thought to act as an initiator of cardiac hypertrophic response at the level of the sarcolemma, but the pathways underpinning this effect have not been examined. Methods and Results— To determine the mechanistic role of STIM1 in cardiac hypertrophy and during the transition to heart failure, we manipulated STIM1 expression in mice cardiomyocytes by using in vivo gene delivery of specific short hairpin RNAs. In 3 different models, we found that Stim1 silencing prevents the development of pressure overload–induced hypertrophy but also reverses preestablished cardiac hypertrophy. Reduction in STIM1 expression promoted a rapid transition to heart failure. We further showed that Stim1 silencing resulted in enhanced activity of the antihypertrophic and proapoptotic GSK-3&bgr; molecule. Pharmacological inhibition of glycogen synthase kinase-3 was sufficient to reverse the cardiac phenotype observed after Stim1 silencing. At the level of ventricular myocytes, Stim1 silencing or inhibition abrogated the capacity for phosphorylation of AktS473, a hydrophobic motif of Akt that is directly phosphorylated by mTOR complex 2. We found that Stim1 silencing directly impaired mTOR complex 2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTOR complex 2. Conclusions— These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiomyocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2, which further results in repression of GSK-3&bgr; activity.

Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression.

Nanoparticle-based delivery of nucleotides offers an alternative to viral vectors for gene therapy. We report highly efficient in vivo delivery of modified mRNA (modRNA) to rat and pig myocardium using formulated lipidoid nanoparticles (FLNP). Direct myocardial injection of FLNP containing 1-10 μg eGFPmodRNA in the rat (n = 3 per group) showed dose-dependent enhanced green fluorescent protein (eGFP) mRNA levels in heart tissue 20 hours after injection, over 60-fold higher than for naked modRNA. Off-target expression, including lung, liver, and spleen, was <10% of that in heart. Expression kinetics after injecting 5 μg FLNP/eGFPmodRNA showed robust expression at 6 hours that reduced by half at 48 hours and was barely detectable at 2 weeks. Intracoronary administration of 10 μg FLNP/eGFPmodRNA also proved successful, although cardiac expression of eGFP mRNA at 20 hours was lower than direct injection, and off-target expression was correspondingly higher. Findings were confirmed in a pilot study in pigs using direct myocardial injection as well as percutaneous intracoronary delivery, in healthy and myocardial infarction models, achieving expression throughout the ventricular wall. Fluorescence microscopy revealed GFP-positive cardiomyocytes in treated hearts. This nanoparticle-enabled approach for highly efficient, rapid and short-term mRNA expression in the heart offers new opportunities to optimize gene therapies for enhancing cardiac function and regeneration.

Effectiveness of gene delivery systems for pluripotent and differentiated cells

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases, both to study them and to explore therapies. However, a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study, we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV), adenoviruses and lentiviral vectors in hESC, hiPSC, and the derived cardiomyocytes. In undifferentiated cells, adenoviral and lentiviral vectors were superior, whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes, 1, 2, 6, and 9, of which 2 and 6 were superior in their transduction efficiency. Interestingly, we observed that AAVs severely diminished the viability of undifferentiated cells, an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore, we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally, adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion, adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines, whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells.

Effect of bortezomib on the efficacy of AAV9.SERCA2a treatment to preserve cardiac function in a rat pressure-overload model of heart failure

Adeno-associated virus (AAV)-based vectors are promising vehicles for therapeutic gene delivery, including for the treatment for heart failure. It has been demonstrated for each of the AAV serotypes 1 through 8 that inhibition of the proteasome results in increased transduction efficiencies. For AAV9, however, the effect of proteasome inhibitors on in vivo transduction has until now not been evaluated. Here we demonstrate, in a well-established rodent heart failure model, that concurrent treatment with the proteasome inhibitor bortezomib does not enhance the efficacy of AAV9.SERCA2a to improve cardiac function as examined by echocardiography and pressure volume analysis. Western blot analysis of SERCA2a protein and reverse transcription-PCR of SERCA2a mRNA demonstrated that bortezomib had no effect on either endogenous rat SERCA2a levels nor on expression levels of human SERCA2a delivered by AAV9.SERCA2a. Similarly, the number of AAV9 genomes in heart samples was unaffected by bortezomib treatment. Interestingly, whereas transduction of HeLa cells and neonatal rat cardiomyocytes by AAV9 was stimulated by bortezomib, transduction of adult rat cardiomyocytes was inhibited. These results indicate an organ/cell-type-specific effect of proteasome inhibition on AAV9 transduction. A future detailed analysis of the underlying molecular mechanisms promises to facilitate the development of improved AAV vectors.