Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.This work was supported by Genethon, the French Muscular Dystro-phy Association (AFM), and Spark Therapeutics. It was also sup-ported by the European Union’s Research and Innovation Programunder grant agreement number 667751 (to F.M.), the EuropeanResearch Council Consolidator Grant under grant agreement number617432 (to F.M.), and Marie Skłodowska-Curie Actions-IndividualFellowship (MSCA-IF) grant agreement number 797144 (to U.C.)S

Analysis of PERV-C superinfection resistance using HA-tagged viruses

Abstract Background Using pigs as organ donors has advanced xenotransplantation to the point that it is almost ready for clinical use. However, there is still a zoonotic risk associated with xenotransplantation, and the potential transmission of porcine endogenous retroviruses needs to be surveyed. Despite significant attempts to eliminate this risk, by the selection of PERV-C free pigs with low expression of PERV-A, -B, and by the genome-wide inactivation of PERV using CRISPR/Cas9, the impact of superinfection resistance (SIR) was not investigated. SIR is a viral trait that prevents reinfection (superinfection). For PERV, the underlying mechanism is unclear, whether and how cells, that harbor functional PERV, are protected. Using PERV-C(5683) as a reference virus, we investigated SIR in a newly developed in vitro model to pursue the mechanism and confirm its protective effect. Results We developed three PERV-C constructs on the basis of PERV-C(5683), each of which carries a hemagglutinin tag (HA-tag) at a different position of the envelope gene (SP-HA, HA-VRA, and RPep-HA), to distinguish between primary infection and superinfection. The newly generated PERV-C(5683)-HA viruses were characterized while quantifying the viral RNA, reverse transcriptase activity, protein expression analysis, and infection studies. It was demonstrated that SP-HA and RPep-HA were comparable to PERV-C(5683), whereas HA-VRA was not replication competent. SP-HA and RPep-HA were chosen to challenge PERV-C(5683)-positive ST-IOWA cells demonstrating that PERV-C-HA viruses are not able to superinfect those cells. They do not integrate into the genome and are not expressed. Conclusions The mechanism of SIR applies to PERV-C. The production of PERV-C particles serves as a defense mechanism from superinfection with exogenous PERV-C. It was demonstrated by newly generated PERV-C(5683)-HA clones that might be used as a cutting-edge tool. The HA-tagging of PERV-C is novel, providing a blueprint for the tagging of other human tropic PERV viruses. The tagged viruses are suitable for additional in vitro and in vivo infection studies and will contribute, to basic research on viral invasion and pathogenesis. It will maintain the virus safety of XTx

Aspergillus fumigatus exoβ(1‐3)glucanases family GH55 are essential for conidial cell wall morphogenesis

International audienceThe cell wall of Aspergillus fumigatus is predominantly composed of polysaccharides. The central fibrillar core of the cell wall is composed of a branched β(1-3)glucan, to which the chitin and the galactomannan are covalently bound. Softening of the cell wall is an essential event during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosyl hydrolases. In this study, we characterised the role of the glycosyl hydrolase GH55 members in A. fumigatus fungal morphogenesis. We showed that deletion of the six genes of the GH55 family stopped conidial cell wall maturation at the beginning of the development process, leading to abrogation of conidial separation: the shape of conidia became ovoid, and germination was delayed. In conclusion, the reorganisation and structuring of the conidial cell wall mediated by members of the GH55 family is essential for their maturation, normal dissemination, and germination

Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells

The architecture of functional TNTs is still under debate. Here, the authors combine correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structure of TNTs in neuronal cells, showing that they form structures that are distinct form other membrane protrusions

Gene therapy with secreted acid alpha-glucosidase rescues Pompe disease in a novel mouse model with early-onset spinal cord and respiratory defects.

Pompe disease (PD) is a neuromuscular disorder caused by deficiency of acidalpha-glucosidase (GAA), leading to motor and respiratory dysfunctions. Available Gaa knock-out (KO) mouse models do not accurately mimic PD, particularly its highly impaired respiratory phenotype. Here we developed a new mouse model of PD crossing Gaa KOB6;129 with DBA2/J mice. We subsequently treated Gaa KODBA2/J mice with adeno-associated virus (AAV) vectors expressing a secretable form of GAA (secGAA). Male Gaa KODBA2/J mice present most of the key features of the human disease, including early lethality, severe respiratory impairment, cardiac hypertrophy and muscle weakness. Transcriptome analyses of Gaa KODBA2/J, compared to the parental Gaa KOB6;129 mice, revealed a profoundly impaired gene signature in the spinal cord and a similarly deregulated gene expression in skeletal muscle. Muscle and spinal cord transcriptome changes, biochemical defects, respiratory and muscle function in the Gaa KODBA2/J model were significantly improved upon gene therapy with AAV vectors expressing secGAA. These data show that the genetic background impacts on the severity of respiratory function and neuroglial spinal cord defects in the Gaa KO mouse model of PD. Our findings have implications for PD prognosis and treatment, show novel molecular pathophysiology mechanisms of the disease and provide a unique model to study PD respiratory defects, which majorly affect patients. This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics.This work was supported by Genethon and the French Muscular Dystrophy Association (AFM, to F.M.). It was also supported by the European Union’s research and innovation program under grant agreement no. 667751 (to F.M.), the European Research Council Consolidator Grant under grant agreement no. 617432 (to F.M.), Marie Skodowska-Curie Actions Individual Fellowship (MSCA-IF) grant agreement no. 797144 (to U.C.), and by Spark Therapeutics under a sponsored research agreement.S

THE PALISADE LAYER OF THE POXVIRUS CORE IS COMPOSED OF FLEXIBLE A10-TRIMERS

Although vaccinia virus (VACV) is the best studied poxvirus, the structure of the mature virus (MV) remains poorly understood. Its asymmetric shape, size and compactness poses a major challenge for electron microscopy (EM) analysis, including cryoEM. Sub-viral particles, in particular membrane-free viral cores, may overcome these limitations. We compare cores obtained by detergent-stripping MVs with cores in the cellular cytoplasm, early in infection. By combining cryo-electron tomography (cryoET), subtomogram averaging (STA) and AlphaFold2 (AF2), abundant core-structures are analyzed, focusing on the prominent palisade layer on the core surface. On detergent-stripped cores, the palisade is composed of densely packed trimers of the major core protein A10. On the core surface they display a random order and their classification indicate structural flexibility. On cytoplasmic cores A10 is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. CryoET and STA also uncover unexpected details of the layers beneath the palisade both on in vitro and in situ cores, that are compared to AF2 structure predictions of known VACV core-associated proteins. Altogether, our data identify for the first time the structure and molecular composition of the palisade units. The results are discussed in the context of the VACV replicative cycle, the assembly and disassembly of the infectious MV

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase

International audiencePompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA

Intracellular offspring released from SFB filaments are flagellated

International audienceThe gut commensal segmented filamentous bacterium (SFB) attaches to the ileal epithelium and potently stimulates the host immune system. Using transmission electron microscopy (TEM), we show that mouse and rat SFB are flagellated above the concave tip at the unicellular intracellular offspring (IO) stage and that flagellation occurs prior to full IO differentiation and release of IOs from SFB filaments. This finding adds a missing link to the SFB life cycle

MybA, a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA‐sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane‐associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus

Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates

International audiencePompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach