Masayuki Endo

Pseudoxanthoma Elasticum Is a Metabolic Disease

Pseudoxanthoma elasticum (PXE) is a pleiotropic multisystem disorder affecting skin, eyes, and the cardiovascular system with progressive pathological mineralization. It is caused by mutations in the ABCC6 gene expressed primarily in the liver and kidneys, and at very low levels, if at all, in tissues affected by PXE. A question has arisen regarding the pathomechanism of PXE, particularly the metabolic versus the PXE cell hypotheses. We examined a murine PXE model (Abcc6(-/-)) by transplanting muzzle skin from knockout (KO) and wild-type (WT) mice onto the back of WT and KO mice using mineralization of the connective tissue capsule surrounding the vibrissae as an early phenotypic biomarker. Grafting of WT mouse muzzle skin onto the back of KO mice resulted in mineralization of vibrissae, whereas grafting KO mouse muzzle skin onto WT mice did not. Thus, these findings implicate circulatory factors as a critical component of the mineralization process. This mouse grafting model supports the notion that PXE is a systemic metabolic disorder with secondary mineralization of connective tissues and that the mineralization process can be countered or even reversed by changes in the homeostatic milieu.

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

Correction of murine ADAMTS13 deficiency by hematopoietic progenitor cell–mediated gene therapy

ADAMTS13, a metalloprotease primarily synthesized in liver and endothelial cells, cleaves von Willebrand factor (VWF) at the central A2 domain, thereby reducing the sizes of circulating VWF multimers. Genetic or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). To date, plasma infusion or exchange is the only proven effective therapy for TTP. In search for a better therapy, an autologous transplantation of hematopoietic progenitor cells transduced ex vivo with a self-inactivating lentiviral vector encoding a full-length murine Adamts13 and an enhanced green fluorescent protein (GFP) reporter gene was performed in Adamts13(-/-) mice after irradiation. All recipient mice showed detectable ADAMTS13 antigen and proteolytic activity in plasma despite only low levels of bone marrow chimerism. The levels of plasma ADAMTS13 were sufficient to eliminate the ultralarge VWF multimers and offered systemic protection against ferric chloride-induced arterial thrombosis. The data suggest that hematopoietic progenitor cells can be genetically modified ex vivo and transplanted in an autologous model to provide adequate levels of functional ADAMTS13 metalloprotease. This success may provide the basis for development of a novel therapeutic strategy to cure hereditary TTP in humans.

The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors

Gene transfer after intra-amniotic injection has, in general, been of low efficiency and limited to epithelial cells in the skin, pulmonary and gastrointestinal system. We have recently shown that early gestational administration results in a more efficient gene transfer to developmentally accessible stem cell populations in the skin and eye. In this study we present a comprehensive analysis of patterns of tissue expression seen after early intra-amniotic gene transfer (IAGT) using lentiviral vectors. To assess the influence of developmental stage on tissue expression, injections were administered from the late head fold/early somite stage (E8) to E18. In early gestation (E8–10), green fluorescent protein (GFP) expression was observed in multiple organs, derived from all three germ layers. Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells of ectoderm- and endoderm-derived organs after E11. The amount and duration of gene expression was much higher after IAGT at early gestational time points. The observed temporal patterns of gene expression correspond to the predicted developmental accessibility of organ-specific cell populations. This model may be useful for the analyses of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.

Early gestational gene transfer of IL-10 by systemic administration of lentiviral vector can prevent arthritis in a murine model

Gene therapy has been applied to murine models of rheumatoid arthritis (RA) using a number of different strategies to downregulate inflammation in synovial joints. However, prolonged joint expression has been problematic. Our laboratory has found that early gestational intravascular injection of lentiviral vector leads to efficient transduction and sustained transgene expression in articular cartilage and synovium. In this study, we show that in utero gene transfer of IL-10 can prevent and decrease pathology in a murine model of RA. Following prenatal injection of lentiviral vector containing murine IL-10 gene, the cytokine was detectable in the serum, and the green fluorescent protein reporter gene was detectable in chondrocytes and synoviocytes of adult mice up to 21 weeks of age. Adult mice that had been treated prenatally were later immunized against type II collagen to induce an autoimmune arthritis. Compared with controls, prenatally treated mice demonstrated delayed onset of arthritis, decreased frequency of arthritis and markedly decreased severity of disease, by both clinical and histological criteria. This effect was directly related to levels of IL-10 expression, but no immunosuppressive effects of the therapy were observed. This study demonstrates proof of principle for the prenatal prevention and amelioration of RA by early gestational gene transfer of the anti-inflammatory cytokine, IL-10.

Robust In Vivo Transduction of Nervous System and Neural Stem Cells by Early Gestational Intra Amniotic Gene Transfer Using Lentiviral Vector

Presently, in vivo methods to efficiently and broadly transduce all major cell types throughout both the central (CNS) and peripheral adult nervous system (PNS) are lacking. In this study, we hypothesized that during early fetal development neural cell populations, including neural stem cells (NSCs), may be accessible for gene transfer via the open neural groove. To test this hypothesis, we injected lentiviral vectors encoding a green fluorescent protein (GFP) marker gene into the murine amniotic cavity at embryonic day 8. This method (i) efficiently and stably transduced the entire nervous system for at least 80% of the lifespan of the mice, (ii) transduced all major neural cell types, and (iii) transduced adult NSCs of the subventricular zone (SVZ) and subgranular zones (SGZs). This simple approach has broad applications for the study of gene function in nervous system development and adult NSCs and may have future clinical applications for treatment of genetic disorders of the nervous system.

Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa

Mutations of the LAMB3 gene cause a lethal form of junctional epidermolysis bullosa (JEB). We hypothesized that early intra-amniotic gene transfer in a severe murine model of JEB would improve or correct the skin phenotype. Time-dated fetuses from heterozygous LAMB3IAP breeding pairs underwent ultrasound guided intra-amniotic injection of lentiviral vector encoding the murine LAMB3 gene at embryonic day 8 (E8). Gene expression was monitored by immunohistochemistry. The transgenic laminin-β3 chain was shown to assemble with its endogenous partner chains, resulting in detectable amounts of laminin-332 in the basement membrane zone of skin and mucosa. Ultrastructually, the restoration of ∼60% of hemidesmosomal structures was also noted. Although we could correct the skin phenotype in 11.9% of homozygous LAMB3IAP mice, none survived beyond 48u2009h. However, skin transplants from treated E18 homozygous LAMB3IAP fetuses maintained normal appearance for 6 months with persistence of normal assembly of laminin-332. These results demonstrate for the first time long-term phenotypic correction of the skin pathology in a severe model of JEB by in vivo prenatal gene transfer. Although survival remained limited due to the limitations of this mouse model, this study supports the potential for treatment of JEB by prenatal gene transfer.

Animal Models for Prenatal Gene Therapy: Rodent Models for Prenatal Gene Therapy

Fetal gene transfer has been studied in various animal models, including rabbits, guinea pigs, cats, dogs, and nonhuman primate; however, the most common model is the rodent, particularly the mouse. There are numerous advantages to mouse models, including a short gestation time of around 20 days, large litter size usually of more than six pups, ease of colony maintenance due to the small physical size, and the relatively low expense of doing so. Moreover, the mouse genome is well defined, there are many transgenic models particularly of human monogenetic disorders, and mouse-specific biological reagents are readily available. One criticism has been that it is difficult to perform procedures on the fetal mouse with suitable accuracy. Over the past decade, accumulation of technical expertise and development of technology such as high-frequency ultrasound have permitted accurate vector delivery to organs and tissues. Here, we describe our experiences of gene transfer to the fetal mouse with and without ultrasound guidance from mid to late gestation. Depending upon the vector type, the route of delivery and the age of the fetus, specific or widespread gene transfer can be achieved, making fetal mice excellent models for exploratory biodistribution studies.

SFCP-011 – Chirurgie viscérale – Une thérapie génique in utero par ecSOD permet-elle de prévenir les lésions pulmonaires hyperoxiques néonatales ?

Objectifs L’oxygene est la principale therapeutique utilisee pour traiter les nouveaux-nes atteints d’un syndrome de detresse respiratoire neonatal. Certains enfants conservent des sequelles de bronchodysplasie pulmonaire secondaires a l’exposition prolongee et a forte dose a l’oxygene. La superoxide dismutase extracellulaire (ecSOD) est une enzyme jouant un role central dans les mecanismes naturels de defense contre le stress oxidatif. Nous avons teste dans un modele murin l’hypothese suivante : une therapie genique in utero par le gene de ecSOD peut-elle prevenir les lesions pulmonaires hyperoxiques neonatales ? Materiel et methode Des souris C57/B6 gestantes ont ete traitees par injection intra-amniotique d’un vecteur adenoviral comportant un gene rapporteur (gfp) seul ou associe au gene de ecSOD. Des fœtus non traites ont servi de fœtus temoins. Les nouveaux-nes ont ete repartis en 2 groupes : groupe O2 = exposes a une FiO2 de 90 % durant 7 jours, ou groupe AA = maintenus en air ambiant (FiO2 21 %). A l’issue de l’exposition, les nouveaux-nes ont ete peses et euthanasies, puis les poumons preleves, le volume pulmonaire a ete mesure et les lesions parenchymateuses ont ete analysees surcoupes histologiques. Resultats Les poumons des souris du groupe O2 presentaient des lesions hyperoxiques caracterisees par une diminution de volume pulmonaire, une desorganisation des alveoles, une diminution du rapport tissue/air, une diminution du volume tissulaire et de la surface d’echange. Il n’a pas ete observe d’amelioration des lesions dans le groupe traite par Ad-ecSODgfp. Le volume pulmonaire moyen des nouveaux-nes exposes a l’hyperoxie etaient de 133 microl, 152 microl, et 120 microl dans les groupes non traitees, traites par Adgfp et Ad-ecSODgfp respectivement, tandis que les volumes pulmonaires des nouveaux-nes maintenus en AA etaient respectivement de 142 microl, 196 microl, et 174 microl (differences non significatives entre les souris traites par Ad-ecSODgfp vs les autres groupes-One wayANOVA). Conclusion Dans notre modele, nous n’avons pas mis en evidence d’efficacite du traitement par therapie genique in utero par ecSOD. Nos resultats suggerent que la transfection in utero de ecSOD serait insuffisante pour prevenir les lesions pulmonaires hyperoxiques neonatales.

445. Developmental Stage Determines Distribution of Organ Transduction after Intra-Amniotic Injection of Lentiviral Vector

Gene transfer after intra-amniotic injection has in general been of low efficiency, and limited to epithelial cells in the skin, pulmonary, and gastrointestinal system. We hypothesized that early gestational administration might result in broader and more efficient gene transfer due to developmental accessibility of specific cell populations. To test this hypothesis, we injected lentiviral vector carrying the green fluorescent protein (GFP) reporter gene into the fetal murine amniotic space from the late head fold/early somite stage (E8) to E18. From E8 to E12 amniotic space injections were performed under ultrasound image guidance using a Visualsonics@ Vevo 660 system with a 40 mHz scanhead. After E12, injections were performed under direct vision using a stereoscopic microscope. We observed the injected mice under fluorescence stereo microscopy at sequential time points after birth and confirmed GFP expression by immunohistochemistry. We injected 584 fetal mice from E8 to E18 and the total survival rate was 52.2% (305/584). In early gestation (E8-10), significant GFP expression was observed in multiple organs (Table). Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells after E11. The observed temporal patterns of gene expression correspond to the expected embryologic accessibility of organ specific cell populations.