L Lawrence

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding β-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.

Distinct Patterns of Microvascular Endothelial Cell Morphology Are Determined by Extracellular Matrix Composition

Endothelial interactions with the extracellular matrix (ECM) play important roles in angiogenesis but whether specific ECM signals can determine specific cellular morphologies is unclear. The authors compared in vitro ECM-induced morphological responses of the phenotypically distinct human placental microvascular endothelial cells (HPMECs) with large vessel endothelial cells (HUVECs). HPMECs showed distinct patterns of reorganization in response to collagen-I or collagen-IV (monolayer disruption, sprouting, migration) and Matrigel or laminin-A (intussusception, cord formation, tubulogenesis), and an intermediate response to fibrin; whereas HUVECs responded similarly to collagen-1 and Matrigel (elongation, lattice formation, vacuolation) and showed little response to fibrin. Although the extent of collagen and Matrigel responses of HPMECs were increased by serum, acidic or basic fibroblast growth factor (aFGF, bFGF), or vascular endothelial growth factor (VEGF), and varied with matrix protein concentration, the basic patterns were matrix specific, and were independent of fibronectin. The collagen responses correlated with disruption of adherens and tight junctions and the formation of filopodial protrusions. Matrigel responses were associated with up-regulated junctional localization of VE-cadherin, and tubulogenesis developed mainly through paracellular remodeling rather than intracellular vacuolation. Overall, these findings suggest that distinct ECM interactions stimulate specific morphological responses. These signals may regulate morphological behaviour in the angiogenesis cycle, switching endothelial cells between migratory and vasculogenic phenotypes.

Enhancement of adenovirus-mediated gene transfer to the airways by DEAE dextran and sodium caprate in vivo

Gene transfer to the trachea and airways by adenoviral vectors is limited by the basolateral localization of viral receptors, resulting in relatively low levels of transduction. Modification of paracellular permeability by sodium caprate, which opens tight junctions, enhances gene transfer from the apical side of cultured human airway epithelial cells. Based on this observation we investigated whether Na-caprate could also increase gene transfer when applied to the luminal surface of the airway epithelia in vivo and compared these results with EGTA, which has previously been shown to enhance adenovirus transduction. Transgene expression in the trachea and upper airways was increased 25-fold by a 10-min pretreatment with 50 mM Na-caprate, corresponding to a 3-fold improvement over EGTA. In the more peripheral airways EGTA had no effect, whereas expression of beta-gal was increased 3-fold by Na-caprate. When the adenovirus was complexed with DEAE dextran, transduction of the airway epithelia after Na-caprate pretreatment was increased 45-fold over virus alone. In conclusion, Na-caprate facilitates gene transfer to airway epithelia, particularly when adenovirus is complexed with DEAE dextran, and may in future be used in a clinical setting to enhance the efficiency of vectors for gene therapy of cystic fibrosis via airway delivery.

Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy

Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied β-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.