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

Abstract

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 [email protected] 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.TableGFP positive organs by different gestational IAGTDay of injection E08E09E10E11E12E13–16E17–18Ectoderm-derived organsPineal gland ((+))(−)(−)(−)(−)(−)(−)Spinal cord ((+))(−)(−)(−)(−)(−)(−)Brain ((+))((+))(−)(−)(−)(−)(−)Olfactory bulb ((+))((+))(−)(−)(−)(−)(−)Pituitary ((+))((+))((+))(−)(−)(−)(−)Retina ((+))(−)(−)(−)(−)(−)(−)Lens ((+))((+))((+))(−)(−)(−)(−)Cornea ((+))((+))((+))((+))((+))((+))(−)Parotid gland ((+))((+))((+))(−)(−)(−)(−)Submaxillary gland ((+))((+))((+))(−)(−)(−)(−)Mammary gland ((+))((+))(−)(−)(−)(−)(−)Skin ((+))((+))((+))((+))((+))(−)(−)Inner ear ((+))((+))(−)(−)(−)(−)(−)Teeth ((+))((+))((+))(−)(−)(−)(−)Tongue ((+))((+))((+))((+))(−)(−)(−)Mesoderm-derived organsKidney ((+))(−)(−)(−)(−)(−)(−)Muscle ((+))(−)(−)(−)(−)(−)(−)Endoderm-derived organsThymus ((+))((+))((+))(−)(−)(−)(−)Thyroid ((+))((+))((+))(−)(−)(−)(−)Esophagus ((+))((+))(−)(−)(−)(−)(−)Paranasal sinus ((+))((+))((+))((+))((+))((+))((+))Lung (−)(−)(−)(−)((+))((+))((+)) Open table in a new tab We conclude from this study that early gestational intra-amniotic gene transfer has higher efficiency and a broader distribution of transduction than what has been previously observed later in gestation. This model may be useful for investigation of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders. 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 [email protected] 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. We conclude from this study that early gestational intra-amniotic gene transfer has higher efficiency and a broader distribution of transduction than what has been previously observed later in gestation. This model may be useful for investigation of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.