Gene Transfer to Ocular Stem Cells by Early Gestational Intraamniotic Injection of Lentiviral Vector

Abstract

Ocular gene transfer has generally been approached by direct intraocular injection. In this study, we hypothesized that an opportunity exists during early gestation when specific ocular stem cell populations are accessible for gene transfer. These include the stem cell populations that maintain the cornea, lens, and retina throughout life. To test this hypothesis, we injected lentiviral vector encoding the green fluorescent protein (GFP) reporter gene into the murine amniotic space from the late head fold/early somite stage postcoital day 8 (E8) to E18 and performed sequential analysis of GFP expression in ocular tissues. Depending on the timing of vector exposure, significant GFP expression was observed in all ectoderm-derived tissues in the eye. With injection at early gestational time points, GFP expression persisted long term, with evidence of high efficiency stem cell transduction in the cornea, lens, and retina. The observed patterns and duration of gene expression confirm the accessibility of ocular stem cell populations for lentiviral vector-based gene transfer at specific developmental time points in early gestation. This model may be useful for the investigation of mechanisms of genetic and/or developmental ocular disease and for the development of prenatal gene therapy for specific ocular disorders.

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

“…5,12,19,24 However, in general, gene transfer after intra-amniotic vector injection has been inefficient and transient because of the relatively late developmental stage at which these experiments have been performed. 5,26,27 In contrast to previous studies, we have recently reported that early IAGT results in efficient transduction of skin stem cell populations 28 and the ectoderm-derived stem cell populations of the eye, 29 including stem cells in the neuroectoderm-derived retina. However, in the course of these studies we observed a broad range of transduction events involving multiple tissues of all three germ layers.…”

“…These mice are inclusive of the animals reported in our previous two reports of IAGT that focused on ocular and skin stem cell transduction. 28,29 From E8 to E12, we used an ultrasound-guided injection system to visualize and inject the amniotic space as previously described. 29 After E13, we injected under stereomicroscopic magnification.…”

“…28,29 From E8 to E12, we used an ultrasound-guided injection system to visualize and inject the amniotic space as previously described. 29 After E13, we injected under stereomicroscopic magnification. The overall survival rate was 50.1% (349/696).…”

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

“…24 The lentivector transfer plasmid contained the enhanced GFP reporter (Clontech Laboratories, Mountain View, CA, USA) under the control of the CMV promoter. The GP64 was cloned from the Autographa californica derived AcMNP baculovirus plasmid.…”

“…In contrast, HIV-1-based lentiviral vectors (LVs) have increased packaging capacity (optimal transgene insert B5-7 Kb 23 ) and, following in utero delivery, are able to integrate into the host genome to provide life-long stable transgene expression. 16,[24][25][26][27] Fetal lung gene transfer has been investigated in non-human primates, 20,28 rabbits, 29 rats 30,31 and sheep 32 using vesicular stomatitis virus glycoprotein (VSVg) pseudotyped LV. Long-term luciferase transgene expression (that is 12-15 months after fetal gene transfer) has been reported 31,33 although there is a paucity of quantitative information on degree of epithelial transduction and cell types transduced.…”