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

Abstract

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.

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

“…57 LVs were generated by transient co-transfection of 293T cells with the packaging, transfer and envelope plasmids. 26 Titer of all LV was determined by p24 antigen ELISA (Center for AIDS Research, The University of Pennsylvania, Philadelphia, PA, USA). Titer of AAV2/6.2 was determined via qPCR using a primer set matching the polyadenylation signal in the vector genome as previously described.…”

“…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.…”

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

“…First, although we have chosen to focus on skin gene transfer in this study, IAGT is not specific for skin. 26,35 While some selectivity occurs due to the limited interface between amniotic fluid and other fetal tissues, we have documented other tissues, including neuroectoderm, 35,37 that can be transduced by IAGT. This raises obvious concern about the potential for insertional mutagenesis, developmental effects and the potential for germ line alteration that exists for lentiviral vector-based approaches.…”

Genetic Therapy for the Fetus: A Once in a Lifetime Opportunity

“…Because AAV genomes are not replicated during cell division, which occurs at a rapid rate during early fetal/postnatal development, one would anticipate a rapid decline in transgene expression (Boyle et al, 2001). HIV-1-based lentiviral vectors (LVs) are able to integrate into the host genome to provide lifelong stable transgene expression Stitelman et al, 2010). An undesirable consequence of LVs for gene therapy is the risk of activation of oncogenes and/or insertional mutagenesis, which may be heightened for FGT because of rapid fetal growth.…”