All ETDs from UAB

Advisory Committee Chair

Thomas M Ryan

Advisory Committee Members

Casey D Morrow

Hengbin Wang

John C Kappes

Peter J Detloff

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Gene therapy for hematopoietic disorders using viral vectors has achieved significant clinical benefit. However this approach has been held back by adverse genotoxic events due to random viral integration into the genome. A better understanding of viral transgene chromosomal position effects is critical for the design of safer viral vectors and clinical protocols. However, detailed analysis of viral transgenes is hampered by the heterogeneity of the viral-transduced bone marrow cell populations. I hypothesized that viral transduction of pluripotent stem cells would enable the clonal analysis of viral transgene expression, safety, and efficacy both in vitro and in vivo. Here I report the high-resolution clonal analysis of recombinant human beta globin lentiviral gene therapy for a mouse model of beta-thalassemia. Embryonic stem (ES) cells from beta-globin knockout (KO) mice were transduced with lentivirus. In six subclones the precise proviral chromosomal integration sites were mapped; human beta-globin transgene expression was determined after in vitro differentiation; and 100% ES cell derived mice were established by tetraploid embryo complementation or eight-cell embryo injection. Individual transgenes in multiple copy number animals were segregated by breeding. The chromosomal position effects on transgene and flanking gene expression were analyzed for each integration sites. I discovered that: 1) Lentiviral transduction did not compromise the pluripotency of the ES cell subclones. 2) A single recombinant human beta-globin lentiviral transgene could cure beta-thalassemia. 3) Expression levels of lentiviral transgenes positively correlated with their proximity to nearby genes. 4) Recombinant self-inactivating (SIN) lentivirus, could still induce insertional dysregulation of flanking genes in vivo. 5) In multiple copy number animals the total transgene expression level was less than the combined sum of expression levels observed in their single copy offspring. 6) Lentiviral transgene expression after in vitro differentiation of ES cells was similar to that observed in vivo and could be a useful predictor of therapeutic efficacy. The methodology used in this study is not restricted to gene therapy for hematopoietic disorders, but can be applied more generally to the delivery of any therapeutic gene to any cell type, tissue, or organ at any stage of development.

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