Microsomal triglyceride transfer protein (MTP) is made up of two subunits, a large 97-kDa subunit, designated eMTP, and a small subunit, protein disulfide isomerase. Mutations in eMTP are the underlying cause of abetalipoproteinemia, an autosomal recessive disorder characterized by the absence of apoB-containing lipoproteins and fat malabsorption. Conventional inactivation of eMTP is embryonic lethal. We have inserted loxP sequences flanking exons 5 and 6 of the eMTP gene to produce a conditional knockout by gene targeting. Mice with liver-specific inactivation of the gene fail to produce any very low density, intermediate density or low density lipoproteins. We will use the floxed mice as a model to study eMTP and apoB-lipoprotein physiology and somatic gene therapy.
In Specific Aim 1, we will create intestine-specific knockout of eMTP by intraluminal intestinal delivery of a Cre adenovirus (AdCre1), a novel strategy for the production of intestine-specific knockouts. We will also produce combined liver and intestine-specific eMTP inactivation by cross-breeding the mice with Cre transgenic mice and inducing Cre expression by pI:pC treatment.
In Specific Aim 2, the liver-specific, intestine-specific and combined knockout animals will be characterized with respect to their general health and lipoprotein metabolism.
In Specific Aim 3, we will use the eMTP-deficient mice as a model for intestinal gene therapy, an exciting but under-investigated area. There are many advantages in using the intestine as a target organ for the treatment of local as well as systemic genetic and acquired diseases. We will deliver the eMTP gene into the intestine by 3 types of vectors: retrovirus, VSV-G pseudotyped retrovirus, and adeno-associated virus. Response to therapy will be monitored in vivo and in vitro. Finally, in Specific Aim 4, we will restore eMTP expression in knockout mice and induce eMTP overexpression in wild-type mice using a gutless adenoviral vector. This vector, developed at Baylor, produces long-term (greater than 1 year) expression of transgenes in the liver following a single injection. Wild-type eMTP-/- and apobec-1-/- mice will be studied. In summary, this application utilizes state-of-the-art gene delivery technology plus a conditional knockout model to examine important issues in MTP biology, tissue-specific gene targeting, and intestinal gene therapy.
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