SOMATIC GENE THERAPY FOR PHENYLKETONURIA

本提案涉及遗传转移方法的发展

This proposal deals with the development of methods for genetic transfer of cloned human phenylalanine hydroxylase (PAH) into cultured and primary cells and whole animals. A series of retroviral vectors will be evaluated as vehicles for genetic transfer of PAH and their ability to direct high level of PAH expression and produce high titers of the recombinant virus. We will explore the use of recombinant retroviruses to introduce PAH activity into a variety of cell types including mouse NIH3T3, which is a standard host for characterization of retrovirus function, primary and immortalized human cell lines, which will allow characterization of the ability to infect human cells, and various mouse and rat hepatoma cell lines which are known to be deficient in PAH expression and can be used to characterize the ability of the recombinant retroviruses to reconstitute PAH holoenzyme activity. We will explore several methods for infection of live animals and infection of primary cells in vitro prior to transplantation into host animals. Of particular importance is the development of methods for infection of hepatocytes in vitro and implantation of these cells into host animals. In order to constitute PAH holoenzyme activity in cells other than hepatocytes, we will construct retroviral vectors which express both PAH and dihydropteridine reductase (DHFR). The expression of recombinant PAH will be investigated in whole animals and the biochemical effects of the gene transfer will be evaluated in the hyperphenylalaninemic (PH-/PH-) mouse. These experiments are designed to provide the experimental groundwork for genetic therapy of PKU. PKU represents an attractive model for the study of somatic gene therapy for a common disorder which is a recognized medical and public health concern.