Harbhajan S. Paul, Ph.D. of Biomed Research & Technologies, Inc. has provided a short review and an update on the research project that was reported in the last issue (Spring/Summer 2003) of the MSUD Newsletter. The MSUD Family Support Group has been supplying bridge funding for one year for this project that could lead to gene therapy for MSUD. Dr. Paul reports that his company was advised to submit a new grant to the NIH by April 1, 2004. Dr. Paul will give an update on this research at the MSUD Symposium 2004 in July.

We had initially created a classic MSUD mouse which carries a knock-out gene of the E2 subunit of the branched-chain ketoacid dehydrogenase (BCKDH) complex. As expected, 25% of the offspring of such mice were completely deficient in BCKDH and died a few days after birth. This neonatal lethality severely limited the usefulness of this model system as it was impossible to perform studies with these mice, such as, developing gene therapy.

To overcome this problem and to increase the usefulness of the MSUD mouse model, we created a conditional transgenic rescue of the lethal phenotype. In this new model, the rescuing transgene is under the control of a tetracycline-responsive promoter, and the investigator controls the level of transgene expression by adding or removing tetracycline from the drinking water and/or food.

For the tetracycline-regulated system, it is essential to create and screen several transgenic lines of mice in order to find those that perform optimally and truly represent the disease model. This required extensive breeding and testing of a large number of mice - a very time-consuming process.

We have now produced several transgenic lines. One line looks most promising, because these mice survive to adulthood. These mice represent an intermediate MSUD mouse model. They have branched-chain amino acid levels in the blood that are intermediate between control values and those of the classic MSUD model. Four additional lines have been tested, and these mice did not survive into adulthood. Four other lines are currently being analyzed for their ability to survive to adulthood. Several other lines remain to be tested.

Our goal now is to complete the screening and characterization of as many lines as possible, and to select one to establish a stable mouse colony to provide a continuous and predictable source of animals to allow for long-term studies. Our characterization of the mouse model has four components: a) screen for long-term and high frequency of survival, b) confirm reproductive ability, c) characterize blood amino acid levels, and d) monitor efficiency and reproducibility of transgene regulation by tetracycline. At a later date, characterization may also include histo-pathological analysis of selected organs, such as the brain.

The progress of this project has been slow, but substantial. Because of the complex biochemistry involved in MSUD, the project has proved to be much more complicated than initially anticipated. Secondly, limited funds have precluded us from adding additional resources, such as, research staff and support. In spite of these difficulties, we have made significant progress and expect to establish a large and stable colony of MSUD mice.

Funds provided by the MSUD Support Group are being used for the following three categories:

  1. Cost of animal care at the University of Pittsburgh. (Most of the funds are being used for housing the mice.)
  2. Costs related to amino acid analysis of blood from the mice.
  3. Some limited lab supplies.

In addition to funds provided by the MSUD Support Group, Biomed Research & Technologies, Inc. has continued to contribute funds and resources toward this project.