Kristen J. Skvorak, Ph.D.
University of Pittsburgh, Pediatrics Department
Editor's Note: Dr. Kristen Skvorak recently received a doctoral degree from the University of Pittsburgh. Her research focused on treatment of MSUD. In this article she describes her work and the possible implications for those living with this disease.
As a University of Pittsburgh graduate student studying under Dr. Gregg Homanics, my main focus was to correct a mouse model of intermediate MSUD (iMSUD). I last reported my progress on achieving that goal in the MSUD Family Support Group newsletter in August of 2007. Now, only two short years later, I have a great deal of exciting results to share!
Hepatocyte (Liver Cell) Transplantation
As you know, transplanting a healthy liver into a patient can essentially cure MSUD. Therefore, it was reasonable to think that transplanting healthy cells into an MSUD liver may also improve disease symptoms. Cell transplant has many advantages over whole organ transplant: it is less invasive with fewer and less serious complications, less expensive, repeated treatments are possible, and cryopreservation of donor cells could allow them to be ready “on demand.” For these studies, I collaborated closely with Dr. Stephen Strom of the University of Pittsburgh, a leading expert in the field of hepatocyte (i.e., liver cell) transplant (HTx). Prior to this, Dr. Strom had mediated the clinical application of HTx to patients of liver failure and liver-based metabolic disease, though not MSUD. This new direction proved very fruitful, and in August of 2008 I was awarded my doctorate for my research in the field of MSUD.
Our animal studies were conducted on mice bred to have intermediate MSUD. Since MSUD patients typically begin treatment at birth, we also treated iMSUD mice at this time. Two separate injections of 100,000 healthy liver cells were injected directly into the livers of newborn mice. The mice were then allowed to develop normally and without any other treatments. Although only ~3% of the MSUD liver was repopulated by healthy cells, there were many improvements in the health of these animals when compared to untreated iMSUD mice. Circulating BCAAs were reduced an astounding 75% and body weight was increased 30% at 20 days of age (weaning). At 37 days of age (early adult), liver BCKDH activity, the enzyme which is compromised in MSUD, was more than doubled from 6% (untreated) to 14% (HTx), and improvements were found in some neurotransmitters, neurotransmitter metabolites, and amino acids (most notably dopamine and alloisoleucine) in the brains of HTx-iMSUD mice. Excitingly, these neurological improvements are the first demonstration that HTx can have therapeutic effects on extrahepatic organs outside of the liver. Finally, the survival of transplanted iMSUD animals was greatly improved; however, long term survival was not determined since the experiment was terminated at 37 days. At this time, experiments are planned to improve liver repopulation and determine long term survival.
One of the major causes of brain injury in MSUD is thought to be related to leucine accumulation. Dr. William Zinnanti, a collaborator at The Penn State University, hypothesized if movement of leucine into the brain was impeded, disease symptoms would improve. Norleucine is an atypical amino acid shown to compete with leucine at the blood-brain barrier for brain access. To accomplish this, norleucine, an atypical amino acid shown to compete with leucine at the blood-brain barrier for brain access, was supplemented in mouse chow. Mice with intermediate MSUD (iMSUD) were fed a high protein diet with or without norleucine. When norleucine was supplemented with a high protein diet, survival was improved and the onset of encephalopathy was delayed in iMSUD mice compared to those only receiving a high protein diet. Norleucine-supplemented feed given to nursing mothers also resulted in improved survival in classic MSUD (cMSUD) mouse pups. Based on these data, norleucine should be further tested as a potential treatment for MSUD children admitted during catabolic stress. Dr. Zinnanti hopes to start a clinical trial sometime next year. (Editor: see Dr. Zinnanti's article)
In addition to norleucine studies, Dr. Zinnanti characterized a great deal of neurological changes in the brains of both iMSUD and cMSUD mice. These new data have greatly contributed to our general understanding of the pathophysiology of MSUD. Dr. Zinnanti proposes two convergent mechanisms of brain injury resulting from BCKA accumulation:
(1) neurotransmitter deficiencies and growth restriction, and
(2) Kreb's cycle disruption and disruption of energy metabolism leading to energy deprivation. This new brain injury proposal could illuminate new solutions to prevent such injuries from occurring.
Getting the Word Out
Scientific meetings are an excellent venue for a young scientist. It's a place to establish connections and collaborations, share and discuss new data, and discover new technologies from other like-minded researchers from around the world. I have had the incredible opportunity to present my HTx data at two international meetings in the past year. Two talks were given at the Society for the Study of Inborn Error of Metabolism (SSIEM) Symposium in Lisbon, Portugal, and one talk at the Cell Transplantation Society Symposium in Okayama, Japan. All presentations were very well received, and I have been contacted many times by investigators interested in applying cell therapies to their own research. Dr. Zinnanti has presented his norleucine data at the Grand Rounds at Kennedy Krieger Institute (Johns Hopkins University) and will present at the SSIEM Symposium in La Jolla, CA on August 29th, 2009. In addition, an in depth characterization paper on the iMSUD mouse was recently published which describes its remarkable similarity to the human disease. The iMSUD mouse is currently maintained at The Jackson Laboratories, an animal model “zoo” allowing open access to researchers around the world.
We hope the public availability of the iMSUD mouse, as well as the newly published characterization and therapeutic data, will serve to encourage others to forge ahead into the study of MSUD. A greater number of analytical minds studying a problem should yield a more complete understanding of the pathophysiology behind the human disease. In time, this will undoubtedly lead to new therapies to treat or possibly cure the disorder.
- KJ Skvorak, et al. Hepatocyte transplantation (HTx) corrects selected neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD). BBA - Molecular Basis of Disease. Submitted
- Skvorak KJ, et al. Hepatocyte transplantation improves phenotype and extends survival in a murine model of intermediate maple syrup urine disease. Molecular Therapy. May 2009; 17(7): 1266-1273
- Skvorak KJ. Animal Models of Maple Syrup Urine Disease. Journal of Inherited Metabolic Disease. Apr 2009; 32(2): 229-246
- Zinnanti WJ, et al. Dual mechanism of brain injury and novel treatment strategy in maple syrup urine disease. Brain. Apr 2009; 132(Pt 4): 903-18