|Michael A. Barry
August 2006 Progress Update from PAF Newsletter
|The Barry Laboratory at the Mayo Clinic is working on a project to test if gene therapy can be used to treat propionic acidemia. To test this, PA mice from Dr. Miyazaki are being used as subjects for delivery of the PCCA gene to their livers. Sean Hofherr, a graduate student in Dr. Barry’s laboratory is pursuing this project for his Ph.D. thesis. To date, Sean has generated a series of gene therapy vectors expressing either the human or the mouse PCCA gene for testing in the PA mice. Preliminary experiments in the mice indicate that the vectors can be used to deliver PCCA gene to the liver to express amplified amounts of the protein. Work is underway to determine how this modifies the blood levels of propionate metabolites and to what degree this rescues the whole body and neurological symptoms of the disease in the mice. In the process of this work, Dr. Barry’s group generated antibodies against different parts of the PCCA protein to help in tracking where, when, and how much of the PCCA protein was being produced by their gene therapy vectors. With these tools in hand, as a side project, their group has also used them to probe some of the basic biology of the PCCA protein. While much is known about the genetics and disease symptoms of PA, little data can be found in the literature regarding the distribution of PCCA protein in different tissues. For example, the level of protein expression in different tissues may explain (in part) some of the tissue damage and symptoms due to loss of PCCA. Likewise, knowing where PCCA is and is not expressed might better guide how transplantation and gene therapies need to be applied and how this might differ between a mouse model and humans. For example, one might predict that the liver expresses the highest level of PCCA given its role in metabolizing excess amino acids and fatty acids. Conversely, one might predict that the brain or the basal ganglion might express lower amounts of PCCA, since many of the symptoms of the disease are manifested in these sites, particularly if these are due to effects within individual cells rather than due to metabolite overload. Given these issues, Dr. Barry’s group used these new antibodies to screen for PCCA protein production in mouse and human tissue panels. While they expected PCCA to be either ubiquitously expressed or expressed at highest levels in the liver, to their surprise, they observed a marked variation in amount of PCCA in different tissues. In both mouse and human tissues, the kidney appeared to have the highest levels of PCCA protein, in fact higher than in the liver per unit protein. In contrast, in the brain, PCCA was undetected in mouse (but not necessarily zero), and was detectable, but at low levels in the human brain samples. These data suggest PCCA is not ubiquitously expressed at high levels in all tissues and that the kidney may play a significant role in elimination of propionic metabolites. While the kidney had higher levels of PCCA when equalized for protein in the different tissues, it should be noted that the liver is still substantially larger in size and so likely “handles” substantially more metabolites. However, better knowledge of the locations of PCCA and cross-talk between organs may assist in optimizing therapeutics and to avoid mis-steps when translating between mouse models and PA patients. Work is underway to screen more specific regions of the brain for PCCA expression and to track how the protein’s expression may change over time in the PCCA mutant mice.|
Progress Update as seen in the August 2006 PAF Newsletter
|The strength of our research lies in the finding that some forms of mutant PCC are very responsive to an addition of small chemicals called chemical chaperones. These altered forms of the enzyme are not deficient in their ability to carry out the enzymatic reaction but rather in their ability to form the proper structure and assume the correct shape. The chaperone helps them to fold with a large increase in activity. We have carried out the initial experiments on normal and three mutant forms of PCC in a bacterial system in which the human enzyme can be manufactured. Later, we have used the chaperones in skin cell cultures derived from controls and propionic acidemia patients. Again, in some cases we saw large increases in PCC activity. We will continue to screen different chemicals and different mutations for their ability to yield more active PCC. The hope is that this approach can be introduced in clinical practice and help some patients to overcome their metabolic disease. The real promise is that some of the drugs, which gave us the best results, are already in use in clinical practice to treat other disorders.
PCC Website: http://www.uchsc.edu/cbs/pcc/about_pcc.htm
|Research Studies in Propionic Acidemia|
|Dr. Jan Kraus’ laboratory, Dept. of Pediatrics, University of Colorado School of Medicine – Update 11/2011
Propionic acidemia (PA) is a serious life threatening inherited disorder of metabolism. The disease is caused by deficiency of an enzyme called Propionyl CoA Carboxylase or PCC for short. PCC is a large enzyme consisting of six alpha and six beta subunits. The enzyme deficiency in turn is caused by mutations in either the PCCA or PCCB gene. My laboratory is currently supported by PAF for two projects associated with this disease.
The first project deals with the determination of the mutations or inherited changes in the DNA of propionic acidemia patients from USA whose DNA samples have been submitted to the Corriell Institute. Most of these patients are members of the PA foundation. Knowing the mutations will lead to better understanding of the disease and lead to improved treatment for the affected patients. We will also determine which of the two mutations in each patient came from which parent. This determination, in turn, will enable diagnoses of mutation carrier status in both parents’ families. The second project is entitled Enzyme Replacement Therapy for Propionic Acidemia. The main objective of this research proposal is to develop a therapeutic treatment of propionic acidemia (PA) by enzyme replacement therapy. Hurdles with enzyme replacement therapy include the delivery of the active enzyme into the patient cells as well as directing it to the correct location within the cell. In the case of PCC it needs to be delivered to the mitochondria. The mitochondria are separate membrane enclosed organelles within the cell that mainly supply the energy for cells. One promising way to deliver the PCC subunits across both the cell and mitochondrial membrane is the use of what is known as the TAT peptide. This peptide can cross cellular membranes and will also take along anything that is attached to it. Thus we propose to use the TAT peptide and a mitochondrial targeting sequence as a way to deliver the functioning PCC subunits to cells. The peptide and the targeting sequence will then be removed from PCC by another enzyme already present in mitochondria.
None of this work would have been possible without generous support from the Propionic Acidemia Foundation. Please give money to the foundation to support these and other studies on this devastating disease.
“This project deals with the determination of the mutations or inherited changes in the DNA of Propionic Acidmia patients from USA whose DNA samples have been submitted to the Coriell Institute. Most of thse patients are members of the PA Foundation. Knowing mutations will lead to better understanding of the disease and lead to improved treatment for the affected patients. We will also determine which of the two mutations in each patient came from which parent. This determination, in turn will enable diagnoses of mutation carrier status in both parents’ families.”
Pancreatitis is an inflammation of the pancreas. It can be a complication of Propionic Acidemia. Symptoms may include vomiting, nausea, tender abdomen, and fever. Blood tests of amylase and lipase (pancreatits enzymes) will confirm a diagnosis of pancreatitis.
Carbaglu (potential medication for treatment of elevated ammonia)
Carnitine – also see Sites of Interest Supplementation