|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.