DESCRIPTION (provided by applicant): Pompe disease is a neuromuscular disorder resulting from mutations in the gene for acid a- glucosidase (GAA) - an enzyme necessary to degrade lysosomal glycogen. Hypoventilation is a hallmark feature of all forms of Pompe disease that has historically been attributed to respiratory muscle pathology. The experiments proposed in this R21 grant application will provide fundamental, mechanistic information about the clinical problem of respiratory insufficiency in Pompe disease. Most importantly, we propose a direct test of the hypothesis that central nervous system dysfunction is a primary contributor to respiratory insufficiency in Pompe disease. Evidence is mounting in support of this hypothesis, but definitive proof is lacking. To accomplish this goal we propose to use a "site-specific" Cre-Lox recombination approach to knockout the GAA gene in spinal and medullary respiratory neurons of mice while leaving skeletal and cardiac muscle gene expression unaltered. If the hypothesis is confirmed it will inform the clinical community about the underlying causes of respiratory insufficiency in Pompe patients. More importantly, confirmation of our hypothesis would necessitate a shift from the current emphasis on purely muscle directed therapies towards approaches which would impact on both muscle and neural function. Thus, overarching goal of the proposed studies is to determine if the respiratory control system becomes dysfunctional when GAA gene expression is "knocked out" in respiratory neurons. We also propose to compare and contrast the role of spinal motoneurons vs. medullary respiratory control neurons with regard to impaired respiratory motor output in Pompe disease. An initial clinical trial of GAA gene transfer to the diaphragm of Pompe patients is underway (ClinicalTrials.gov: NCT00976352). This work will complement the ongoing trial by examining the importance of motoneurons vs. medullary neurons to respiratory insufficiency. This is important since phrenic motoneurons can be transduced via retrograde viral transport post-diaphragm injection whereas medullary neurons will not. Thus, the clinical trial is not likely to result in transduction of medullary respiratory neurons. In developing this application we obtained a mouse colony with a "floxed" GAA gene. We propose to use stereotaxic and/or retrograde delivery of AAV vectors driving Cre recombinase expression to selectively knockout the GAA gene in spinal respiratory (phrenic) motoneurons (Aim 1) and brainstem respiratory control neurons (Aim 2). This work is a collaborative effort between a respiratory control scientist (Fuller), an AAV specialist and clinician working with Pompe patients (Byrne), and an AAV specialist with expertise in stereotaxic delivery (Mandel).

Pompe disease is a lysosomal storage disorder associated with systemic deficiency of an enzyme (acid alpha glucosidase) which is required to degrade glycogen. We hypothesize that part of the reason that breathing problems are common in Pompe disease is that brainstem neurons and spinal cord motoneurons fail to adequately control the respiratory muscles. To test this hypothesis, we propose to use genetic approaches to "knockout" the acid alpha glucosidase gene in spinal and medullary respiratory neurons of mice while leaving skeletal and cardiac muscle gene expression unaltered.