Duchenne Muscular Dystrophy (DMD) is a degenerative disease leading to fatal respiratory and cardiac complications. It is caused by loss of functional dystrophin through mutation of the dystrophin gene. Possible treatments could use Adeno-associated virus (AAV) to introduce genes into muscle cells. This strategy requires muscle specific regulatory cassettes containing sequences specific for transcribing the therapeutic gene in muscle cells. My studies seek to increase the activity of one such cassette, MHCK7. Since AAV vectors can package only ~5000 basepairs of DNA, the dystrophin cDNA and regulatory cassette must be miniaturized. I am modifying MHCK7 by adding a 95 bp intronic enhancer (SIE) from the muscle creatine kinase (MCK) gene that is important for MCK expression in slow muscle fibers and cardiac muscle. The SIE has been placed ~1kb 3’ of the MHCK7 luciferase reporter cDNA to mimic its endogenous genomic location. SIE addition was performed by introducing a HindIII site 3’ of the luciferase polyA sequence by site directed mutagenesis, and the subsequent opening of DNA at that site. The SIE was transferred from a previously made plasmid and ligated into the opened MHCK7. Restriction digests and gel electrophoresis were used to assess successful introduction of HindIII and to confirm digests for opening the DNA and isolating the SIE. New recombinant MHCK7-3’-SIE will be transfected into skeletal and cardiac muscle cultures, and its activity will be assessed through luciferase activity. The SIE’s contribution to expression will be compared to that of the original MHCK7 plasmid as well as to MHCK7 with the SIE located immediately 5’ of the cassette. If the new regulatory cassette is more active than MHCK7 and 5’-SIE-MHCK7, further studies will test the cassette in conjunction with a microdystrophin cDNA. If activity is not increased, I will test alternative locations of the SIE.

Vascular calcification (VC) is an indicator of atherosclerotic plaque burden and is a strong, independent predictor for future cardiovascular complications, significantly increasing the risk for cardiovascular morbidity and mortality. Type II diabetes (T2D), the 7th leading cause of death in the US, is associated with increased incidence of VC clinically, as well as in our recent animal study. Although VC was considered a degenerative process involving passive accumulation of calcium-phosphate salts associated with tissue necrosis, numerous studies have indicated that VC is an active, cell-mediated process that resembles embryonic bone formation and remodeling. Smooth muscle cells (SMCs) have been implicated in giving rise to bone- and cartilage-like cells in calcifying blood vessels by undergoing lineage reprogramming in response to local environmental cues. We investigated the association of S100A11, a calcium binding protein, with a multi-ligand receptor, the receptor for advanced glycation end products (RAGE), as a possible signaling mechanism involved in the lineage reprogramming of SMCs under T2D settings. We used a genetic fate mapping strategy to trace SM-derived cells in the model T2D mice that developed vascular calcification. Immunofluorescent dual staining for S100A11 and RAGE, along with immunohistochemical staining for Runx2/Cbfa1, an early osteochondrogenic marker, was performed on aortic sections of the T2D mice. The results revealed that S100A11-RAGE signaling is most likely to be involved in cartilage lesion development. This is supported by co-localization of S100A11-RAGE markers within cells of chondrocyte morphology in cartilaginous and calcific areas of blood vessels. Interestingly, almost all S100A11-RAGE positive cells were derived from SMCs that have been labeled by specific transgenes. These results highlight the significance of S100A11-RAGE signaling in VC and possibly provide for the development of preventative or therapeutic targets against T2D-related VC.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases (GOLD, 2011). There are two classes of medications to treat COPD, bronchodilators and corticosteroids. Some patients are prescribed multiple inhalers to maximize treatment effectiveness. Correct inhaler technique is important to ensure proper deposition of medications in the airways; however, administration techniques vary across devices. This raises the question as to if there is a relationship between the number of prescribed inhalers with patients’ confidence in their inhaler technique and medication behavior. We hypothesized that patients using multiple inhalers would have decreased confidence in their inhaler technique and adherence to inhaler use may be lower. To answer this question, we used data collected from an ongoing longitudinal observational study looking at biological causes and functional consequences of depression in COPD patients. Our analyses showed that patients who were on 3 or more inhalers had the same level of confidence in taking their inhalers correctly as patients who were on 2 or fewer inhalers. They also did not differ in forgetting to take or being careless about their medications compared to patients who took fewer inhalers. In addition, they were no less likely to take their inhalers when there were changes in their daily routine. These results refute our hypothesis, and instead, demonstrate that there is no relationship between the number of prescribed inhalers and inhaler technique confidence and medication behavior.

Duchenne Muscular Dystrophy (DMD) patients have mutated dystrophin genes and suffer from muscular degeneration, which leads to cardiac, respiratory and general skeletal muscle disabilities. Potential treatment of DMD patients could involve gene therapy in which Adeno-Associated Viruses (AAV) deliver functional dystrophin cDNA ligated to muscle specific regulatory cassettes that express dystrophin exclusively in striated muscle tissues. The Hauschka lab has been designing regulatory cassettes (e.g. MHCK7) that contain control regions from genes that are highly active in skeletal muscles such as the muscle Creatine Kinase (MCK) and α-Myosin Heavy Chain (α-MHC) genes. Due to the limited size of DNA that can fit into AAV capsids, the cassettes need to be as compact as possible. My goal is to test whether adding a small intronic enhancer (SIE) from the MCK gene, that is known to have high activity in skeletal muscle in vitro, will improve transcriptional activity in cardiac and skeletal muscle cultures compared to MHCK7. The SIE was isolated and cloned immediately 5’ of MHCK7. After sequencing to confirm the SIE orientation, we carried out a pilot study in which 5’-SIE-MHCK7 was transiently transfected into skeletal muscle cultures. 5’-SIE-MHCK7 activity, as measured by the reporter gene firefly luciferase, was several fold higher than MHCK7. This suggests that the addition of the SIE to two known muscle specific enhancers increases transcriptional activity in skeletal muscle cells. Future studies will focus on confirming this result, and comparing 5’-SIE-MHCK7 activity to aMHCK7-SIE cassette in which the SIE is located 3’ of the luciferase reporter, so as to mimic its normal location in the MCK gene. The 5’-SIE-MHCK7 cassette will also be tested in cardiomyocyte cultures, and smaller cassette versions will be designed that eliminates additional non-conserved DNA sequences from the SIE and MHCK7 components.