Attempts to develop gene therapy for Duchenne muscular dystrophy (DMD) have been complicated by the enormous size of the dystrophin gene. We have performed a detailed functional analysis of dystrophin structural domains and show that multiple regions of the protein can be deleted in various combinations to generate highly functional mini- and micro-dystrophins. Studies in transgenic mdx mice, a model for DMD, reveal that a wide variety of functional characteristics of dystrophy are prevented by some of these truncated dystrophins. Muscles expressing the smallest dystrophins are fully protected against damage caused by muscle activity and are not morphologically different from normal muscle. Moreover, injection of adeno-associated viruses carrying micro-dystrophins into dystrophic muscles of immunocompetent mdx mice results in a striking reversal of histopathological features of this disease. These results demonstrate that the dystrophic pathology can be both prevented and reversed by gene therapy using micro-dystrophins.

The breast and ovarian cancer susceptibility gene BRCA1 plays a major role in the DNA damage response pathway. The lack of well-characterized human BRCA1-null cell lines has limited the investigation of BRCA1 function, particularly with regard to its role in ovarian cancer. We propagated a novel BRCA1-null human ovarian cancer cell line UWB1.289 from a tumor of papillary serous histology, the most common form of ovarian carcinoma. UWB1.289 carries a germline BRCA1 mutation within exon 11 and has a deletion of the wild-type allele. UWB1.289 is estrogen and progesterone receptor negative and has an acquired somatic mutation in p53, similar to the commonly used BRCA1-null breast cancer cell line HCC1937. We used ionizing radiation to induce DNA damage in both UWB1.289 and in a stable UWB1.289 line in which wild-type BRCA1 was restored. We examined several responses to DNA damage in these cell lines, including sensitivity to radiation, cell cycle checkpoint function, and changes in gene expression using microarray analysis. We observed that UWB1.289 is sensitive to ionizing radiation and lacks cell cycle checkpoint functions that are a normal part of the DNA damage response. Restoration of wild-type BRCA1 function in these cells partially restores DNA damage responses. Expression array analysis not only supports this partial functional correction but also reveals interesting new information regarding BRCA1-positive regulation of the expression of claudin 6 and other metastasis-associated genes and negative regulation of multiple IFN-inducible genes. (Mol Cancer Res 2007;5(1):35 -45)

Defective cardiac muscle relaxation plays a causal role in heart failure. Shown here is the new in vivo application of parvalbumin, a calcium-binding protein that facilitates ultrafast relaxation of specialized skeletal muscles. Parvalbumin is not naturally expressed in the heart. We show that parvalbumin gene transfer to the heart in vivo produces levels of parvalbumin characteristic of fast skeletal muscles, causes a physiologically relevant acceleration of heart relaxation performance in normal hearts, and enhances relaxation performance in an animal model of slowed cardiac muscle relaxation. Parvalbumin may offer the unique potential to correct defective relaxation in energetically compromised failing hearts because the relaxation-enhancement effect of parvalbumin arises from an ATP-independent mechanism. Additionally, parvalbumin gene transfer may provide a new therapeutic approach to correct cellular disturbances in calcium signaling pathways that cause abnormal growth or damage in the heart or other organs.J. Clin. Invest. 107:191-198 (2001).questions: (a) Could physiologically relevant levels of parvalbumin be achieved in the left ventricle at 6 days after gene transfer? (b) Under physiological conditions, can parvalbumin alter mechanical relaxation properties of the left ventricle? (c) Can parvalbumin improve heart performance in an experimental model of abnormally slow myocardial relaxation? MethodsCardiac gene transfer in vivo. Adult, female Sprague-Dawley rats (250 g) were anesthetized with sodium pentobarbital (30 mg/kg body weight intraperitoneally). Animals were intubated and placed on artificial ventilation (50-60 breaths per minute). A thoracotomy was performed over the third intercostal space of the left thorax to expose the heart. An insulin syringe was inserted into the LV free wall (apical to base), using the left anterior descending coronary artery as a guide, and then slowly injecting 50-100 µl solution (parvalbumin titer, 1.87 × 10 10 PFU/ml; AdlacZ titer, 1.00 × 10 10 PFU/ml, each in PBS/10% glycerol; vehicle was PBS/10% glycerol) while withdrawing the syringe. The chest was closed and negative pleural pressure reestablished before extubation. Animals were given antibiotics via the drinking water. At day 6 after gene (or vehicle) transfer, heart parvalbumin expression and/or heart mechanical function was evaluated. This procedure has been approved by the University Committee on the Use and Care of Animals at the University of Michigan.Myocardial relaxation performance. At day 6 after gene transfer, hearts were isolated, retrogradely perfused with oxygenated Krebs-Henseleit buffer warmed to 37°C, and electrically paced at 4 Hz (supramaximal, 5-millisecond pulses; Model S5 Stimulator; Grass Instruments, Quincy, Massachusetts, USA). Cardiac twitch contractile parameters (see Figure 3) were obtained from Langendorff perfused hearts by placing monofilament sutures at the apical and the base regions of the LV wall (between the circumflex and lateral descending coronary arteries). The base sutu...