Transfer of Therapeutic Genes into Fetal Rhesus Monkeys Using Recombinant Adeno-Associated Type I Viral Vectors.

Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa-/- mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 1012 particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.

Diaphragm Pacing as a Rehabilitative Tool for Patients With Pompe Disease Who Are Ventilator-Dependent: Case Series.

BACKGROUND AND PURPOSE: Pompe disease is an inherited disorder notable for severe, progressive ventilatory compromise. Although ventilatory failure has been attributed to myofiber dysfunction secondary to diaphragmatic glycogen accumulation, neural involvement of the phrenic motor system is also a prominent feature. Direct diaphragm pacing supplements respiratory function in other disorders of the phrenic motor system. Accordingly, it is hypothesized that augmented neuromuscular activity via diaphragm pacing would promote weaning from mechanical ventilation in patients with Pompe disease who are unresponsive to conventional, muscle-directed treatments. CASE DESCRIPTION: Three patients with Pompe disease developed diaphragm paresis that resulted in chronic mechanical ventilation dependence. After preoperative inspiratory muscle strengthening exercises failed to improve function, fine-wire pacing electrodes were laparoscopically implanted into the diaphragm. Diaphragm conditioning was initiated the first postoperative week and consisted of gradual increases in stimulation parameters, lengthening of stimulation sessions, and ventilator weaning. Ventilation and intramuscular electromyographic activity were recorded periodically during conditioning to quantify diaphragm neuromuscular function. OUTCOMES: During paced breathing without mechanical ventilation, tidal volumes increased, and 2 patients were weaned from daytime ventilator dependence within the first 3 months of pacing, which has been sustained over the long-term. A third patient reduced reliance on daytime ventilation, but weaning was delayed by malacia of the large airways. In all patients, pacing appeared to facilitate spontaneous phrenic motor unit activity during independent breathing without ventilator or pacer support. DISCUSSION: The findings are consistent with the view that diaphragm pacing has potential rehabilitative value to reduce reliance on mechanical ventilation in people with Pompe disease, but further study is needed. Diaphragm pacing represents a paradigm shift in the management of respiratory insufficiency for Pompe disease that warrants further controlled examination.

Altered activation of the diaphragm in late-onset Pompe disease.

Pompe disease is an inherited neuromuscular disorder that affects respiratory function and leads to dependence on external ventilatory support. We studied the activation of the diaphragm using bilateral phrenic magnetic stimulation and hypothesized that diaphragm compound muscle action potential (CMAP) amplitude and evoked transdiaphragmatic pressure (Twitch PDI) would correlate to disease severity. Eight patients with late onset Pompe disease (LOPD, aged 14-48 years) and four healthy control subjects completed the tests. Maximal Twitch PDI responses were progressively reduced in patients with LOPD compared to control subjects (1.4-17.1cm H2O, p<0.001) and correlated to voluntary functional tests (p<0.05). Additionally, CMAP amplitude (mA) was lower in the patients who used nighttime or fulltime ventilatory support, when compared to controls and patients who used no ventilatory support (p<0.005). However, the normalized (%peak) Twitch PDI and CMAP responses were similar between patients and controls. This suggests a loss of functional phrenic motor units in patients, with normal recruitment of remaining motor units.

Stimulation of Respiratory Motor Output and Ventilation in a Murine Model of Pompe Disease by Ampakines.

Pompe disease results from a mutation in the acid α-glucosidase gene leading to lysosomal glycogen accumulation. Respiratory insufficiency is common, and the current U.S. Food and Drug Administration-approved treatment, enzyme replacement, has limited effectiveness. Ampakines are drugs that enhance α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor responses and can increase respiratory motor drive. Recent work indicates that respiratory motor drive can be blunted in Pompe disease, and thus pharmacologic stimulation of breathing may be beneficial. Using a murine Pompe model with the most severe clinical genotype (the Gaa(-/-) mouse), our primary objective was to test the hypothesis that ampakines can stimulate respiratory motor output and increase ventilation. Our second objective was to confirm that neuropathology was present in Pompe mouse medullary respiratory control neurons. The impact of ampakine CX717 on breathing was determined via phrenic and hypoglossal nerve recordings in anesthetized mice and whole-body plethysmography in unanesthetized mice. The medulla was examined using standard histological methods coupled with immunochemical markers of respiratory control neurons. Ampakine CX717 robustly increased phrenic and hypoglossal inspiratory bursting and reduced respiratory cycle variability in anesthetized Pompe mice, and it increased inspiratory tidal volume in unanesthetized Pompe mice. CX717 did not significantly alter these variables in wild-type mice. Medullary respiratory neurons showed extensive histopathology in Pompe mice. Ampakines stimulate respiratory neuromotor output and ventilation in Pompe mice, and therefore they have potential as an adjunctive therapy in Pompe disease.

Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5.

Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. Transgene detection 80 days post AAV treatment suggests transduction of crypt progenitor cells. This study shows the feasibility of AAV-mediated intestinal gene delivery, applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.