RESUMEN
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 (â¼7 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.
Asunto(s)
Proteínas Portadoras/genética , Dependovirus/genética , Distrofina/genética , Terapia Genética , Vectores Genéticos/administración & dosificación , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , alfa-Glucosidasas/genética , Adolescente , Animales , Niño , Preescolar , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Diafragma , Evaluación Preclínica de Medicamentos , Femenino , Técnicas de Transferencia de Gen , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Humanos , Macaca mulatta , Masculino , RatonesRESUMEN
BACKGROUND: Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B(12), adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). METHODS: The human ATR cDNA was cloned into a recombinant adeno-associated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/Bl6 mice at a dose of 1 x 10(10) and 1 x 10(11) particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. RESULTS: Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken beta-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. CONCLUSIONS: These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments.