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.

Comparison of growth and differentiation of fetal and adult rhesus monkey mesenchymal stem cells.

The goal of this study was to compare the growth and differentiation potential of fetal and adult rhesus monkey (Macaca mulatta) mesenchymal stem cells (rhMSCs). rhMSCs were obtained from healthy early third-trimester fetal (n = 3) and adult (n = 3) rhesus monkey bone marrow. Fetal rhMSCs were plated at 10, 50, 100, or 1,000 cells/cm(2) in medium containing 10% or 20% infant monkey serum (IMS) or fetal bovine serum (FBS). Fetal rhMSCs grown at 1,000 cells/cm(2) in 20% FBS showed faster growth rates and differentiation toward adipogenic, chondrogenic, and osteogenic lineages when compared to other culture conditions and to adult cells (p < 0.05). Fetal rhMSC showed higher population doubling times (11.3 +/- 0.5) when compared to adult cells (7.3 +/- 0.8) during the first three passages. Adult rhMSC did not grow beyond the third passage under all culture conditions, including those supplemented with insulin-like growth factor (IGF)-I, IGF-II, platelet-derived growth factor (PDGF), and fibroblast growth factor-2 (FGF-2). After the third passage, adult rhMSC cultures were observed with large syncytia and with evidence of apoptosis. Cells obtained from these cultures tested positive for simian foamy virus (SFV) by PCR, RT-PCR, and immunofluorescent assay. Adult rhMSCs cultured with 10 microM tenofovir, an antiviral agent, showed normal growth and differentiation for over 20 population doublings. These findings suggest that: (1) fetal rhMSCs possess greater self-renewal and differentiation potential when compared to adult cells; and (2) SFV can inhibit proliferation of adult rhMSCs in culture, whereas the addition of tenofovir can successfully suppress SFV replication in vitro and result in resumed growth.