Intrapulmonary and intramyocardial gene transfer in rhesus monkeys (Macaca mulatta): Safety and efficiency of HIV-1-derived lentiviral vectors for fetal gene delivery

Abstract

Fetal gene transfer was studied using intrapulmonary and intramyocardial transfer of SIN HIV-1-derived lentiviral vectors expressing EGFP in rhesus monkeys. Fetuses were monitored sonographically during gestation and tissue analyses performed at term or 3 months postnatal age. Animals remained healthy during the study period as evidenced by normal growth, development, hematology, clinical chemistry, echocardiography, and pulmonary function tests. Strong pulmonary fluorescence was observed postnatally after fetal intrapulmonary delivery of lenti-CMV, but not lenti-SP-C, and compared to nontransferred controls. High EGFP copy numbers were found by quantitative PCR with both vector constructs in lung lobes (<or=15%) and EGFP copies were also detected in the diaphragm, pericardium, and thorax. No differences were found in lung:body weight ratios, percentage lung parenchyma, or overall morphology when compared to controls. For intramyocardial gene delivery, strong transgene expression was found within the myocardium and pericardium, and high EGFP copy numbers were found by quantitative PCR (3-36%). EGFP was also detected in the aorta, thorax, and diaphragm. These studies indicate that postnatal heart and lung development and function were not altered after fetal intraorgan gene transfer and subsequent transgene expression prenatally and postnatally, and gene transfer was restricted to the thoracic cavity with intrapulmonary and intramyocardial lentiviral vector-mediated gene delivery.

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

“…5 Studies have also shown the efficiency of lentiviral vector-mediated gene transfer in fetal monkeys using both systemic and organ-targeting approaches. [6][7][8][9] These studies have indicated that intraperitoneal (i.p.) administration of reporter genes (e.g., enhanced green fluorescent protein [eGFP] or firefly luciferase) in early gestation results in long-term transgene expression in the muscular component of the diaphragm and peritoneum.…”

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

“…In contrast, HIV-1-based lentiviral vectors (LVs) have increased packaging capacity (optimal transgene insert B5-7 Kb 23 ) and, following in utero delivery, are able to integrate into the host genome to provide life-long stable transgene expression. 16,[24][25][26][27] Fetal lung gene transfer has been investigated in non-human primates, 20,28 rabbits, 29 rats 30,31 and sheep 32 using vesicular stomatitis virus glycoprotein (VSVg) pseudotyped LV. Long-term luciferase transgene expression (that is 12-15 months after fetal gene transfer) has been reported 31,33 although there is a paucity of quantitative information on degree of epithelial transduction and cell types transduced.…”

Fetal Gene Therapy

“…It was not surprising, therefore, when we examined other tissues of the recipients, to find that gene transfer was not limited to cells of the hematopoietic system, but had occurred in essentially all of the organs we examined, including numerous cell types within the liver, lung, and brain [79,81,82,86]. Concomitantly, in utero gene transfer studies performed by other investigators in sheep, rodent, and non-human primate models employing a variety of viral-based gene delivery vectors produced similar results [78][79][80][81][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105], raising the exciting possibility that in utero gene therapy could potentially be used to treat not only hematologic disorders, but also numerous genetic disorders that affect tissues other than the hematopoietic system. For example, in the case of the hemophilias, this method could likely be used with success to delivering genes for the missing coagulation factors to the developing liver at levels that would covert patients with severe hemophilia to a moderate or even mild phenotype [79].…”