Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers.

Vascular cells are an important target for gene transfer because of their potential to deliver gene products both locally and systemically. Direct retroviral gene transfer to vascular cells in vivo has been limited by inefficient rates of transduction. We hypothesized that vascular cell transduction efficiency (TE), during short retroviral incubation periods, is significantly improved in vitro and in vivo using centrifugation to increase viral titer. Furthermore, we hypothesized a linear relationship between concentration of viable viral particles (measured as colony-forming units (CFUs)/cell) and retroviral TE during short incubation periods. Cultured rat pulmonary artery endothelial cells (RPAECs), rat aortic smooth muscle cells (RSMCs), and human iliac artery endothelial cells (HIAECs) demonstrated a strong correlation between TE and high concentrations of virus (> 100 CFU/cell) during retroviral incubation periods of 10 to 60 minutes. High titers, and thereby high concentrations, were achieved by centrifugation and resuspension in a fraction of the original volume. Titers was consistently increased tenfold, for a twentyfold increase in concentration by volume. A 20-minute incubation with a Moloney murine leukemia-derived retroviral vector coding for human placental alkaline phosphatase, pLJhpAP, at a concentration of 1150 CFU/cell yielded TEs of 10.6% +/- 0.7%, 40.4% +/- 1.6%, and 15.1% +/- 2.0% for RPAECs, RSMCs, and HIAECs, respectively. A similar effect was shown using the Moloney murine leukemia-derived MFGlacZ retroviral vector, coding for Escherichia coli beta-galactosidase. Increased titer and concentration had no effect on target cell viability, as shown by trypan blue exclusion. Although RSMCs had the most cells transduced in a given incubation period (p < 0.05), RPAECs had the highest replication rate (p < 0.05), suggesting the importance of factors other than cell cycle on retroviral TEs during short, clinically relevant incubation periods. In subsequent in vivo experiments, gene transfer was achieved in the rat carotid artery during a 20-minute incubation period infusing the concentrated pLJhpAP retrovirus after carotid balloon injury. Rats infused with virus 2 days after balloon injury exhibited hpAP activity (0 to 10 cells/section/rat) in the neointima of five out of six rats. Rats infused 4 days after balloon injury exhibited hpAP activity (0 to 25 cells/section/rat) in the media and adventitia of five out of five rats. Control rats that received the balloon injury alone or the balloon injury and unconcentrated retrovirus exhibited zero hpAP activity. We conclude that the TE of retroviral-mediated gene transfer to vascular cells in vitro and in vivo can be improved during short, clinically relevant incubation periods using centrifugation to increase retroviral titer, and thereby concentration of viable viral particles.

Induction of angiogenesis by lidocaine and basic fibroblast growth factor: a model for in vivo retroviral-mediated gene therapy.

A strategy of direct, in vivo retroviral-mediated gene therapy targeting capillary endothelial cells must provide an environment of active angiogenesis. Both lidocaine and basic fibroblast growth factor (bFGF) promote angiogenesis, but the angiogenic response invoked by these substances in normal skeletal muscle has not been fully characterized. We sought to characterize these agents' angiogenic effects in anterior tibialis muscles of male Sprague-Dawley rats. An injection of either 1% lidocaine with 1:100,000 epinephrine or alternate-day injections of bFGF (0.025 or 0.25 microgram) with or without heparin were tested (n = 6 muscles/condition). Rats were sacrificed 4, 7, 10, or 12 days later and muscles were evaluated histologically to determine the number of proliferating cells using 5-bromo-2'-deoxycytidine (BrdC) and evaluated for capillary density using Griffonia simplicifolia I (GSI) lectin. At all time points, lidocaine produced at least 20-fold greater capillary density and cellular proliferation than PBS control (P < 0.0001). Injections of high-dosage bFGF produced more than fivefold greater capillary density than control injections at 7 and 10 days (P < 0.001), and more than twofold greater proliferation at 4, 7, and 12 days (P < 0.001). Capillary density returned to control levels 12 days following bFGF administration, whereas it remained well above control levels for 12 days after lidocaine administration. To confirm that lidocaine can be utilized in gene therapy strategies targeting vascular endothelium and skeletal muscle fibers, concentrated pLJ retrovirus containing cDNA for the heat-stable human placental alkaline phosphatase (hpAP) marker gene was infused into the rat hindlimb vasculature 4 days post-lidocaine administration. Rats receiving pLJhpAP retrovirus demonstrated significant hpAP transgene expression in endothelial cells and myocytes 21 days after the lidocaine injection (n = 6 muscles). In contrast, controls receiving pLJhpAP infusion without prior lidocaine administration failed to demonstrate any hpAP transgene expression. Lidocaine treatment evokes a substantially higher proliferative response than bFGF and, importantly, a durable angiogenic response in skeletal muscle. Thus, lidocaine is an ideal agent to induce angiogenesis in preparation for direct in vivo retroviral-mediated gene therapy targeting vascular endothelium.

Systemic delivery of the interleukin-1 receptor antagonist protein using a new strategy of direct adenoviral-mediated gene transfer to skeletal muscle capillary endothelium in the isolated rat hindlimb.

Current gene therapy strategies using adenoviral vectors to target the lung or liver have been complicated by an acute inflammatory response that can result in loss of transgene expression as well as tissue injury and necrosis. Skeletal muscle comprises 40% of total body weight; it possesses a high density, accessible capillary network that is resistant to injury and thus may be a logical target for adenoviral vectors. We hypothesized that adenoviral transduction of the rat skeletal muscle capillary bed during vascular isolation would achieve efficient gene transfer sufficient to achieve systemic serum levels of a recombinant protein without significant tissue injury. During vascular isolation of the hindleg, a replication-incompetent adenovirus (Ad) encoding for either the marker gene, human placental alkaline phosphatase (hpAP), or interleukin-1 receptor antagonist (IL-1ra) was infused and subsequently flushed from the circulation after a 30-min dwell period. Gene transfer over a 10(9)-10(12) particle/ml range to the gastrocnemius capillary endothelium and muscle fibers was highly efficient and titer-dependent, reaching maximum transduction rates of 71 +/- 7% and 25 +/- 5%, respectively, 5 days after gene transfer (n = 3-8 rats/group, p < 0.05). hpAP transgene expression was barely detectable at 14 days. No significant tissue injury or necrosis of the skeletal muscle was observed at 5 and 14 days, and distant organ gene transfer was minimal or absent. Gastrocnemius muscle from rats (n = 4) given Ad-IL-1ra had 241 +/- 66 pg IL-1ra/mg protein at 5 days, while those given Ad-hpAP, negative control (n = 3) had 35 +/- 14 pg IL-1ra/mg protein (p < 0.05). Ad-IL-1ra rats (n = 4) had serum levels of 185 +/- 20 pg/ml IL-1ra at 5 days whereas Ad-hpAP control rats (n = 5) had no IL-1ra detectable (p < 0.0001). Athymic rats given Ad-IL-1ra (n = 6) had serum levels of 493 +/- 62 pg/ml IL-1ra 14 days after transduction, and IL-1ra was detected for up to 98 days. Sera from Ad-IL-1ra athymic rats significantly inhibited IL-1 beta-induced (1 ng/ml) prostaglandin E2 (PGE2) production from cultured endothelial cells by 82 +/- 2% (p < 0.001). Thus, this gene transfer strategy is the first to result in substantial transduction of both skeletal muscle capillary endothelium and fibers, sufficient to achieve pharmacologic levels of IL-1ra. Although no acute tissue injury or necrosis was observed, persistence of transgene expression in athymic rats suggests that loss of expression in normal rats was by an immune-mediated mechanism.