Intramuscular Injection of an Adenoviral Vector Expressing Hepatocyte Growth Factor Facilitates Hepatic Transduction with a Retroviral Vector in Mice

Abstract

Retroviral vectors can result in therapeutic andstable levels of expression of proteins from the liver. However, m ost retroviral vectors transduce only dividing cells, and hepatocytes are norm ally quiescent. The goal of this study was to determ ine if an adenoviral vector could transiently express hepatocyte growth factor (HG F) in order to induce hepatocyte replication and facilitate retroviral vector transduction of the liver. Intramuscular injection of an adenoviral vector that expressed human HGF from the cytomegalovirus promoter (Ad.C M V.HGF) resulted in m oderate levels of H GF in blood and liver, and replication of 3 to 12% of hepatocytes. No cytopathic effect was observed in the liver, and a control adenoviral vector indu ced no or lower levels of replication. W hen a retroviral vector expressing b -galactosidase cD NA was injected into a peripheral vein during the peak period of hepatocyte replication induced by intram uscularly adm inistered Ad.C M V.HG F, 8% of hepatocytes were transduced. W e conclude that intram uscular injection of Ad.C M V.HG F is a safe and effective way to induce transient systemic expression of HG F and hepatocyte replication, and to facilitate transduction of hepatocytes with a retroviral vector. 911 OVERVIEW SU M M A RY Retroviral vectors can resu lt in stable and therapeutic levels of expression of proteins from the liver. H owever, m ost retroviral vectors transduce the liver in vivo only when the norm ally quiescen t hepatocytes are induced to replicate. Although 70% partial hepatectom y, 70% portal branch occlusion, or adm inistration of a liver toxin have been used to induce hepatocyte rep lication for gene therapy, their potential m orbidity m akes them less attractive for use in hum ans. G rowth factors that induce hepatocyte rep lication are a prom ising approach for facilitating retro viral vector transduction. H owever, they are expensive, and optim al results req uire continuous infusion or m ultiple injections. W e dem onstrate here that intram uscular injection of Ad.C M V.H G F resu lted in the transient appearance of H G F in the seru m and liver, hepatocyte rep lication, and retro viral vector transduction without apparent toxicity. This approach will be a useful tool for perform ing gene therapy experim ents in large num bers of animals with a specific genetic disorder.

Lipopolysaccharide potentiates the effect of hepatocyte growth factor on hepatocyte replication in rats by augmenting AP-1 activity

“…An adenoviral vector designated Ad. CMV.HGF that contains the 2.3-kb human HGF complementary DNA downstream of the immediate early cytomegalovirus promoter 25 was used to infect 293-N3S cells (Microbix Biosystems, Toronto, Ontario) that were at 5 ϫ 10 5 cells/mL in previously described media 25 at a multiplicity of infection of 10. Cell debris was pelleted 40 to 44 hours later by centrifugation at 800g for 10 minutes at 4°C and up to 8 L of supernatant containing 0.5 to 2 µg/mL of HGF was applied to a 140-mL heparin sepharose CL-6B column (Pharmacia, Uppsala, Sweden).…”

“…26 To obtain highly purified HGF, HGF was eluted from the heparin sepharose column with 2 mol/L NaCl, followed up by purification over a 25-mL SP-Sepharose (Pharmacia) column and elution with a 0.15 to 1.0 mol/L NaCl gradient. Fractions containing HGF were identified by enzyme-linked immunosorbent assay, 25 concentrated by an Amicon filter (Millipore Corporation, Bedford, MA) with a 30-kd cutoff, and stored at Ϫ70°C in phosphate-buffered saline (PBS; 139 mmol/L NaCl, 2.7 mmol/L KCl, 10.1 mmol/L sodium phosphate, 1.8 mmol/L potassium phosphate). Reducing and non-reducing sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 27 were performed.…”

“…The concentration of HGF that induced 50% scattering of Madin-Darby canine kidney cells was determined as described. 25 HGF standards were single-chain HGF obtained from Becton Dickinson Labware (Bedford, MA). Liver HGF levels were determined as described previously 25 and normalized to total protein levels as determined using a Bradford assay kit obtained from Bio-Rad.…”

“…25 HGF standards were single-chain HGF obtained from Becton Dickinson Labware (Bedford, MA). Liver HGF levels were determined as described previously 25 and normalized to total protein levels as determined using a Bradford assay kit obtained from Bio-Rad. Both preparations of HGF resulted in delivery of a cumulative dose of 3 ng/kg of endotoxin (low-dose HGF), or 30 ng/kg (high-dose HGF), as determined by a limulus amebocyte lysate gel-clot assay.…”

Fas/Cd95 Pathway Induces Mouse Liver Regeneration and Allows for Highly Efficient Retrovirus–Mediated Gene Transfer

“…[14][15][16][17][18][19] To be efficient, these strategies necessitate very high doses of HGF and a continuous retrovirus infusion for several days. The percentage of retroviral vector-transduced cells would otherwise remain below 10%.…”