Hydroxyethyl starch-based preservation solutions enhance gene therapy vector delivery under hypothermic conditions

Abstract

Isolated liver perfusion offers a unique prospect for safe, effective targeting of gene therapies that can be directed against allograft rejection or recurrent diseases such as reinfection by hepatitis C virus (HCV). We aimed to examine the effect of organ preservation solutions on vector-based gene therapy delivery under hypothermic conditions. University of Wisconsin (UW) solution, histidine tryptophan ketoglutarate (HTK), EloHaes, sodium-poly(ethylene glycol)-UW solution [Institut Georges Lopez 1 solution (IGL-1)], and Dulbecco's modified Eagle's medium (DMEM) culture medium (control) were tested at 2°C or 37°C for lentiviral vector transduction efficiencies to the hepatoma cell line Huh-7 and primary human or mouse hepatocytes. Lentiviral vectors expressing short hairpin RNA were used to target HCV replication. With a potent short hairpin RNA vector, transductions were directly correlated to the therapeutic effect, with low transduction yielding low knockdown and vice versa. Green fluorescent protein (GFP) reporter gene expression was observed with vector incubation times as short as 10 minutes. The highest transductions were seen, after 2-hour 37°C incubation, in UW (62% Ϯ 6 SEM); they were significantly higher than those in HTK (21% Ϯ 7 SEM). Neither adenosine nor glutathione, present in UW, provided any increase in transduction when supplemented to HTK, although the addition of hydroxyethyl starch (HES) significantly improved transductions. To rule out size exclusion as a mechanism of HES, IGL-1 was tested but did not result in better transductions than HTK or DMEM. When supplemented to UW, anionic compounds reduced transduction, and this indicated a charge interaction mechanism of HES. In conclusion, this study demonstrates that effective vector delivery can be achieved under conditions of hypothermic liver perfusion. UW provides superior transduction to hepatocytes over nonstarch solutions.

Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi)

“…We used a subgenomic HCV replication model based on a Huh7 hepatoma cell line containing the non-structural sequence of the HCV genome with a luciferase reporter gene (Huh7-ET), mimicking viral replication without virus particle production 24. As reported previously, LV-shNS5b resulted in a mean±SD maximum inhibition of HCV replication of 98±0.5% (n=8, p<0.001) at the highest transduction efficiency 8 25. However, at suboptimal transduction efficiency, the percentage inhibition of viral replication as measured by luciferase activity significantly exceeded the percentage of transduced cells as measured by GFP expression (figure S1 in online supplement).…”

Organ Preservation: Current Concepts and New Strategies for the Next Decade

“…ing cold preservation [127] and in protocols for ex vivo modification. In the recipient, the therapeutic gene will be expressed after transplantation [128].…”

Infectivity and stability of hepatitis C virus in different perfusion solutions

“…Hydroxyethyl starch (HES), a component of UW Belzer, could account for the boosted HCV entry. In fact, it was shown that the entry of VSV pseudotyped lentiviral particles was facilitated in the presence of UW Belzer suggesting its use in gene therapy during cold storage of liver transplants . Even though HES is a non‐ionic compound, it appears to neutralize repulsive forces between pseudoparticles and the cell surface.…”