Gene Gun Bombardment with DNA-Coated Gold Particles Is a Potential Alternative to Hydrodynamics-Based Transfection for Delivering Genes into Superficial Hepatocytes

Abstract

Although in vivo nonviral gene delivery to the liver is critical for hepatic gene therapy, there are a number of technical obstacles. Enhanced green fluorescent protein (EGFP)-encoding DNA was coated onto gold particles (gold-DNA), dissolved in phosphate-buffered saline (pure DNA), and prepared as a polymer adjuvant (jetPEI)-galactosidase solution (polymer-DNA). Murine liver transfection was attempted by nonviral approaches, which included hydrodynamics-based transfection (HBT) of pure DNA, transport and transhepatic injection of polymer-DNA, and gene gun bombardment with pure DNA, gold-DNA, and polymer-DNA. Only HBT and gene gun bombardment yielded significant numbers of EGFP(+) hepatocytes. With the exception of the edge of the liver, HBT had a whole-liver transfection rate of 20% under optimized conditions. HBT resulted in marked hepatic infarctions, most prominently at the edge of the liver. For gene gun bombardment, the transfection rate was pressure dependent and limited to 15% for gold-DNA. Triple or quadruple bombardment at 30 psi resulted in a transfection rate comparable to that of a single bombardment at higher pressure, but was associated with minimal scattered hepatic necrosis. The EGFP(+) hepatocytes were located mainly in the superficial layers. We conclude that both HBT and gene gun bombardment yielded efficient murine hepatocyte transfection in vivo. Severe hepatic infarction impedes foreign gene expression in the superficial hepatocytes after HBT. Repeated bombardment with gold-DNA, using an accelerated particle gene gun at 30 psi, is a potential alternative to HBT for delivering genes to superficial hepatocytes in vivo, although gold-related hepatic necrosis is a persistent problem.

Hydrodynamics-based Transfection of Pancreatic Duodenal Homeobox 1 DNA Improves Hyperglycemia and is Associated with Limited Complications in Diabetic Mice

“…after HBT, the aLT levels increased initially but decreased afterward in the case of . The results corroborated that the effects of gene delivery using HBT are usually transient, and the peak effects of gene delivery are observed 2-3 d after HBT and they diminish afterward [16]. The transfection efficiency in the case of the pEGFP-and pcdna-Pdx1-transfected Hepa1-6 cells was 55% and 5%, respectively.…”

“…No significant difference in the ALT levels were demonstrated between the mice that received pcdnaPdx1 and those that received pcdna prior to and after HBT. The transient increase in the aLT levels after HBT can be due to hydrodynamic pressure-related hepatic injury [16]. Except some peripheral hepatic necrosis, which is a common complication secondary to HBT [16], no definite cell aggregation due to hepatic inflammation was documented within 2 wk after HBT, regardless of the emergence of Pdx1-positive hepatocytes.…”

“…Furthermore, fetal hepatitis was developed after helper-dependent-adenovirus (Hda) delivery of Pdx1 to the liver of sTZinduced diabetic mice [10]. Naked deoxyribonucleic acid (dna) counters the potential biohazards of viral gene delivery and is considered useful because it can be manipulated using standard recombinant dna techniques and be delivered using both physical and chemical methods [16]. Our studies and those of others have shown the feasibility and efficiency of nonviral hepatic gene delivery in mice by using hydrodynamics-based transfection (HBT) [16,17].…”

“…Naked deoxyribonucleic acid (dna) counters the potential biohazards of viral gene delivery and is considered useful because it can be manipulated using standard recombinant dna techniques and be delivered using both physical and chemical methods [16]. Our studies and those of others have shown the feasibility and efficiency of nonviral hepatic gene delivery in mice by using hydrodynamics-based transfection (HBT) [16,17]. Therefore, our study aimed to determine the prognosis when Pdx1 was delivered to the liver of diabetic mice via HBT and to assess the efficiency of this method.…”

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs

“…This technique, also referred to as ballistic DNA delivery, has been used to deliver transgenes to skin, liver and muscle tissues of rats and mice (Yang et al, 1990) and DNA vaccines to skin, muscles and tumours in animal models and in human clinical trials. Recently, is has been shown to be efficient at delivering a small reporter gene to mouse liver in vivo (Chang et al, 2008). Almost no data is available on the ability of gene gun to deliver large DNA constructs in vivo.…”