Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection

Abstract

ABSTRACT.Mitochondrial genetic disorders are a major cause of mitochondrial diseases. It is therefore likely that mitochondrial gene therapy will be useful for the treatment of such diseases. Here, we report on the possibility of mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. The HLV injection procedure, a useful method for transgene expression in skeletal muscle, involves the rapid injection of a large volume of naked plasmid DNA (pDNA) into the distal vein of a limb. We hypothesized that the technique could be used to deliver pDNA not only to nuclei but also mitochondria, since cytosolic pDNA that is internalized by the method may be able to overcome mitochondrial membrane. We determined if pDNA could be delivered to myofibrillar mitochondria by HLV injection by PCR analysis. Mitochondrial toxicity assays showed that the HLV injection had no influence on mitochondrial function. These findings indicate that HLV injection promises to be a useful technique for in vivo mitochondrial gene delivery.

Condensation of Plasmid DNA Enhances Mitochondrial Association in Skeletal Muscle Following Hydrodynamic Limb Vein Injection

“…At 2 min after the injection, the tourniquet was released. A schematic diagram of this methodology, which was previously reported for the HLV injection of naked pDNA [5], is shown in Figure 1.…”

“…pDNA was condensed with a polycation (Protamine) and the condensed pDNA was intravenously injected into the dorsalis pedis vein via HLV injection, which is similar to a previously reported method [5]. It is generally thought that the condensed pDNA is internalized into cells via hydrodynamic force, and mitochondrial localization of pDNA may be achieved.…”

“…Therefore, mitochondrial gene therapy and diagnosis could be expected to offer substantial medical benefits, however, the utility of this strategy has not yet been realized because the technology for in vivo mitochondrial gene delivery is a bottleneck. We previously reported on the potential for using hydrodynamic limb vein (HLV) injection for achieving mitochondrial gene delivery targeted to mammalian skeletal muscle tissue [5]. Skeletal muscle represents an attractive target tissue for mitochondrial gene therapy, because mitochondrial genomic dysfunctions in skeletal muscle are largely associated with various mitochondrial diseases [2,4].…”

“…The HLV injection procedure, a useful method for nuclear transgene expression in skeletal muscle, involves the rapid injection of a large volume of naked plasmid DNA (pDNA) into the distal vein of a limb [6,7,8,9]. Using PCR analysis, we demonstrated the HLV injection technique could be used to deliver naked pDNA into myofibrillar mitochondria and that it had no influence on mitochondrial function [5]. …”

Efficient Packaging of Plasmid DNA Using a pH Sensitive Cationic Lipid for Delivery to Hepatocytes

“…Despite the advantages of gene delivery therapy, recent advances in gene delivery, including plasmid DNA (pDNA), have been relatively restricted compared to the delivery of artificially synthesized oligonucleotides, including small interfering RNA (siRNA), anti-sense oligonucleotides and antimicro RNA oligonucleotides because of difficulty associated with developing a safe, effective non-viral vector for pDNA. 1) Hydrodynamic tail vein (HTV) injection is an established method for the successful gene delivery to hepatocytes, 2) myocytes, 3) mitochondria of limb muscles 4) and so on. Jacobs et al reported that transgene expression in the liver as the result of HTV injection equaled that of adenovirus injection.…”