Opening the Floodgates: Clinically Applicable Hydrodynamic Delivery of Plasmid DNA to Skeletal Muscle (2004)

A number of issues hinder the translation of the concept of gene therapy into clinical practice. Of these, perhaps the most fundamental issue is the efficiency of gene delivery. Considerable effort has been and is being expended on developing the optimal vector and method of administration. The use of plasmid DNA as a vector system is an attractive prospect, as it is the simplest vector system to manufacture in the large quantities required for human treatment, its quality is relatively easy to control compared to complex viruses, and it can be stored for long periods of time in a lyophilized form. In addition, the use of plasmid DNA does not involve the same concerns associated with the use of replication-deficient viral vectors, such as the possibility of generating replication-competent virus by recombination with other viruses, the risk of insertional mutagenesis, and pre-existing and induced immune responses to viral proteins. However, plasmid DNA is significantly less efficient at carrying therapeutic genes into target cells than are recombinant viral vectors, such as those based on adenovirus, retrovirus and adeno-associated virus. Thus, provided the efficiency of delivery in vivo can be improved substantially, plasmid DNA is inherently one of the safer vectors for clinical application. The method of administration will also be very important for successful clinical application. For diseases like Duchenne muscular dystrophy (DMD), where all the skeletal muscles are affected to a greater or lesser extent, local intramuscular injections will not be of any real therapeutic value. Regardless of the vector used, intramuscular delivery is limited to about one centimeter surrounding the injection site. Thus, treatment of a large muscle group such as the quadriceps would require an unacceptably large number of injections. In addition, treatment of any individual muscle is unlikely to provide useful function for the patient, as many muscles work in antagonistic pairs. In 1990, Jon Wolff and colleagues demonstrated that skeletal muscle could take up and express naked plasmid DNA following intramuscular injection in the mouse [1Wolff J.A. et al.Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Crossref PubMed Scopus (3182) Google Scholar]. Subsequently, it was demonstrated that other tissues had similar properties, although expression was more sustained in skeletal and cardiac muscle. Indeed, expression of a luciferase reporter could be detected for nineteen months in mouse muscle [2Wolff J.A. et al.Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle.Hum. Mol. Genet. 1992; 1: 363-369Crossref PubMed Scopus (670) Google Scholar]. This long-term expression is not due to integration of the DNA, but is likely a reflection of the stability of postmitotic myonuclei in normal muscle. Thus, there is less need to use integrating vectors such as retroviruses compared to other tissues such as liver. However, the efficiency of naked plasmid gene transfer is very low following simple intramuscular injection. Fortunately, developments over the last several years have dramatically improved the efficiency of plasmid-based gene delivery. These developments are all physical, providing a motive force to drive the plasmid into close association with the muscle fiber membrane and/or transiently increasing membrane permeability. These methods include in vivo electroporation and hydrodynamic (pressure-mediated) vascular delivery. Electroporation dramatically increases the efficiency of plasmid gene transfer by several orders of magnitude, but the effect is limited to the region between the electrodes and thus multiple injections would still be required for each muscle. In contrast, using a combination of large volumes and rapid injection into the iliac artery, Jon Wolff's group demonstrated delivery to multiple muscles of the rat hindlimb at transfection efficiencies exceeding that found locally following intramuscular injection [3Budker V. et al.The efficient expression of intravascularly delivered DNA in rat muscle.Gene Ther. 1998; 5: 272-276Crossref PubMed Scopus (151) Google Scholar]. This work was later extended to high pressure delivery to the limbs via the brachial and femoral arteries in primates [4Zhang G. et al.Efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates.Hum. Gene Ther. 2001; 12: 427-438Crossref PubMed Scopus (143) Google Scholar] and dogs. At the time, it was thought that the use of the arterial system was important, because the elastic and relatively nondistensible nature of the arteries ensured transmission of the solution volume at the level of the arterioles and capillaries. In contrast, it was predicted that delivery via the venous supply would not work because the large veins would merely expand to accommodate the increased volume and thereby fail to increase the pressure significantly within the capillaries. In addition, veins possess valves to prevent the pooling of blood, which might act to block the distal flow of fluid from proximal injections. At about the same time that arterial hydrodynamic delivery to muscle was described, hydrodynamic delivery to the liver via the tail vein in mice was simultaneously published by John Wolff's and Dexi Liu's groups [5Liu F. Song Y. Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.Gene Ther. 1999; 6: 1258-1266Crossref PubMed Scopus (1472) Google Scholar, 6Zhang G. Budker V. Wolff J.A. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.Hum Gene Ther. 1999; 10: 1735-1737Crossref PubMed Scopus (816) Google Scholar]. This method of delivery employed rapid injection of large volumes equivalent to or greater than the blood volume of the mouse. Amazingly, this doubling of the blood volume was remarkably well tolerated by the mice and this technique has since been adopted by a number of groups examining gene expression in the liver. Leaf Huang's group has recently published a variant on tail vein delivery that shows that it is possible to deliver plasmids to multiple skeletal muscles of mice via both the tail artery and vein provided that the aorta and vena cava are temporarily clamped below the level of the kidneys [8Hagstrom J.E. et al.A facile non-viral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs.Mol. Ther. 2004; (this issue.)PubMed Google Scholar]. Arterial delivery resulted in ten-fold higher gene expression compared to intravenous delivery. Large volumes were again used—equivalent to the blood volume of the mouse. Although the above studies [3Budker V. et al.The efficient expression of intravascularly delivered DNA in rat muscle.Gene Ther. 1998; 5: 272-276Crossref PubMed Scopus (151) Google Scholar, 4Zhang G. et al.Efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates.Hum. Gene Ther. 2001; 12: 427-438Crossref PubMed Scopus (143) Google Scholar, 7Liang K.W. et al.Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA.Gene Ther. 2004; 11: 901-908Crossref PubMed Scopus (69) Google Scholar] demonstrated the potential for vascular delivery to multiple muscles, it was clear that delivery would probably need to be repeated a number of times to achieve a clinically useful therapy for limb muscles of patients with DMD. Delivery via the arterial supply is likely to require a minor degree of surgery to expose the artery and raises concerns about damage to the artery itself, thus making repeated administration by this route impractical for routine clinical application. The use of the very large volumes relative to body mass—although tolerated by mice—are very unlikely to be tolerated by humans, and are thus unlikely to be acceptable in a clinical setting. The paper by Hagstrom and colleagues in this issue of Molecular Therapy, the result of collaboration between Jon Wolff's group and Mirus Corporation, is an important step towards clinical realization of the hydrodynamic delivery of plasmid DNA to skeletal muscle. The paper reports studies of intravenous delivery of a range of plasmids to the temporarily isolated limbs of mice, rats, dogs, and monkeys. They show that the intravenous delivery does not require extremely large volumes or rapid injection, possibly as the post-capillary venules are reported to be the most permeable part of the microvasculature in muscle. In addition, the efficiency of delivery was equivalent to or higher than that observed by the arterial route, which used more than three times the volume and twice the injection speed of the venous administrations. More importantly, the intravenous delivery of plasmid was also very effective in dogs and rhesus monkeys, and there was no sign of a decreasing level of expression with increasing body size. Effective repeat administration was also demonstrated in the rat, with an increased proportion of the muscle fibers showing expression after three administrations compared to that seen with a single intravenous injection. The same delivery method also works for other nucleic acids, as shown by delivery of siRNA. The team reported no adverse effects following the intravenous hydrodynamic procedure, except for a small increase in serum levels of muscle creatine kinase in the monkeys, indicating mild muscle damage. Isolation of the limb was achieved using a tourniquet or blood pressure cuff, and the vein for injection was cannulated percutaneously. Thus, the protocol did not require any surgery in the larger species. The intravenous approach, while promising for the development of gene therapies for DMD and the related muscular dystrophies, still requires further work before transfer to a clinical setting. The efficiency of dystrophin delivery in the dystrophic mdx4cv mouse (3-15%) was too low to predict a beneficial clinical effect from a single administration, and further work will be required to improve the efficiency of dystrophin delivery. Increasing the quantity of DNA delivered, using functional mini- and microdystrophin constructs and multiple injections may improve efficiencies to the point where a clinical benefit could be measured. However, it might not prove possible to reach very high efficiencies if the extravasation of fluid containing the plasmid is solely at the postcapillary venules. While all muscle fibers are intimately in contact with capillaries along the whole length of each fiber, it is unlikely that all muscle fibers would have such a close relationship with postcapillary venules. This possible anatomical limitation remains to be explored. Although additional work is required, the paper by Hagstrom and colleagues is the first clear demonstration of a clinically applicable protocol for the nonviral delivery of genetic material to multiple muscles groups. It opens the floodgates for a whole range of possible genetic manipulations of skeletal muscle ranging from basic research to clinical application, including the genetic manipulation of fiber phenotype, the use of a substantial muscle mass as a platform for local and/or systemic expression of a range of therapeutic proteins as well as the direct treatment of muscle diseases.