Post written by Robert L. Kruse, MD, PhD, from the Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Viral-based approaches predominate the field of gene therapy for rare diseases but suffer many limitations including anti-vector immune responses and high cost of manufacturing. Our study focused on developing a novel nonviral method of gene therapy, leveraging endoscopic retrograde cholangiopancreatography (ERCP) to inject plasmid DNA through the biliary system into the liver. We hypothesized that the small volume of the biliary system would enable efficient DNA entry into liver cells when applying hydrodynamic fluid force and that protein expression could be increased through plasmid sequence optimization.

It was important to conduct this study because the application of gene therapy is limited today, being only tested in a small number of rare diseases in the liver that have yet to be approved by the FDA. While rare diseases offer a tremendous proof of principle for gene therapy, there are many more common liver indications, ranging from cancer, autoimmune, cirrhosis, and NAFLD/NASH that could be revolutionized by genetic medicine. Since current viral tools are insufficient, a nonviral approach for DNA expression in the liver that is at least 100 times cheaper than viral vectors would be transformative.

Our study found that biliary hydrodynamic injection could mediate plasmid DNA delivery into 32.7%-51.9% of liver cells in pigs, depending on the DNA dose employed. This was measured by immunohistochemical staining in pig liver for encoded human FIX protein, which is deficient in hemophilia B. Crucially, because we employed human-sized animal models and used clinical equipment, we envision ease in translating our approach into patients. Importantly, this is the first time that nonviral delivery efficiency has surpassed the efficiency of viral vector delivery in large animals, which should encourage more investigators to embrace this approach.

Looking ahead, this study opens the possibility of widely available gene therapy via ERCP, given that clinical-grade manufacturing of plasmid DNA is cheap and widely available. Next steps will focus on optimizing expression and dosing of plasmid DNA for specific disease indications, along with testing the strategy in currently available models of pig disease. In work funded by the NIH for hemophilia B, testing will continue in nonhuman primate models, which will allow for secretion of human Factor IX. Together, these studies should set the stage for potential clinical trials of the approach.

Figure 4. Immunohistochemistry shows efficient human factor IX (hFIX) expression in pigs after hydrodynamic delivery. A, Immunohistochemistry for hFIX in human liver tissue demonstrated homogenous, cytoplasmic expression in hepatocytes. Control tissue from a noninjected pig liver demonstrates only light background staining for porcine factor IX appreciated. Immunostaining in hFIX-injected pig no. 3 revealed abundant hFIX hepatocytes, with similar cytoplasmic staining to human hepatocytes with variable intensity (bar, 50 μm). B, An example of a liver section stained for hFIX at low magnification power is presented from pig no. 3, left medial lobe proximal section, demonstrating that immunostaining can be observed in every single lobule, with intensity highest in the center of the lobule (bar, 1 mm). A magnified image from the same liver biopsy section is also presented, showing intense staining bordering the central vein and radiating to the lobule borders (bar, 200 μm).

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