“…In parallel, we have recently reported that HRII of MC vectors applies to larger animal models such as domestic small pigs, where we found a stable hepatocyte transfection and no acute adverse complications. 47 A further improvement toward mitigating the invasiveness of the procedure for infusion via the bile system could be endoscopic retrograde cholangiopancreatography (ERCP) that was reported for delivering plasmid DNA to adult pigs. 44 Firefly luciferase is a widely used imaging reporter transgene that allows visualization of luciferase expression in real-time bioluminescence imaging of mice.…”
Section: Discussionmentioning
confidence: 99%
“…37 For these reasons, non-viral gene delivery of naked DNA vectors or vector DNA complexes with liposomes or nanoparticles through the biliary tract has been extensively investigated in rodent models, [38][39][40][41][42][43] dogs, 38 and pigs. [44][45][46][47] This application has also been applied to the administration of viral vectors for liver-targeted delivery in rodents and large animals. [48][49][50][51][52][53] However, non-viral gene delivery via intrabiliary injection has so far not been shown to treat any liver-defected murine models.…”
“…In parallel, we have recently reported that HRII of MC vectors applies to larger animal models such as domestic small pigs, where we found a stable hepatocyte transfection and no acute adverse complications. 47 A further improvement toward mitigating the invasiveness of the procedure for infusion via the bile system could be endoscopic retrograde cholangiopancreatography (ERCP) that was reported for delivering plasmid DNA to adult pigs. 44 Firefly luciferase is a widely used imaging reporter transgene that allows visualization of luciferase expression in real-time bioluminescence imaging of mice.…”
Section: Discussionmentioning
confidence: 99%
“…37 For these reasons, non-viral gene delivery of naked DNA vectors or vector DNA complexes with liposomes or nanoparticles through the biliary tract has been extensively investigated in rodent models, [38][39][40][41][42][43] dogs, 38 and pigs. [44][45][46][47] This application has also been applied to the administration of viral vectors for liver-targeted delivery in rodents and large animals. [48][49][50][51][52][53] However, non-viral gene delivery via intrabiliary injection has so far not been shown to treat any liver-defected murine models.…”
“…We used a previously described approach that is based on the split-intein system of Nostoc punctiform (Npu) to split ABEmax at the cysteine 574 of SpCas9 (the two rAAV vectors will be referred to as N-int-ABEmax and C-int-ABEmax; Figure 1D) 35,38 . In this approach coinfection of hepatocytes will lead to the expression of N-int-ABEmax and C-int-ABEmax under the hepatocyte-specific p3 promoter 29,32 , and protein trans-splicing will lead to the reconstitution of full-length and functional ABEmax (Figure 1E) 35,38 .…”
Section: Design and Validation Of Adenine Base Editing For The Knockd...mentioning
confidence: 99%
“…While mice tolerate rAAV vector infusion with high transduction efficacy, large animal models such as pigs and NHP exhibit severe toxicity upon systemic high-dose administration of rAAV, including systemic and local immune responses. Various methods for cell, viral vector or (naked-) DNA infusion directly to the liver were reported for pigs, including open surgery, endoscopic, and/or ultra-sound guided procedures (see: Kamimura et al [28][29][30][31][32][33][34] ). These direct delivery methods facilitate direct injection of vectors into the liver, which can enable higher infection rates at lower vector doses.…”
One challenge for liver-directed gene therapy is sufficient vector delivery to the target tissue while minimizing loss of the applied vector dose to other tissues. Infusion via peripheral veins is the least invasive approach; however, it results in systemic diffusion and substantial vector dilution. Here, we describe a safe and minimally invasive method to deliver adeno-associated virus (AAV) vectors to the liver of small weaned pigs by ultrasound-guided percutaneous trans-hepatic portal vein injection. 4-week-old piglets were infused with ~2.5x1014 vector genomes comprising a dual-rAAV2/9 vector system with a split adenine base editor for in vivo inactivation of PCSK9 to reduce LDL-cholesterol levels. Animals had no signs of discomfort and tolerated the procedure well. However, despite 45% editing of the target site with the applied adenine base editor system in cultivated porcine cells, we only found low amounts of AAV vector genomes and neither detectable transgene-expression nor successful editing in the treated pig livers. We hypothesize that rapid proliferation of pig hepatocytes caused AAV vector dilution, leading to a loss of the vectors from the nucleus, and hence insufficient base editor protein expression for achieving detectable editing rates. Nonetheless, ultrasound-guided percutaneous transhepatic injection to the portal vein is well-tolerated in piglets and has potential for human (neonate) application.
“…Nevertheless, the method is clinically attractive because it could be performed through endoscopic retrograde cholangiopancreatography (ERCP), a method that has been in clinical practice for many years and was previously tested by the authors in a large-animal model. 6 Figure 1 Hepatocytes are arranged in liver lobules that receive oxygenated blood from the heart via the hepatic artery (red) and deoxygenated blood via the portal vein (blue). Bile drains from hepatocytes via the hepatic ducts (green).…”
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