Intrabiliary infusion of naked DNA vectors targets periportal hepatocytes in mice.

Hydrodynamic tail vein injection (HTV) is the "gold standard" for delivering naked DNA vectors to mouse liver, thereby transfecting predominately perivenous hepatocytes. While HTV corrects metabolic liver defects such as phenylketonuria or cystathionine ß-synthase deficiency, correction of spf ash mice with ornithine transcarbamylase (OTC) deficiency was not possible despite overexpression in the liver, as the OTC enzyme is primarily expressed in periportal hepatocytes. To target periportal hepatocytes, we established hydrodynamic retrograde intrabiliary injection (HRII) in mice and optimized minicircle (MC) vector delivery using luciferase as a marker gene. HRII resulted in a transfection efficiency below 1%, 100-fold lower than HTV. While HRII induced minimal liver toxicity compared with HTV, overexpression of luciferase by both methods, but not of a natural liver-specific enzyme, elicited an immune response that led to the elimination of luciferase expression. Further testing of MC vectors delivered via HRII in spf ash mice did not result in sufficient therapeutic efficacy and needs further optimization and/or selection of the corrected cells. This study reveals that luciferase expression is toxic for the liver. Furthermore, physical delivery of MC vectors via the bile duct has the potential to treat defects restricted to periportal hepatocytes, which opens new doors for non-viral liver-directed gene therapy.

Comprehensive characterization of ureagenesis in the spfash mouse, a model of human ornithine transcarbamylase deficiency, reveals age-dependency of ammonia detoxification.

The most common ureagenesis defect is X-linked ornithine transcarbamylase (OTC) deficiency which is a main target for novel therapeutic interventions. The spf ash mouse model carries a variant (c.386G>A, p.Arg129His) that is also found in patients. Male spf ash mice have a mild biochemical phenotype with low OTC activity (5%-10% of wild-type), resulting in elevated urinary orotic acid but no hyperammonemia. We recently established a dried blood spot method for in vivo quantification of ureagenesis by Gas chromatography-mass spectrometry (GC-MS) using stable isotopes. Here, we applied this assay to wild-type and spf ash mice to assess ureagenesis at different ages. Unexpectedly, we found an age-dependency with a higher capacity for ammonia detoxification in young mice after weaning. A parallel pattern was observed for carbamoylphosphate synthetase 1 and OTC enzyme expression and activities, which may act as pacemaker of this ammonia detoxification pathway. Moreover, high ureagenesis in younger mice was accompanied by elevated periportal expression of hepatic glutamine synthetase, another main enzyme required for ammonia detoxification. These observations led us to perform a more extensive analysis of the spf ash mouse in comparison to the wild-type, including characterization of the corresponding metabolites, enzyme activities in the liver and plasma and the gut microbiota. In conclusion, the comprehensive enzymatic and metabolic analysis of ureagenesis performed in the presented depth was only possible in animals. Our findings suggest such analyses being essential when using the mouse as a model and revealed age-dependent activity of ammonia detoxification.

A simple dried blood spot-method for in vivo measurement of ureagenesis by gas chromatography-mass spectrometry using stable isotopes.

BACKGROUND: Clinical management of inherited or acquired hyperammonemia depends mainly on the plasma ammonia level which is not a reliable indicator of urea cycle function as its concentrations largely fluctuate. The gold standard to assess ureagenesis in vivo is the use of stable isotopes. METHODS: Here we developed and validated a simplified in vivo method with [15N]ammonium chloride ([15N]H4Cl) as a tracer. Non-labeled and [15N]urea were quantified by GC-MS after extraction and silylation. RESULTS: Different matrices were evaluated for suitability of analysis. Ureagenesis was assessed in ornithine transcarbamylase (OTC)-deficient spfash mice with compromised urea cycle function during fasted and non-fasted feeding states, and after rAAV2/8-vector delivery expressing the murine OTC-cDNA in liver. Blood (5µL) was collected through tail vein puncture before and after [15N]H4Cl intraperitoneal injections over a two hour period. The tested matrices, blood, plasma and dried blood spots, can be used to quantify ureagenesis. Upon [15N]H4Cl challenge, urea production in spfash mice was reduced compared to wild-type and normalized following rAAV2/8-mediated gene therapeutic correction. The most significant difference in ureagenesis was at 30min after injection in untreated spfash mice under fasting conditions (19% of wild-type). Five consecutive injections over a period of five weeks had no effect on body weight or ureagenesis. CONCLUSION: This method is simple, robust and with no apparent risk, offering a sensitive, minimal-invasive, and fast measurement of ureagenesis capacity using dried blood spots. The stable isotope-based quantification of ureagenesis can be applied for the efficacy-testing of novel molecular therapies.

Hepatocyte Transfection in Small Pigs After Weaning by Hydrodynamic Intraportal Injection of Naked DNA/Minicircle Vectors.

Liver is an attractive organ for gene delivery in order to correct various genetic (metabolic) diseases. Hydrodynamic vein injection of naked DNA/minicircles devoid of viral or plasmid backbones was demonstrated in, for example, murine phenylketonuria to allow sustained therapeutic transduction of hepatocytes. Here we show successful hepatocyte transfusion in domestic small pigs immediately after weaning upon portal vein catheterization and hydrodynamic injection of naked DNA/minicircle vectors expressing the luciferase gene from the CMV or a liver-specific promoter. First, we established a surgical method allowing hydrodynamic portal vein pressurization up to 120 mmHg and infusion of naked DNA in pigs (n = 5) with long-term survival. No acute adverse effects such as changes in liver transaminases or signs of liver cell damage were observed. We then showed efficiency of stable hepatocyte transfection at 10 and 28 days in single experiments (n = 7) where we found that up to 60% of samples (45/75) were polymerase chain reaction (PCR)-positive for minicircle-DNA. Of these samples, 13% of the positive specimen (6/45) showed low but stable luciferase expression when driven by a liver-specific promoter, as well as appropriate copy numbers per diploid genome. In conclusion, we accomplished a safe procedure for stable transfection of liver cells upon hydrodynamic gene delivery using minicircle vectors in small pigs as a prerequisite to potentially treat infants with genetic liver diseases.