In the rat liver, Adenoviral gene transfer efficiency is comparable to AAV

Abstract

Adenoviral (AdV) and Adenovirus-associated viral (AAV) vectors both are used for in vivo gene therapy of inherited liver disorders, such as Crigler-Najjar syndrome type 1. In a relevant animal model, the Gunn rat, both vectors efficiently correct the severe hyperbilirubinemia characteristic of this liver disorder. Although the clinical use of AAV is more advanced, as demonstrated by the successful phase 1 trial in hemophilia B patients, because of its large cloning capacity AdV remains an attractive option. A direct comparison of the efficacy of these two vectors in the liver in a relevant disease model has not been reported. Aim of this study was to compare the efficiency of clinically applicable doses of both vectors in the Gunn rat. AdV or scAAV (self-complimentary AAV) ferrying identical liver-specific expression cassettes of the therapeutic gene, UGT1A1, were injected into the tail vein. As the titration methods of these two vectors are very different, a comparison based on vector titers is not valid. Therefore, their efficacy was compared by determining the amount of vector genomes delivered to the liver required for therapeutic correction of serum bilirubin. Like AAV, the liver-specific first-generation AdV also provided sustained correction in this relevant disease model. UGT1A1 mRNA expression provided per genome was comparable for both vectors. Flanking the expression cassette in AdV with AAV-ITRs (inverted terminal repeats), increased UGT1A1 mRNA expression eightfold which resulted in a significant improvement of efficacy. Compared with AAV, less AdV genomes were needed for complete correction of hyperbilirubinemia.

Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice

“…Genomic DNA was isolated as previously described 37 from at least 8 random pieces from 4 different lobes to minimize sampling error. hABCB4 and GFP copy number were determined by qPCR in a LightCycler480 (Roche Diagnostics USA) using the SensiFAST TM SYBR No-ROX kit (GC Biotech, Waddinxveen, the Netherlands) and the primers mentioned in Table S1.…”

“…Results were processed and analyzed using LinRegPCR software and normalized to the geometric mean of 36b4 and Gapdh. To determine the expression of both the hABCB4 and GFP genes, total liver RNA was isolated as previously described using Trizol, 37 according to the same method to minimize sampling error. Two micrograms of RNA were then reverse transcribed to cDNA using the RevertAid cDNA Synthesis Kit (Thermo Scientific) with random hexamers and oligo dT primers.…”

Gene Replacement Therapy for Genetic Hepatocellular Jaundice

“…Multiple viral vectors showed promising results in vivo [96][97][98], of which the AAV vector seems to have most potential in CN [99]. Administration of AAV containing a liver-specific promoter and the human UGT1A1 gene resulted in a lifelong correction of serum bilirubin in the Gunn rat [100,101].…”

Liver‐targeted gene therapy: Approaches and challenges

“…AAV2 and AAV8 are the most commonly used vectors for gene therapy because of their higher rates of transduction efficiency in comparison with AAV5, which transduces poorly in the liver, as shown recently in rat models of CN1 and hyperbilirubinema . Moreover, the gene transfer efficiency of AAV vectors is comparable to that of adenoviral (Ad) vectors, at least in the rat liver . AAV is a small [4.7 kilobase (kb)], single‐stranded DNA parvovirus that preferentially integrates into the q13.4‐ter arm of human chromosome 19 .…”

“…29 Moreover, the gene transfer efficiency of AAV vectors is comparable to that of adenoviral (Ad) vectors, at least in the rat liver. 30 AAV is a small [4.7 kilobase (kb)], single-stranded DNA parvovirus that preferentially integrates into the q13.4-ter arm of human chromosome 19. 31 Recombinant adenoassociated virus (rAAV) vectors can be generated by the cotransfection of 2 plasmids, one containing the transgene flanked by inverted terminal repeats (ITRs) and the other encoding Rep, Cap, and Ad proteins, into packaging 293 cells ( Fig.…”