RETRACTED: Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA

Abstract

We explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine phenylalanine hydroxylase cDNA in the livers of phenylketonuric (PKU) mice. The phiBT1 phage integrase is an enzyme that catalyses the efficient recombination between unique sequences in the phage and bacterial genomes, leading to the site-specific integration of the former into the latter in a unidirectional manner. Here we showed that this phage integrase functions efficiently in mouse cells, and several naturally occurring pseudo-attP sites located in the intergenic regions of the mouse genome have been identified and molecularly characterized. We further demonstrated that the addition of nuclear localization signal sequences to the C terminus of the phage integrase enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system, we delivered mouse phenylalanine hydroxylase cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that the severity of the hyperphenylalaninemic phenotype in the treated mice decreased significantly. After three applications, serum phenylalanine levels in all treated PKU mice were reduced to the normal range and remained stable thereafter. Their fur color also changed from gray to black, indicating the reconstitution of melanin biosynthesis as a result of available tyrosine derived from reconstituted phenylalanine hydroxylation in the liver. Thus, the phiBT1 bacteriophage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in DNA-mediated gene therapy for a multitude of genetic disorders.

Intracellular Gene Transfer in Rats by Tail Vein Injection of Plasmid DNA

“…It was demonstrated in mice that both macromolecules such as DNA, RNA, and proteins and small molecules can be delivered to hepatocytes through a simple injection into the tail vein (6,7). In gene therapy studies, hydrodynamic delivery of plasmid DNA has generated gene product at the therapeutic level and resulted in complete cure of diseases (8)(9)(10)(11). By combining the technique of image-guided catheter insertion with a computer-controlled injection device (12), we have demonstrated successful gene delivery to cells in the liver (13) and skeletal muscle (14) in pigs, suggesting that site-specific gene delivery can be achieved in large animals and it is feasible to apply hydrodynamic gene delivery to treatment of human diseases.…”

Identification of pharmacological chaperones as potential therapeutic agents to treat phenylketonuria

“…Both these supplementations may allow less (but still) restrictive l-Phe diets (11). Two alternative approaches are currently being investigated to treat PKU, both envisioning complete substitution of the classic low-l-Phe diet, i.e., enzyme replacement therapy using formulations of PEG-modified phenylalanine ammonia lyase (12) and gene therapy (13,14). Both approaches are still in their experimental infancy.…”

Structural impact of hydrodynamic injection on mouse liver

“…In the past few years, the method of hydrodynamic delivery 1,2 has provided a new means for delivery of nucleic acids to hepatocytes in rodents. [3][4][5] Since its establishment, this technique has been widely used for function analysis of coding [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] and non-coding sequences, [23][24][25][26][27][28][29] and for assessment of therapeutic potential of selected DNA and RNA sequences in animal models. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][30][31][32][33] In addition, hydrodynamic delivery of siRNA, short hairpin RNA (shRNA) or plasmid constructs encoding an shRNA has been e...…”