Genome-targeted gene therapy for PKU

18 October 2005

A novel and technique for gene therapy has reportedly cured the genetic disease phenylketonuria (PKU) in mice. PKU is an autosomal recessive disease caused by a defective gene for the enzyme phenylalanine hydroxylase (PAH), which is a crucial for metabolism of the amino acid phenylalanine in the liver. In many countries including the UK, neonatal screening programmes test for PKU; affected children who are diagnosed early are restricted to a phenylalanine-free diet, which prevents the severe, irreversible mental retardation otherwise caused by the condition.

Reporting in the advance online edition of the journal Proceedings of the National Academy of Sciences of the United States of America (PNAS), US scientists outline a procedure whereby a viral (bacteriophage) based system was used to direct integration of a functional mouse PAH gene to a specific region of the mouse genome [Chen L and Woo SLC (2005) PNAS Early Edition October 17, 10.1073/pnas.0503877102]. The PKU mouse model is a fairly accurate recreation of the human disorder, and has been used before for gene therapy studies by different groups.

In this new paper the authors, who have previously reported transient correction of the PKU mouse phenotype by adenovirus-mediated PAH gene delivery, use a phiBT1 phage system to deliver the gene. This system comprises the gene of interest in a DNA construct flanked by attachment sites, and delivered with another construct containing the gene for the viral integrase protein that catalyses insertion of DNA sequences between the attachment sites via recombination with complementary attachment sites in target DNA. It was tested in mouse cells in vitro, and reporter gene expression observed in 27% of samples.

The researchers next identified a total of eight sites in the mouse genome that could function as pseudo-attachment sites for the phiBT1 phage system, by integrating reporter genes to mouse cells and then sequencing the sites of integration. These sites showed up to 51% sequence similarity with the bacterial attachment sites. Groups of nude mice (a strain lacking competent immune systems) were injected with components of the phiBT1 system, including a reporter gene; integration occurred in positive but not negative controls, and more than 95% of gene integration took place at a single site, mpsP3, and a further 4% at two additional sites.

Having established that it was feasible to achieve site-specific integration of DNA sequences into mammalian genomes using the phage system, the researchers then looked at PKU mice to see whether delivery of the PAH gene in this manner could alleviate disease symptoms. Twelve PKU mice were injected in the tail vein with an integrating DNA construct containing the mouse PAH gene; six of these mice also received the integrase gene construct, whilst the other six received an integrase-negative construct (ie. one which would not support integration of the PAH gene to the mouse genome). Injections were repeated in weeks 4 and 12, and blood samples were taken biweekly from the mice and analysed for phenylalanine.

Serum phenylalanine levels dropped significantly in both groups of mice following injection, but in the integrase-negative control group it returned to pre-treatment level (1600

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