Sir Aaron Klug

From the Laboratory to the Clinic: Towards Therapeutic Applications of Engineered Zinc Finger Protein


Abstract

The switching on (or “expression”) of a gene is brought about by the binding of a set of specific proteins, called transcription factors, to the DNA in a special (regulatory) region of the gene. This is the first step in a pathway leading to the eventual product of the gene, a protein (or RNA).

The most frequently used class of transcription factors in the human genome are called zinc fingers, so-called because they contain short stretches of protein folded about a zinc ion and they grip or grasp the DNA double helix. Each finger recognizes the sequence of three successive DNA base pairs. The zinc finger motif was discovered in 1985 during our biochemical studies on a protein called TFIIIA which regulates the expression of certain genes in the frog, but it was subsequently found to be widespread throughout nature.

It has long been the goal of molecular biologists to design and construct DNA binding proteins for the specific control of gene expression. The natural zinc finger design is ideally suited for such purposes, discriminating between closely related DNA sequences both in vitro and in vivo. Whereas other DNA binding proteins generally make use of the 2-fold symmetry of the double helix, zinc fingers do not, and so can be linked linearly in tandem to recognize DNA sequences of different lengths, with high fidelity. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA. By fusing zinc finger peptides to repression or activation domains, genes can be selectively targeted, and switched off and on. More generally, using other effector domains, genes can be manipulated. Over the last twenty years my colleagues and I have been developing the technology for practical applications of gene regulation, whereby virtually any human gene can be specifically targeted.

Several recent applications of such engineered chimeric zinc finger proteins (ZFPs) are described, including the activation of vascular endothelial growth factor (VEGF) in a human cell line and in an animal model. Clinical trials have recently begun on using VEGF-activating ZFPs to treat human peripheral arterial obstructive disease, by stimulating vascular growth.

Also in progress are pre-clinical studies using chimeric zinc finger nucleases to target the defective genes in two single gene disorders, severe combined immunodeficiency disease (SCID) and sickle cell anaemia (SCA). The aim is to replace them in each case by a correct copy from an extrachromosomal DNA donor by means of the natural process of homologous recombination. Promising results have been reported.


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