ZFP Nucleases (ZFNs)
Our engineered ZFPs can also be attached to the cleavage domain of a restriction endonuclease, an enzyme that cuts DNA, thereby creating a ZFN. The ZFN is able to recognize its intended gene target through its engineered ZFP DNA-binding domain.
When a pair of ZFNs is bound to the DNA target site, in the correct orientation and spacing, the DNA sequence is cut between the ZFP binding sites. DNA binding by both ZFNs is necessary for cleavage.
Figure 1: Schematic to demonstrate the position and orientation of the binding of a ZFN dimer to a specific DNA sequence to enable generation of a double stranded break in the DNA.
This break in the DNA triggers a natural process of DNA repair in the cell. The repair process can be harnessed to achieve one of several outcomes that may be therapeutically useful (see below).
NHEJ = Non-Homologous End Joining. HR = Homologous Recombination.
Figure 2: Schematic to demonstrate potential outcomes of a double-strand break in DNA generated by a pair of ZFNs, gene knockout, gene correction or DNA insertion.
If cells are simply treated with ZFNs alone the repair process frequently results in the rejoining of the two broken ends of the DNA. As a consequence there is oftern a loss of a small amount of genetic material that results in the disruption of the original DNA sequence and generates a shortened or non-functional protein (gene knockout). ZFN-mediated genome editing may be used to disrupt a gene that is involved in disease pathology such as the knockout of the CCR5 gene to treat HIV infection.
In contrast, if cells are treated with ZFNs in the presence of an additional “donor” DNA sequence that encodes the correct gene sequence, the cell can use the donor as a template to correct the cell’s gene as it repairs the break. This results in ZFN-mediated gene correction, enabling a corrected gene to be expressed in its natural chromosomal context and may provide a novel approach for the precise repair of DNA sequence mutations responsible for monogenic diseases such as hemophilia, sickle cell disease or X-linked severe combined immunodeficiency (X-linked SCID).
In addition, by making the donor sequence a gene-sized segment of DNA, a new copy of a gene can also be added into the genome at a specific location. The ability to place a gene-sized segment of DNA into a pre-determined location in the genome, with singular specificity, eliminates insertional mutagenesis concerns associated with traditional gene replacement approaches and broadens the range of mutations of a gene that can be corrected in a single step. We are pursing this strategy with our In Vivo Protein Replacement Platform (IVPRP) which uses the Albumin gene in the liver as a "safe harbor" site for therapeutic targeted gene addition. We are using the IVPRP in our Shire-partnered programs in hemophilia A and B and in our proprietary programs in lysosomal storage disorders (LSDs).