Tuesday 18 October 2011
Something closer to the clinic is the zinc-finger nuclease, an enzyme created by the fusion of zinc finger DNA-binding domains with an endonuclease’s cleavage domain.
Such nucleases form the basis for Sangamo’s HIV treatment, SB-728-T. It consists of removing T cells and treating them with the nucleases to introduce a deletion in CCR5, creating the same sort of HIV resistance as seen in the patient who was cured through marrow transplants.
Sangamo presented positive Phase I results in september 2011 and is working on a similar method to treat stem cells.
Zinc-finger nucleases (ZFN) have room for improvement, though; use of other modular DNA-binding proteins called TAL effectors suggests that TAL effector nucleases (TALEN) might be even easier to predict and engineer.
In any case, modular nucleases offer more precision in gene therapy than has previously been possible, reducing the risk of side effects (such as leukemia) and may lead to further applications and treatments.
Gene therapy in situ is still considerably more difficult, so it is not possible to expect a ZFN or TALEN to repair BRCA genes—yet.
Engineered ZFPs can 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 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.
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.
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 disruption of the original DNA sequence and results in the generation of a shortened or non-functional protein (gene disruption).
ZFN-mediated gene modification may be used to disrupt a gene that is involved in disease pathology such as disruption 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 resulting in ZFN-mediated gene correction.
ZFN-mediated gene correction enables 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 anemia 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 specifically into a pre-determined location in the genome eliminates the 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.
Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines. Steentoft C, Vakhrushev SY, Vester-Christensen MB, Schjoldager KT, Kong Y, Bennett EP, Mandel U, Wandall H, Levery SB, Clausen H. Nat Methods. 2011 Oct 9;8(11):977-82. PMID: 21983924