By Nermina Lamadema, Postdoctoral Research Associate at King's College London
Currently there is no effective treatment for many diseases caused by the gene mutations because there is no effective way to make precise alterations in human DNA. Gene therapy strategies to overcome these limitations and attempt to add/edit a working copy of the gene have faced many setbacks over the recent years. Recently however a several new approacheshave been attempted. One approach involves editing rather than replacing a faulty copy of the gene. This approach uses DNA binding proteins called Zinc Fingersthat can be engineered to target the specific genomic regions. Other approach involves using the Chimeric Antigen Receptors (CAR) of the immune cells to insert or make new genes.
Zinc fingers are essential protein components used by cells to switch the genes on and off. They consist of two loops of protein held together to form a “finger” which holds the atom of zinc.Classical zinc finger proteins Cys2His2(C2H2) were first discovered in the DNA transcription factor TFIIIA and are known to exist in 2% of all human naturally occurring transcription factors. These factors which are highly conserved across the species from yeast to humans are involved in gene expression control.
The zinc finger domain typically has two antiparallel beta strands and an alpha helix (ββα) structure of around 30 amino acids in size, stabilized by the presence of a zinc ion bound by two cysteine residues in the β sheets, and two histidine residues in the α helix.Alpha helix residues contain DNA interacting regions capable of contacting three base pairs of DNA so they are the component responsible for DNA recognition specificity.
The specificity of the zinc finger can be manipulated through the variation in the way the helix is presented. As a consequence, the zinc finger motif has been widely used as a template for the rational design and construction of DNA sequence specific proteins, due to its ability to bind to virtually any DNA sequence.
DNA strand in blue and Zinc Finger protein Interaction. Zinc Finger Helix makes contact with the specific bases on the DNA whilst beta strands help stabilize the binding interaction.
The strategy to design and assemble a zinc finger protein (ZFP) employs either combinatorial selection of ZFPs or modular assembly of zinc finger arrays. The combinatorial strategy approach uses phage display to select a protein that binds specific DNA regions being studied.
The rational design of zinc finger proteins involves the generation of modular units where each module of the three ZFP recognizes three base pairs of DNA 5’-GNN-3’, 5’-ANN-3’, 5’-CNN-3’ and 5’-TNN-3’ subunits. ZF proteins can be made more specific by module – linking and recombination to increase the array size e.g. create four or six ZFPs, capable of targeting more extended 12 base pairs (bp) and 18bp DNA contiguous sequences. For in vivo targeting studies this kind of set up should provide the highest level of specificity given that the probability of 18bp sequence occurring more than once in the genome is once in 68×109 bp of sequence.
Zinc Finger Proteins potential uses
Zinc finger proteins can be linked to a variety of different molecules such as gene repressor and activators to turn on or turn off the gene at the site recognized by the fingers.
Most advanced of all approaches is the use of zinc fingers for gene editing. Specific genomic sequences can be cut and pasted using zinc finger proteins fused with specific enzymes capable of cutting the DNA called nucleases.
Fok I nuclease fused with ZFP to target and cut the specific genomic region.
The cell is programmed to attempt to repair the break but this can sometimes lead to the disruption of the gene that has been targeted. AIDS virus for example uses a receptor CCR5 to gain an entry into the white blood cells and this receptor can be targeted and disabled using the zinc finger approach described above.
Zinc finger nucleases genome editing seems to have a promising future given its application in a range of experimental systems such as plant science in major crop species for so called trait stacking which involves physically linking several desired traits to ensure their co-segregation during the breeding processes.
The field of syntheticbiology whichinvolves engineering novel organisms with distinct functions has also benefited from the use of the Zinc Finger nuclease aided genome editing.
One exciting possible application of Zinc Finger gene targeting is in the possible treatment of X-SCID (X-linked Severe Combined Immunodeficiency) which arises as a consequence of a mutation in the IL2-RG gene. An attempt has been made to correct this mutated gene in X-SCID patients using the insertional mutagenesis by retroviral vectors which resulted in the development of leukaemia in treated individuals. This effect is something that could be overcome by replacing viral based vectors with the targeted Zinc Finger platform. However the exact off target effects of this platform will need to be very carefully evaluated before proceeding. Reports so far indicate no significant issues with toxicity or side effects. Currently there is a Phase 2 clinical trial (to evaluate the efficacy of the drug) being conducted on HIV patients using CCR5/ZFN fusion and Phase I study to evaluate safety of another HIV specific ZFP fusion designed to work in Stem Cells and the results are awaited eagerly.
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