zinc Finger Nucleases

[*Content* Introduction to Gene Therapy History of Gene Therapy Gene Editing DNA Double Stranded Break (DSB) Mechanisms and Site-specific Double Stranded Breaks zinc Finger Nucleases Application of Zinc Finger Nucleases Potential Side Effects References [‪*What is Gene Therapy* Gene therapy is an experimental treatment that involves introducing genetic material into a person’s cells to fight or prevent disease. Researchers are studying gene therapy for a number of diseases, such as (1) severe combined immuno-deficiencies (2) hemophilia (3) Parkinson's disease (4) cancer and (5) even HIV, through a number of different approaches. A gene can be delivered to a cell using a carrier known as a “vector” and the most common types of vectors used in gene therapy are viruses. The viruses used in gene therapy are altered to make them safe, although some risks still exist with gene therapy. While the technology is still in its infancy, it has been used with some success. It is a technique for correcting defective genes that are responsible for disease development. *Basic Process of Gene Therapy* Today, several approaches to gene therapy are being tested, including: Replacing a mutated gene that causes disease with a healthy copy of the gene Inactivating, or “knocking out,” a mutated gene that is functioning improperly Introducing a new gene into the body to help fight a disease Change the regulation of gene pairs In general, a gene cannot be directly inserted into a person’s cell. It must be delivered to the cell using a carrier, or vector. Vector systems can be divided into: (i) Viral (ii) Non-viral Viral Vectors *History of Gene Therapy* In the 1980s, Scientists began to look into gene therapy. They would insert human genes into a bacteria cell and then the bacteria cell would transcribe and translate the information into a protein. Then they would introduce the protein into human cells. The first gene therapy was performed on September 14th, 1990. Ashanti DeSilva was treated for Sever combined immuno-deficiency (SCID). Doctors removed her white blood cells and inserted the missing gene into the WBC, and then put them back into her blood stream. This strengthened her immune system but only worked for a few months. A vector delivered the therapeutic gene into a patient’s target cell which became infected with the viral vector; and the vector’s genetic material was inserted into the target cell Functional proteins were synthetized from the therapeutic gene causing the cell to return to a normal state at least temporarily. *Gene Editing* Gene Formatting Gene editing, or genome editing with engineered nucleases is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases, or "molecular scissors." These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('edits'). There are three families of engineered nucleases being used: ( _1) Zinc finger nucleases (ZFNs)_( _2) Transcription Activator-Like Effector-based Nucleases__ _(TALENs) (3) CRISPR-Cas9 system._ [06/04, 8:35 pm] ‪+234 813 872 7278‬: DNA double stranded break (DSB) repair mechanisms* To understand these concepts you need to understand the concept of DNA double stranded break (DSB) repair mechanisms. Two of the known DSB repair pathways that are essentially functional in all organisms are the non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ uses a variety of enzymes to directly join the DNA ends in a double-strand break while in HDR, a homologous sequence is utilized as a template for regeneration of missing DNA sequence at the break point. The natural properties of these pathways form the very basis of nucleases based genome editing. NHEJ is error-prone, and has been shown to cause mutations at the repair site, so if one is able to create a DSB at a desired gene in multiple samples, it is very likely that mutations will be generated at that site in some of the treatments because of errors created by the NHEJ infidelity. On the other hand, because HDR depends on a homologous sequence to repair DSBs, this can be exploited by inserting a desired sequence within a sequence that is homologous to the flanking sequences of a DSB. This when used as a template by HDR system, would lead to the creation of the desired change within the genomic region of interest. Despite the distinct mechanisms, the concept of the HDR based gene editing is in a way similar to that of homologous recombination based gene targeting. However, the rate of recombination is increased by at least three orders of magnitude when DSBs are created and HDR is at work thus making the HDR based recombination much more efficient and eliminating the need for stringent positive and negative selection steps. So based on these principles if one is able to create a DSB at a specific location within the genome, then the cell’s own repair systems will help in creating the desired mutations. *Site-specific double stranded breaks* Creation of a DSB in DNA is easy by using restriction enzymes. However, if genomic DNA is treated with a particular restriction endonuclease many DSBs will be created. This is a result of the fact that most restriction enzymes recognize a few base pairs on the DNA as their target and very likely that particular base pair combination will be found in many locations across the genome. To overcome this challenge and create site-specific DSB, three distinct classes of nucleases have been discovered and bioengineered to date. These are the Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALEN) and meganucleases. The concept behind ZFNs and TALEN technology is based on a non-specific DNA cutting enzyme, which can then be linked to specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors (TALEs). The key to this was to find an endonuclease whose DNA recognition site and cleaving site were separate from each other, a situation that is not common among restriction enzymes. Once this enzyme was found, its cleaving portion could be separated which would be very non-specific as it would have no recognition ability. This portion could then be linked to sequence recognizing peptides that could lead to very high specificity. Zinc Finger Nucleases* Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside Cas9 and TALEN proteins, ZFN is becoming a prominent tool in the field of genome editing. A zinc finger nuclease is a site-specific endonuclease designed to bind and cleave DNA at specific positions. There are two protein domains. The first domain is the DNA binding domain, which consists of eukaryotic transcription factors and contain the zinc finger; and the second domain is the nuclease domain, which consists of the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA. *DNA-binding domain* The DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 base-pairs. If the zinc finger domains are perfectly specific for their intended target site then even a pair of 3-finger ZFNs that recognize a total of 18 base-pairs can, in theory, target a single locus in a mammalian genome. The most straightforward method to generate new zinc-finger arrays is to combine smaller zinc-finger "modules" of known specificity. The most common modular assembly process involves combining three separate zinc fingers that can each recognize a 3 base pair DNA sequence to generate a 3-finger array that can recognize a 9 base pair target site. *DNA-cleavage domain* The non-specific cleavage domain from the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFNs. This cleavage domain must dimerize in order to cleave DNA and thus a pair of ZFNs are required to target non-palindromic DNA sites. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. In order to allow the two cleavage domains to dimerize and cleave DNA, the two individual ZFNs must bind opposite strands of DNA with their C-termini a certain distance apart. Gene Therapy Application of Zinc Finger Nucleases* Zinc finger nucleases are useful to manipulate the genomes of many plants and animals and are also used to create a new generation of genetic disease models called isogenic human disease models. Zinc finger nucleases have also been used in a clinical trial of CD4+ human T-cells with the CCR5 gene disrupted by zinc finger nucleases to be saved as a potential treatment for HIV/AIDS. Custom-designed ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc-finger proteins (ZFPs) offer a general way to deliver a site-specific DSB to the genome, and stimulate local homologous recombination by several orders of magnitude. This makes targeted gene correction or genome editing a viable option in human cells. Since ZFN-encoding plasmids could be used to transiently express ZFNs to target a DSB to a specific gene locus in human cells, they offer an excellent way for targeted delivery of the therapeutic genes to a pre-selected chromosomal site. The ZFN-encoding plasmid-based approach has the potential to circumvent all the problems associated with the viral delivery of therapeutic genes. The first therapeutic applications of ZFNs are likely to involve ex vivo therapy using a patients own stem cells. After editing the stem cell genome, the cells could be expanded in culture and reinserted into the patient to produce differentiated cells with corrected functions. The initial targets will likely include the causes of monogenic diseases such as the IL2Rγ gene and the b-globin gene for gene correction and CCR5 gene for mutagenesis and disablement. Potential Side Effects* If the zinc finger domains are not specific enough for their target site or they do not target a unique site within the genome of interest, off-target cleavage may occur. Such off-target cleavage may lead to the production of enough double-strand breaks to overwhelm the repair machinery and, as a consequence, yield chromosomal rearrangements and/or cell death. Off-target cleavage events may also promote random integration of donor DNA. As with many foreign proteins inserted into the human body, there is a risk of an immunological response against the therapeutic agent and the cells in which it is active. Since the protein will need to be expressed only transiently, however, the time over which a response HB develop is short In conclusion, about 4,000 human diseases are thought to be inherited. Scientists are making good progress figuring out where genes are located on chromosomes. Genetic diseases are caused by mutations, or incorrect sequences, in the normal form of the gene. Gene Therapy via Gene Editing can use overcome some of these deadly diseases such as Huntington’s Disease, Cystic fibrosis etc References: Dean J.A.C, Robert D.S and D.S and Bernd M., Pharmaceutical Biotechnology: Fundamentals and Applications, chapter 21....