Gene Editing: A Ray of Hope for Patients with Genetic Diseases

In spite of so much progress being done in the field of medical science, researchers and doctors are still searching for a cure for genetic diseases. Recent developments in the field of gene editing have helped to improve our ability to make changes to the genomes. Nucleases are the enzymes having the ability to break the bonds between the DNA- and RNA-forming nucleotides. Targeted nucleases have provided the scientists with an opportunity to change almost all kinds of genomic sequences.

Gene Editing Technique:

Gene or genome editing is the insertion, deletion, or replacement of DNA at a specific location in the genome of the cell or an organism. This technique is realized in labs using engineered nucleases. These engineered nucleases are also called molecular scissors.

The following nucleases are engineered to edit different genomes and can possibly be used clinically to correct or introduce genetic mutations for treating diseases that are not responding to traditional therapies:

  • Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)
  • Transcription Activator-Like Effector Nucleases (TALENs)
  • Zinc-Finger Nucleases (ZFNs)

The viral systems such as recombinant adeno-associated virus (rAAV) and transposons are also used.

Wide varieties of useful results in fields such as synthetic biology, disease modelling human gene, and drug discovery can be mainly attributed to the ability of these technologies to induce targeted DNA double-strand breaks (DSBs). These breaks stimulate activation of cellular DNA repair pathways making it possible to introduce site-specific gene modifications. The precision of DSB is the key to gene editing and these molecular scissors need to precisely remove the damaged sequence for the therapy to be successful.

Experiments demonstrated that a ZFN could indeed stimulate recombination at its recognition site after DSB formation in a fruit fly or mammalian genome leading to intensive development of ZFNs. The major challenge faced with ZFNs with respect to gene editing is their low specificity leading to off-target mutations, thereby reducing their potential as a therapeutic option for genetic diseases.

TALENs proved to have better specificity and lesser toxicity as compared to ZFNs because of their affinity toward target DNA. However, their large size had an adverse effect on efficient delivery through a single adeno-associated virus (AAV).

CRISPER-Cas9 system is the latest entrant into gene editing and has emerged as the most flexible and user friendly tool for gene editing. It does not involve any protein engineering and involves a simpler target DNA matching technique. However, this system is also not perfect with respect to specificity. Substantial effort has been dedicated to the improvement of the specificity of this system.

Applications of Gene Editing:

Engineering cell lines and organisms: Prior to the advent of gene editing tools discussed above, genetic modification of mammalian cell lining was a cumbersome and costly affair. However, with these recent techniques, cell lines with any genomic modifications can be created timely and in a cost-effective manner.

Synthetic biology and genome-scale engineering: Gene editing tools help in producing modified bacteria and yeast strains for synthetic biology including metabolic pathway engineering.

Therapeutic genome editing: Gene editing has a tremendous potential and generates maximum interest for its ability to cure genetic diseases. The most successful example of its therapeutic use is ZFN-mediated disruption of HIV co-receptor CCR5 leading to HIV resistance in both CD4+ T cells and CD34+ hematopoietic stem cells. These targeted nucleases can be combined with viral vectors to mediate genome editing in situ.

Recent in vivo gene editing has shown encouraging results in dystrophin expression in the muscles of mouse model of Duchenne muscular dystrophy.

Delivery of an AAV vector encoding a ZFN pair intended to target a defective copy of the factor IX gene, along with its repair template, was able to efficiently cause gene correction in mouse liver, leading to increased production of factor IX protein.

There have been recent reports of successful therapeutic gene editing in a mouse model of human hereditary tyrosinaemia with CRISPER-Cas9 system.

Now there is sufficient proof that gene correction in animal models can also be achieved using multiple AAV particles.

Although these early success stories point toward a very promising therapeutic approach for treating genetic diseases, many challenges still remain before the full potential of genome editing can be realized.

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