Gene Therapy

Gene therapy is an experimental technique that uses genes to treat or prevent disease. This technique allow doctors to treat a disorder by inserting or correcting a gene into a patient’s cells instead of using drugs or surgery. Several approaches exist for gene therapy such as:

  • Compensating a mutated gene that causes disease with a healthy copy of the gene.
  • Correcting or inactivating a mutated gene that is functioning improperly.
  • Introducing a new gene into the body to help fight a disease.

Genome editing

Genome editing is a genetic engineering method in which DNA is inserted, deleted, modified or replaced at specific locations in the genome of a living organism.

The most common methods for such editing use 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 nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations (‘edits’).

Four families of engineered nucleases are used: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system. The last three techniques are prominent tools in the field of genome editing.

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Homology / Integration based strategies

The methods target endogenous genes to certain sites within a genome (called gene targeting) relying on homologous recombination (HR) to achieve integration. They can be used to delete or to insert a specific sequence, an entire gene or exons or modify individual base pairs (introducing point mutations).

By creating DNA constructs that contain a template that matches the targeted genome sequence, it is possible that the HR processes within the cell will insert the construct at the desired location.

Gene targeting requires the creation of a specific vector for each gene of interest. Viral vectors and non-viral vectors can be used to deliver the transgene into cells. Their great efficiency of gene targeting in human somatic cell-types makes Recombinant Adeno-Associated Viruses (rAAV) the most common vector used for transgenes delivery.

The frequency of gene targeting can be significantly enhanced through the use of engineered endonucleases.

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Non integration based strategies

Non-integrating vectors can provide stable transgene expression in quiescent cells and transient or stable expression in proliferating cells. Viral vectors and non-viral vectors can be used to deliver the episomal transgene into cells.

Recombinant Adeno-Associated Viruses (rAAV) the most common vector used for transgenes delivery due to its high titer, mild immune response, ability to infect a broad range of cells, and overall safety. AAV infects a broad range of cell types and infection is not dependent upon active cell division. One concern when using other viruses, such as retrovirus or lentivirus, is the random integration events that can disrupt gene function. Because AAV does not integrate into the host cell genome, the risk of insertional mutagenesis is low. This is why Non-integrating vectors are used to develop alternative strategies, to minimize the side effects of gene therapies.

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Oligonucleotides strategies

The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease process. There are several methods by which this is achieved. One strategy uses antisense specific to the target gene to disrupt the transcription of the faulty gene.

Another uses small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. A further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression.

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The critical importance of validation

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