Up to 4,000 human diseases (including Duchenne Muscular Dystrophy, cystic fibrosis, Parkinson’s and Alzheimer’s diseases, as well as some types of cancer) originate from errors and changes in an individual’s DNA sequence. By repairing or replacing these defective sequences, instead of only treating symptoms, the rising field of genomic medicine aims at treating these pathological conditions at their source, stably replacing the need for drugs or surgery.
Gene therapy is a revolutionary treatment in which new genetic material is introduced in a person’s cells to treat or prevent rare genetic disorders. 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 or introducing a new gene into the body to help fight a disease.
Genome editing allows to introduce specific permanent changes to the DNA of a cell. This addition, removal or modification of specific DNA sequences is obtained using specific enzymes called “engineered nucleases”, which act as molecular scissors able to cut the DNA at specific sites. By editing the genome, the characteristics of a cell or an organism can be changed.
With cell therapy, new, healthy cells are injected or transplanted into a patient to replace the diseased or missing ones and restore long-term tissue function. These therapeutic cells are usually obtained from stem cells, derived directly from the patient (autologous) or from a healthy donor (allogeneic). This includes chimeric antigen receptor (CAR) T cells, genetically engeneering T-cells capable of specifically recognize and destroy cancer cells.
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 at desired genomic locations, that are consequently repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted modifications.
HOMOLOGY / INTEGRATION BASED STRATEGIES
These methods target specific genes to certain sites within a genome, relying on homologous recombination (HR) to achieve integration. They can be used to delete or insert a specific sequence, an entire gene or to modify individual base pairs (introducing point mutations).
By creating DNA constructs that contain a template that matches the targeted genome sequence, the HR processes within the cell will insert the desired modifications at a specific location.
NON INTEGRATION BASED STRATEGIES
The genetic material of non-integrating vectors remains in the cytoplasm in an episomal form. Both viral and non-viral vectors can be used to deliver the episomal transgene into cells, which can provide stable transgene expression in quiescent cells and transient or stable expression in proliferating cells.
Recombinant Adeno-Associated Viruses (rAAV) are the most commonly used non-integrative vectors for transgene delivery due to the high titer, mild immune response, ability to infect a broad range of cells, and overall safety. Since rAAV do not integrate into the host cell genome, the risk of insertional mutagenesis is low, minimising the side effects of gene therapies.
Synthetic oligonucleotides are used in gene therapy to inactivate the genes involved in the disease process. This can be achieved by using antisense oligonucleotides specific to the target gene to disrupt the transcription and/or the translation of the faulty gene. An additional strategy uses double stranded oligonucleotides as a decoy for the transcription factors required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, thus reducing the target gene expression.