Gene editing works by using genetic engineering technologies to replace or inactivate genes, or introduce new or modified genes to treat disease.
A cell and gene therapy product comprises a nuclease, DNA targeting elements, and a donor DNA template.
Nucleases play a key role in cell and gene therapy. Practitioners use them to introduce targeted DNA breaks, which then undergo repair.
DNA targeting elements dictate sequences for gene editing. These elements may include guide RNAs or zinc finger proteins, both of which bind to specific sequences. The donor DNA template is a genetic sequence which provides a target repair sequence.
There are three types of nuclease-dependent genetic engineering approaches which can introduce site-specific breaks in the DNA.
CRISPR-CAS9 is a powerful and popular genetic engineering tool. It deploys an RNA molecule (the “guide RNA” or “gRNA”) to direct the Cas9 enzyme to a specific genetic sequence. The enzyme cuts the DNA strand, initiating a repair process that modifies the DNA sequence at the break site. The CRISPR-CAS9 system has treated diseases such as sickle cell anemia and cystic fibrosis.
TALEN, or transcription activator-like effector nucleases, is another tool that relies on DNA-binding proteins called transcription activator-like effectors (“TALEs”). Practitioners fuse TALEs to the FokI endonuclease enzyme, which then cuts the DNA at a target site. Researchers have used TALENs to modify genes in HIV and cancer treatments.
Similarly, ZFNs, or zinc-finger nucleases, are DNA-binding proteins fused to the FokI endonuclease enzyme that can induce a site-specific break in the DNA. ZFNs have also been used in HIV and cancer treatments.
After the nucleases cut a DNA strand, the therapies rely on a genome’s repair to deliver the desired therapeutic effect. Many products rely on the innate DNA damage repair pathways to complete the modification.
Gene therapies most commonly utilize two DNA repair pathways: homology directed repair and non-homologous end-joining repair.
Homology directed repair (“HDR”) uses homologous DNA sequences to repair DNA breaks. HDR is most active during the S/G2 phase, and the donor DNA template can be supplied as a plasmid or using a viral vector, such as adeno-associated virus.
Non-homologous end-joining repair (“NHEJ”) repairs DNA breaks by joining two ends of cleaved DNA directly together without the need for a homologous template. NHEJ is considered error-prone compared to homologous recombination because it simply rejoins the ends without using a homologous template to ensure that the original sequence is restored. This means that NHEJ can lead to insertions or deletions at the site of repair, which can disrupt genes if the break occurs within a coding region.
The risks of gene editing, such as off-target editing where healthy DNA modifies to ill effect, undercut the promise of genetic engineering. This can include chromosomal rearrangements, insertions, deletions, and the potential for oncogenicity due to insertional mutagenesis. Given the novelty of the field, the long-term effects of on- and off-target editing are still unknown.
Gene therapy products are subject to extensive regulation by the FDA, which has issued recommendations for the development of new genetic products.
To minimize the risk of off-target editing, the FDA recommends thorough characterization of gene therapy products. This may include limiting the in vivo persistence of gene editing components, restricting the distribution of gene editing components to specific sites using tissue-specific promoters, and providing clear instructions on evaluating off-target activity. These instructions may also include the insertion/deletion/conversions, frequency, location, and biologic consequence (when available) of off-target editing, among other considerations.
The FDA also recommends evaluating the genomic integrity of gene therapy products. This includes looking for chromosomal rearrangements, insertions, and deletions, as well as the integration of exogenous DNA. Researchers should also evaluate the potential oncogenicity of insertional mutagenesis. Currently, the FDA has not approved any in vivo gene editing products.
How Does Gene Editing Work?
