Expanding the Genome Engineering Toolbox: How programmable integrases could change the future of genomic medicines

Monogenic diseases are health conditions that are caused by a mutation in a single gene. Cystic fibrosis, Duchenne muscular dystrophy, and Rett syndrome are just a few well-known examples, but there are many more. Individually, these diseases are rare, but together they impose a substantial burden on human health. Monogenic diseases also often affect a diverse patient population. For example, patients suffering from Rett syndrome may have varying symptoms, but they have one thing in common: a damaged gene called MECP2. However, there are more than 800 different MECP2 gene mutations linked to disease progression, according to the International Rett Syndrome Foundation. This makes the development of genomic medicines for the potential treatment of such diseases very challenging.

Over the past decades, researchers have developed a broad variety of tools in an attempt to cure monogenic diseases. One strategy has been to insert a correct copy of the damaged gene into the patient’s genome. This can be achieved either by using viral vectors that randomly integrate into the genome, or by using a more targeted approach, where the integration site is defined by a nuclease (e.g. Zinc Finger Nuclease or CRISPR/Cas9). A nuclease acts like a pair of molecular scissors and can be designed to recognize the desired integration site. It cuts the patient’s DNA, and natural cellular DNA repair mechanisms then assist in the integration of the correct gene into the DNA break. However, numerous DNA repair pathways often compete with each other to repair the DNA break in a cell, thereby restricting the nuclease-mediated insertion approach to cell types that naturally prefer the desired pathway, and limiting the overall editing efficiency. Another strategy that utilizes Base or Prime Editing aims to revert individual disease-causing DNA mutations back to their healthy state. However, each of the more than 800 disease-causing mutations would then likely require a different custom-made genomic medicine. The most practical approach to potentially treat a patient population with diverse mutations in a single gene would be to integrate a healthy copy of the damaged gene directly into its natural location, without relying on the cell’s DNA repair machinery. It is anticipated that this might result in a single genomic medicine that would insert a healthy copy of the gene in front of any of the disease-causing mutations and thus permanently deactivate the mutation while allowing the healthy correct copy to be active only when needed – just as it would be in a healthy individual.

Notably, there is a group of enzymes called Large Serine Integrases (LSI) that have the potential to do exactly that. LSIs have naturally evolved to rearrange DNA in bacteria and viruses through site-specific recombination between two well-defined recognition sites. The value of LSIs has long been recognized by researchers across the world but thus far the usage in human cells has been limited to situations where the LSI’s natural target sequence can be artificially installed in the human genome.

Sangamo’s newly discovered Modular Integrase (MINT) platform doesn’t have this limitation and can be retargeted to novel sites in the human genome without requiring the pre-installation of a target sequence. The MINT platform is a proprietary genome engineering tool that allows Sangamo to reprogram an LSI called Bxb1. The natural Bxb1 enzyme has been shown to successfully target artificially introduced recognition sites in the genomes of many eukaryotic species, including mammals and plants. Recently, we have demonstrated how the MINT platform can be used to retarget Bxb1 to clinically relevant loci in the human genome. Click the links to learn more about how we do this in a manuscript published on bioRxiv, as well as the presentations delivered at the American Society of Cell and Gene Therapy (ASGCT) Conference and the Federation of American Societies for Experimental Biology (FASEB).

Benefits of a modular integrase approach

Traditional gene-editing approaches typically address only one specific genetic mutation or depend on the target cell's DNA repair machinery to correct them all, which makes developing treatments for monogenic diseases challenging. This is where the MINT platform offers a potentially groundbreaking solution: it supports the development of a single potential genomic medicine capable of potentially treating a wide range of mutations across different patients. Specifically, it might be used to insert the correct copy of a gene into its natural position upstream of most, if not all, disease-causing mutations, possibly resulting in a single potential cure for all patients (see figure below). This new genome editing platform has the promise to bring us closer to providing more inclusive and effective treatments for those living with monogenic diseases.

Importantly, the MINT platform has the capability to extend beyond monogenic diseases. It can also be applied to ex vivo cell engineering, which enables new possibilities for developing more advanced cellular therapies. For example, our recent manuscript highlights the retargeting of Bxb1 towards two well-established genomic targets (AAVS1 and TRAC) that are relevant for ex vivo applications ). Beyond the development of genomic medicines, this platform has potential applications in engineering crops to enhance agricultural productivity and drive synthetic biology innovations.

As we continue to push the development of genome editing technologies, we look forward to unlocking new opportunities that could potentially revolutionize genomic medicines and beyond. Follow us to stay updated on our new developments.