GeneBites

In a recent study by Geilenkeuser et al., researchers developed a new system to help different types of gene editing molecules get into cells efficiently. Experiments treating visually-impaired mice with gene editing using this delivery system show promise for potential future clinical development.

In the past, medicine could only treat the symptoms of genetic diseases. But now, with advances in genetics research and technologies, we may be able to treat or cure these diseases at the cause, if we can edit the gene back to a regularly functional state.

Some early cases of gene therapies involved introducing a whole new functional gene copy into cells. For example, in the first recorded successful gene therapy in 1990, doctors put a functional gene encoding an enzyme called adenosine deaminase into the patient’s blood cells to treat an immune deficiency. Today, however, there is much interest in making therapies that can instead make specific changes within the mutated genes of patients. In fact, just this year, the first ever patient of a personalized CRISPR base editing gene therapy, which can be used to make a specific sequence change at a single spot in the genome, was treated for a metabolic disease. 

However, gene therapies, including gene editing, still face several challenges (1,2). For example, the molecules that do the editing have to be able to get into cells. Just like packages need to be properly assembled and labeled to get to their destination and deliver their contents unharmed, gene therapies also need to be packaged for efficient and effective delivery. Often, engineered viruses, such as adenoviruses, adeno-associated viruses, or lentiviruses, are used to carry genetic cargo needed for editing. These viruses can bind to receptors on the surface of target cells and enter the cells to deliver genetic material. However, some non-viral systems also exist, including virus-like particles (VLPs, particles engineered to be structurally similar to viruses, but that don’t have their own genome and can’t replicate once they get into a target cell). 

Some delivery systems can elicit immune reactions, which can be severe or even fatal. Additionally, some gene-editing proteins have off-target effects (they may edit more than just the target gene), so it can be a problem to have the proteins persist in the patient’s cells long-term. So, researchers are working on developing safer and more effective ways to get gene-editing tools into cells.

In a new study, scientists developed a system called engineered nucleocytosolic vehicles for loading of programmable editors (abbreviated to ENVLPE), which allows different types of CRISPR-based tools to be delivered into cells using VLPs. 

For CRISPR technologies, VLPs were designed to carry a CRISPR-associated (Cas) protein itself along with a guide RNA, which tells the Cas protein where to edit the DNA; together these components make up a ribonucleoprotein (RNP) complex. Carrying the RNP rather than a gene encoding the Cas protein means that no genes need to be put into the host cell’s genome. This is beneficial because it avoids the risks of problematic mutations and long-term expression that are associated with putting a gene into host DNA. 

Multiple types of CRISPR systems were delivered using ENVLPE. Initial tests of prime editing, where a Cas protein cuts only one strand of the DNA that then gets repaired based on a mRNA template with the chosen edit, resulted in successful editing in up to 35% of stem cell-derived neurons. Later experiments with modifications to protect prime editing guide RNAs, which are more susceptible to being degraded, showed increased editing efficiency. ENVLPE could also be used for CRISPR base-editing, which modifies the chemical structure of a particular DNA base, changing it from one base to another, without breaking the DNA. Different cell types were base-edited with different efficiencies, with the highest hitting 90%. Additionally, in a proof-of-concept experiment, ENVLPE was also used in combination with a lentivirus to deliver a system to make larger edits through a mechanism called homology-directed repair, or HDR; however, the scientists needed to block a different way that cells repair DNA to make HDR more efficient. 

The scientists tested more modifications and variations of their delivery system, resulting in an optimized version that they called ENVLPE⁺. Although base editing with ENVLPE⁺ was not more efficient than a previously-developed VLP system, prime editing delivered with ENVLPE⁺ was more efficient at editing compared to delivery with another VLP system published in 2024.

The researchers then tested ENVLPE⁺ for clinically-relevant uses, like editing patient cells and treating vision loss. They demonstrated that ENVLPE⁺-delivered base editors could edit receptors on T cells taken from humans, functionally knocking out these receptors to a similar extent as a previous base editing VLP system. Notably, ENVLPE⁺ achieved this effect with a lower VLP dose, indicating an improvement on the previous system. Additionally, ENVLPE⁺ showed promise in delivering treatments in multiple mouse models of inherited retinal degeneration, which causes vision loss. For example, prime editing treatments delivered by ENVLPE⁺ resulted in more efficient editing than a previous VLP system. For one of the mouse models, the ENVLPE⁺-treated mice had a higher retinal response to light compared to the other VLP system, when treated with the same VLP dose (much more VLP was required for the other system to show a similar outcome as ENVLPE⁺).

So, why does all this matter? In short, the flexibility of ENVLPE to carry various types of CRISPR-based treatments means it may be broadly applicable for future clinical applications. Although CRISPR human therapies are still in infancy, many recent advancements have been made in engineering Cas proteins to manipulate the genome in specific ways. Yet, no matter how many CRISPR tools scientists develop in labs, they can’t reach their full potential unless we have reliable ways to get the treatments into the target cells in living people. Outperforming even a VLP system published only last year for prime editing, ENVLPE represents a step forward in delivery of gene-editing technology. 

Edited by Mandy Eckhardt and Jayati Sharma