Will Zinc Finger Nuclease Gene Editing Technologies Compete with CRISPR/Cas9 Over the Next Decade?

Gene editing technologies are rapidly advancing, and two of the most notable are Zinc Finger Nuclease (ZFN) and CRISPR/Cas9. As we look into the next decade, one pressing question is whether ZFN will be able to compete effectively with CRISPR/Cas9 in various applications, particularly in the field of chimeric antigen receptor T-cell (CAR-T) therapy.

Potential Advantages of Zinc Finger Nucleases

ZFN is a protein that can be delivered via an mRNA, offering a distinct advantage over CRISPR/Cas9, which operates as a ribonucleoprotein complex (RNP) that requires an mRNA encoding Cas9 and a guide RNA (gRNA). This mRNA needs to be delivered via a DNA vector, which can be either episomal or integrated. The key difference lies in the transient nature of mRNA versus the long-term stability of DNA.

The transient expression of mRNA in ZFN makes it a better fit for gene editing applications, where the goal is a one-time, permanent correction. Moreover, the potential for prolonged expression is a risk factor, given the possibility of off-target editing, as predicted by Murphy's Law. Therefore, the stable and integrated forms of DNA vectors, such as lentiviral vectors, often represent the worst-case scenario for gene editing.

Accurate Editing and In Vivo Delivery in CAR-T Therapy

Two significant factors may influence whether ZFNs will be more likely than CRISPR/Cas9 to play a role in next-generation CAR-T therapy: accuracy and in vivo delivery.

Accuracy: CRISPR is currently more prone to off-target modifications. However, the situation may change with more research and improvements. On the other hand, ZFN has been shown to have a higher level of accuracy, making it a promising candidate for precise gene editing.

In Vivo Delivery: The next generation of CAR-T therapy, known as CAR-T 2.0, may involve the intelligent design of gene expression chassis. However, the ultimate goal is to achieve in vivo delivery to reduce production costs and increase throughput. ZFN-based systems, like ZFNs, are based on a human scaffold and are expected to be less immunogenic compared to Cas9-based systems. The Fok1 nuclease used in ZFNs and TALENs is derived from bacteria and might induce an immune response. Similarly, immunogenicity concerns are also present for Cas9/Cas12a, which could slow down their in vivo therapeutic applications.

In vivo delivery is also easier for ZFNs due to the smaller expression cassettes that can be inserted into various vectors, while Cas9 systems pose more challenges in viral vector integration due to their larger size.

Future Prospects and Research Directions

The race is on for the most clinically-ready method of gene editing. While CRISPR/Cas9 holds long-term cost advantages, its first trial in humans did not begin until late 2016. In contrast, ZFN has shown a higher accuracy and may offer a more practical solution for clinical applications in the near future.

As research continues, both ZFN and CRISPR/Cas9 technologies will likely evolve. Importantly, the evaluation of their applicability in in vivo settings will be crucial for determining their long-term success in various therapeutic applications.

The next decade will likely see ongoing comparisons and advancements in both technologies. Whether ZFN will be able to compete with CRISPR/Cas9 will depend on continued innovation, regulatory approval, and the ultimate effectiveness of these gene editing tools in numerous medical applications.

Key Takeaways:Transient expression of mRNA in ZFN offers an advantage for gene editing applications.ZFN has shown higher accuracy compared to CRISPR/Cas9, making it suitable for precise vivo delivery is easier for ZFN systems, which can be integrated into various expression vectors.

Will ZFNs take the lead in future gene editing applications, or will CRISPR/Cas9 maintain its dominance? Only time will tell, but ongoing research will undoubtedly shape the future landscape of gene editing technologies.