NovaIscB: A Compact New Tool for Gene Therapy via Rational Engineering

In a groundbreaking advancement, scientists at MIT’s McGovern Institute for Brain Research and the Broad Institute of MIT and Harvard have successfully re-engineered a compact RNA-guided enzyme derived from bacteria into a highly efficient and programmable tool for editing human DNA. This newly developed protein, named NovaIscB, holds significant promise for gene therapies aimed at treating and preventing various diseases due to its ability to make precise genetic code alterations and its simplified delivery to cells.

The research, spearheaded by Feng Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT, an investigator at the McGovern Institute and the Howard Hughes Medical Institute, and a core member of the Broad Institute, was recently published in the journal Nature Biotechnology. This open-access study details the creation and capabilities of NovaIscB, marking a significant leap forward in gene editing technology.

NovaIscB is derived from the IscB family of proteins, which are bacterial DNA cutters discovered by Zhang’s lab in 2021. These IscBs are a subset of OMEGA systems, the evolutionary predecessors of Cas9, a component of the CRISPR system that Zhang and others have transformed into powerful genome-editing tools. Similar to Cas9, IscB enzymes target and cut DNA at specific sites dictated by an RNA guide. By modifying this guide, researchers can redirect these enzymes to target specific sequences.

The appeal of IscBs lies not only in their shared characteristics with Cas9 but also in their significantly smaller size—only a third of Cas9’s size. This compactness is a crucial advantage for gene therapy applications, facilitating easier delivery into cells. The smaller size provides researchers with more flexibility to add new features without making the tool too large for clinical applications.

Early studies of IscBs showed that some could edit DNA targets within human cells. However, these bacterial proteins were not efficient enough for therapeutic use. Thus, the team needed to modify an IscB to ensure it could efficiently edit targets in human cells without causing unintended changes elsewhere in the genome.

Soumya Kannan, a graduate student in Zhang’s lab and now a junior fellow at the Harvard Society of Fellows, along with postdoc Shiyou Zhu, initiated the engineering process by screening almost 400 different IscB enzymes found in bacteria. Ten of these enzymes showed the capability to edit DNA in human cells.

The primary challenge was to enhance the enzyme’s activity exclusively at the sequences specified by its RNA guide. As Zhu explained, it was critical to balance the improvement of both activity and specificity to prevent indiscriminate DNA cutting at unintended locations. The team addressed this by leveraging insights from graduate student Han Altae-Tran, now a postdoc at the University of Washington, regarding the evolution and diversity of bacterial IscBs.

The team discovered that IscBs effective in human cells contained a segment known as REC, which was absent in other IscBs. Structural modeling suggested that expanding a portion of the protein with REC might enable IscBs to recognize longer RNA guides, thereby improving specificity.

Through strategic modifications and swapping parts of REC domains from different IscBs and Cas9s, the team optimized efficiency and specificity, resulting in NovaIscB. This new protein demonstrated over 100 times greater activity in human cells compared to its IscB predecessor, while maintaining good specificity for its intended targets.

Kannan and Zhu strategically engineered NovaIscB, guided by the natural evolution of IscBs and predictions from the artificial intelligence tool AlphaFold2, which helped assess how each change would affect the protein’s structure. This rational engineering approach significantly sped up the identification of a protein with the desired characteristics.

The team successfully used NovaIscB to replace specific DNA letters in human cells and to modulate the activity of targeted genes, demonstrating its versatility as a genome editing tool. The compact size of NovaIscB-based tools allows them to be easily packaged inside a single adeno-associated virus (AAV), the most common vector for safe gene therapy delivery.

To demonstrate its therapeutic potential, Zhang’s team developed OMEGAoff, a NovaIscB-based tool that adds chemical markers to DNA to reduce the activity of specific genes. They programmed OMEGAoff to repress a gene involved in cholesterol regulation and delivered it to the livers of mice using AAV, which led to lasting reductions in cholesterol levels.

The researchers are optimistic that NovaIscB can be adapted to target most human genes and encourage other laboratories to explore this new technology. They also advocate for the adoption of their evolution-guided approach to rational protein engineering, emphasizing the wealth of diversity in nature that can inspire better engineered systems.