MIT Scientists Engineer Compact NovaIscB Tool for Gene Therapy

In a groundbreaking advancement for gene therapy, 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 into a highly efficient tool for editing human DNA. This newly developed protein, named NovaIscB, holds significant promise for precise genetic modifications, gene activity modulation, and other sophisticated editing tasks. Its compact size facilitates easier delivery into cells, positioning NovaIscB as a leading candidate for developing innovative gene therapies to combat various diseases.

The pioneering study, 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 published this month in Nature Biotechnology.

NovaIscB is derived from a bacterial DNA cutter, part of the IscB protein family discovered by Zhang’s lab in 2021. These IscBs belong to the OMEGA system family, evolutionary predecessors to Cas9, a component of the bacterial 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 direct the enzymes to target any desired DNA sequence.

The appeal of IscBs lies not only in their shared characteristics with Cas9 but also in their smaller size—only a third of Cas9’s size. This compactness is advantageous for gene therapy applications, making cell delivery simpler and providing researchers with more flexibility to add new functionalities without exceeding clinical size constraints.

Initial studies confirmed that certain IscB family members could cut DNA targets in human cells. However, these bacterial proteins lacked the efficiency needed for therapeutic deployment. The team recognized the need to modify an IscB to ensure efficient editing in human cells without disrupting other parts of 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 nearly 400 different IscB enzymes found in bacteria. This extensive screening identified ten enzymes capable of editing DNA in human cells.

Further enhancement was required to transform even the most active enzyme into a practical genome editing tool. The challenge was to boost the enzyme’s activity specifically at the RNA-guided target sequences, avoiding indiscriminate DNA cutting. Zhu emphasized the importance of balancing enhanced activity with high specificity.

The team leveraged insights from graduate student Han Altae-Tran, now a postdoc at the University of Washington, regarding the diversity and evolution of bacterial IscBs. They identified a segment called REC, present in IscBs effective in human cells but absent in others, suggesting its importance for DNA interaction in human cells. Structural modeling indicated that expanding the REC region could enable IscBs to recognize longer RNA guides, enhancing specificity.

Experimenting with REC domain segments from various IscBs and Cas9s, the team evaluated the impact of each modification on protein function. Guided by their understanding of IscB and Cas9 interactions with DNA and RNA guides, they made additional changes to optimize both efficiency and specificity, ultimately creating NovaIscB.

NovaIscB demonstrated over 100 times greater activity in human cells compared to its IscB predecessor, with excellent target specificity. Kannan and Zhu’s rational engineering approach, integrating knowledge of IscB evolution and AI-driven structural predictions via AlphaFold2, significantly accelerated the identification of a protein with the desired features.

The team demonstrated NovaIscB’s versatility as a scaffold for various genome editing tools. With different modifications, they successfully replaced DNA letters and altered gene activity in human cells. The resulting NovaIscB-based tools are compact enough for easy packaging into adeno-associated viruses (AAV), the preferred vectors for safe gene therapy delivery.

Demonstrating therapeutic potential, Zhang’s team developed OMEGAoff, a NovaIscB-based tool that uses chemical markers to reduce the activity of specific genes. By programming OMEGAoff to repress a gene involved in cholesterol regulation and delivering it to the livers of mice via AAV, they achieved lasting reductions in blood cholesterol levels.

The team anticipates that NovaIscB can be adapted to target genome editing tools to most human genes, inviting other labs to explore the technology’s potential. They also advocate for the broader adoption of their evolution-guided rational protein engineering approach. Zhu emphasized the value of learning from nature’s diversity to improve engineered systems.