NovaIscB: A Compact New Tool for Gene Therapy Developed Through Rational Engineering

In a groundbreaking advancement, scientists at MIT’s McGovern Institute for Brain Research and the Broad Institute have successfully re-engineered a compact RNA-guided enzyme, initially discovered in bacteria, into a highly efficient and programmable editor of human DNA. This innovative protein, named NovaIscB, holds immense potential for precise genetic code alterations, gene activity modulation, and a spectrum of other editing functions.

The compact size of NovaIscB simplifies its delivery to cells, positioning it as a promising candidate for the development of gene therapies aimed at treating or preventing a wide array of diseases. This study, spearheaded by Feng Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT and a core member of the Broad Institute, was recently published in Nature Biotechnology.

NovaIscB is derived from a bacterial DNA cutter belonging to the IscB protein family, discovered by Zhang’s lab in 2021. These IscBs are part of the OMEGA system, the 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 guided by an RNA sequence, allowing researchers to redirect the enzymes by reprogramming the guide.

The team was particularly drawn to IscBs due to their smaller size, approximately one-third that of Cas9. This compactness is advantageous for gene therapies, facilitating easier cell delivery and providing researchers with greater flexibility to incorporate new functionalities without creating tools too large for clinical applications.

Initial studies indicated that certain IscB family members could cut DNA targets in human cells. However, none of the bacterial proteins were sufficiently effective for therapeutic deployment, necessitating modifications to ensure efficient editing in human cells without disrupting the rest of the genome.

Soumya Kannan and Shiyou Zhu spearheaded the engineering process, screening nearly 400 different IscB enzymes found in bacteria, identifying ten capable of editing DNA in human cells. Enhancing the activity of the most promising candidates was crucial, but only at the sequences specified by the RNA guide, to avoid unintended DNA cuts.

The team optimized IscB for human genome editing by leveraging insights into the diversity and evolution of bacterial IscBs. They noted the presence of a segment called REC in IscBs effective in human cells, which was absent in others. Structural modeling suggested that expanding part of the protein might enable REC to recognize longer RNA guides, enhancing specificity.

Through strategic experimentation and informed by AlphaFold2, an artificial intelligence tool for protein structure prediction, the team created NovaIscB. This new protein demonstrated over 100 times greater activity in human cells than the original IscB, with excellent target specificity.

The team demonstrated NovaIscB as a versatile scaffold for various genome editing tools. With different modifications, they used NovaIscB to replace specific DNA letters and alter the activity of targeted genes in human cells. The resulting tools are compact enough to be easily packaged inside a single adeno-associated virus (AAV), simplifying delivery compared to the bulkier Cas9-based tools.

Demonstrating 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. When programmed to repress a gene involved in cholesterol regulation and delivered to the livers of mice via AAV, OMEGAoff led to lasting reductions in blood cholesterol levels.

The researchers anticipate that NovaIscB can be used to target genome editing tools to most human genes, inviting other labs to explore and expand upon this new technology. They also advocate for their evolution-guided approach to rational protein engineering, emphasizing the wealth of diversity in nature and the potential for continuous improvement of engineered systems.