NovaIscB: A Compact New Tool for Gene Therapy via Rationale Engineering
In a significant advancement for gene therapy, scientists at MIT’s McGovern Institute for Brain Research and the Broad Institute have successfully re-engineered a compact RNA-guided enzyme, originally found in bacteria, into an efficient and programmable editor of human DNA. This innovative protein, named NovaIscB, holds immense potential for precise genetic code alterations, modulation of specific gene activities, and various other editing functions.
The most appealing advantage of NovaIscB is its small size, which streamlines its delivery into cells, positioning it as a strong contender for developing gene therapies aimed at treating or preventing a spectrum of diseases.
The groundbreaking study was 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. Their comprehensive work was published this month in the esteemed journal Nature Biotechnology.
NovaIscB is derived from a bacterial DNA cutter belonging to the IscB protein family, initially discovered by Zhang’s lab in 2021. These IscBs are classified as OMEGA systems, the evolutionary predecessors of Cas9. Like Cas9, IscB enzymes precisely target and cut DNA at sites specified by an RNA guide. By simply reprogramming this guide, researchers can redirect these enzymes to target any desired sequence.
The team focused on IscBs due to their compact size, about one-third the size of Cas9. This compactness is crucial for gene therapies, as smaller tools are more easily delivered to cells. A smaller enzyme also provides more flexibility for modifications, potentially adding new functionalities without making the tool too large for clinical applications.
Despite their promise, the initial IscBs required significant enhancement for effective therapeutic deployment. To address this, Soumya Kannan, a graduate student in Zhang’s lab, and postdoc Shiyou Zhu initiated an extensive engineering process. They screened nearly 400 different IscB enzymes, identifying ten capable of editing DNA in human cells. The challenge then was to enhance the enzyme’s activity specifically at the targeted sequences without causing off-target effects.
The team optimized IscB for human genome editing by leveraging insights into the diversity and evolution of bacterial IscBs. They observed that IscBs effective in human cells included a segment called REC, absent in other IscBs, suggesting its importance for interaction with human DNA. Structural modeling indicated that expanding the REC region could enable IscBs to recognize longer RNA guides, improving specificity.
Through strategic modifications, including swapping REC domain parts and using an artificial intelligence tool called AlphaFold2 for structural predictions, they developed NovaIscB. This new protein demonstrated a 100-fold increase in activity in human cells compared to its predecessor, with maintained specificity for its intended targets.
The team successfully used NovaIscB to replace specific DNA letters and alter targeted gene activity in human cells. Moreover, NovaIscB-based tools are compact enough to be packaged inside a single adeno-associated virus (AAV), the most common vector for safe gene therapy delivery. They also created OMEGAoff, a NovaIscB-based tool that represses specific genes by adding chemical markers to DNA. Delivery of OMEGAoff to mice livers via AAV led to lasting reductions in blood cholesterol levels.
Researchers believe NovaIscB can target most human genes and anticipate its widespread adoption by other labs. They also encourage the use of their evolution-guided approach to rational protein engineering to further improve engineered systems.
