Rationale engineering develops compact gene therapy tool

Researchers at MIT’s McGovern Institute for Brain Research and the Broad Institute of MIT and Harvard have innovatively re-engineered a compact RNA-guided enzyme discovered in bacteria into a highly effective, programmable editor for human DNA.

This newly crafted protein, named NovaIscB, showcases the ability to make precise genetic alterations, modulate gene activities, and perform various editing functions. Its small size streamlines cell delivery, making it a promising candidate for developing gene therapies aimed at treating or preventing diseases.

Leading this groundbreaking study is Feng Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT and a distinguished investigator at the McGovern Institute as well as the Howard Hughes Medical Institute. Zhang’s team recently published their open-access findings in the prestigious journal Nature Biotechnology.

NovaIscB is derived from a bacterial DNA-cutting enzyme known as IscB, part of a protein family identified by Zhang’s lab in 2021. These IscB proteins are considered evolutionary precursors to Cas9, a fundamental component of the CRISPR system, another powerful tool developed for genome editing. Similar to Cas9, IscB enzymes utilize RNA guides to target specific DNA sequences for precise cuts. By reprogramming these guides, researchers can direct the enzymes to desired places within the genome.

The interest in IscBs stems not only from their resemblance to CRISPR’s Cas9 but also from their significantly smaller size, offering distinct advantages for gene therapies. Compact tools enhance delivery efficiency to cells and provide researchers greater flexibility in modifying enzyme functionalities without creating cumbersome clinical tools.

Initial studies revealed that certain IscB proteins could successfully cut DNA within human cells, but these bacterial proteins fell short of therapeutic potential. Therefore, Zhang’s team set out to enhance the enzyme’s efficiency while ensuring minimal disturbance to the wider genome.

Beginning this process, graduate student Soumya Kannan and postdoc Shiyou Zhu screened nearly 400 bacterial IscB enzymes to identify suitable candidates. Remarkably, they found ten capable of effectively editing human DNA.

However, even the most promising enzymes required further enhancement to truly function as effective genome-editing tools. A key challenge was boosting enzyme activity exclusively at targeted RNA-specified sequences. “The critical aspect is maintaining a fine balance between increased activity and specificity,” explains Zhu.

While the bacteria’s IscBs are guided by relatively short RNA sequences, their efficacy in human applications was limited. Zhu and the team aimed to engineer IscBs that could work with longer RNA guides, reducing unintended interactions with non-target DNA sequences.

In optimizing IscBs for human genome editing, the team leveraged insights from graduate student Han Altae-Tran regarding the diversity and evolutionary significance of bacterial IscBs. They identified that IscBs functioning in human cells contained a unique segment known as REC, which was absent from others. Further investigation, aided by structural modeling, suggested that expanding this protein segment might allow IscBs to accommodate longer RNA guides.

Guided by prior knowledge of IscB and Cas9 interactions and predictions from the AI tool AlphaFold2, the team strategically swapped segments of REC from various IscBs and Cas9s, evaluating the impact of these changes on the protein’s functionality.

The culmination of their efforts led to the creation of NovaIscB, a protein that exhibits over 100 times greater activity in human cells compared to its predecessor, while maintaining excellent specificity for its targets.

Kannan and Zhu systematically constructed and screened numerous new IscBs, ensuring that every modification was purposeful. Their methodical approach, rooted in evolutionary understanding and AI-enhanced predictions, significantly shortened the timeline for discovering a new protein conforming to their specifications.

NovaIscB serves as a compelling scaffold for diverse genome editing tools. “Its biochemical functions parallel those of Cas9, facilitating the adaptation of existing tools optimized for the Cas9 framework,” says Kannan. With various modifications, the researchers successfully utilized NovaIscB to precisely substitute specific DNA code segments within human cells and adjust gene activities.

Significantly, the NovaIscB-based tools are compact enough to fit inside a single adeno-associated virus (AAV), the preferred vector for safely delivering gene therapies to patients. In contrast, tools based on Cas9 often necessitate more complex delivery systems due to their size.

Highlighting NovaIscB’s therapeutic promise, Zhang’s team developed a tool named OMEGAoff, which tags DNA with chemical markers to suppress specific gene activities. They programmed OMEGAoff to inhibit a gene integral to cholesterol regulation, subsequently employing AAV to deliver this system to mouse livers, resulting in sustained cholesterol level reductions in the subjects.

The researchers are optimistic about NovaIscB’s ability to target most human genes and anticipate its broad adoption by other laboratories. They’re also eager for fellow researchers to embrace their evolution-guided approach to protein engineering. “Nature presents a wealth of diversity, and different systems have unique strengths and weaknesses,” Zhu notes. “By studying this natural variety, we can continually enhance the systems we aim to engineer.”

This research was partially funded by the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics at MIT, the Broad Institute Programmable Therapeutics Gift Donors, the Pershing Square Foundation, William Ackman, Neri Oxman, the Phillips family, and J. and P. Poitras.

Photo credit & article inspired by: Massachusetts Institute of Technology

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