Rationale engineering generates a compact new tool for gene therapy
In a significant advancement for gene therapy, scientists at the McGovern Institute for Brain Research at MIT and the Broad Institute of MIT and Harvard have successfully re-engineered a compact, RNA-guided enzyme found in bacteria into a highly efficient and programmable editor of human DNA. The breakthrough tool, named NovaIscB, promises to simplify the delivery of gene therapies and enable precise modifications to the genetic code, offering new avenues for treating and preventing diseases.
The research, led by Feng Zhang, a distinguished professor at MIT and investigator at the McGovern Institute and Howard Hughes Medical Institute, builds upon his lab’s 2021 discovery of IscBs, a family of OMEGA systems considered evolutionary ancestors to the revolutionary Cas9 CRISPR system. Like Cas9, IscB enzymes are capable of cutting DNA at specific sites dictated by an RNA guide, allowing for targeted genetic manipulation.
What sets IscBs apart, and drew the team’s attention, is their remarkably small size – approximately one-third the size of Cas9. This compactness is a critical advantage for gene therapies, as smaller tools are considerably easier to package and deliver into human cells, providing greater flexibility for adding new functionalities without making the therapeutic tools overly bulky for clinical application. Despite their potential, the naturally occurring bacterial IscB proteins were not sufficiently efficient or specific for therapeutic deployment in human cells, necessitating extensive engineering.
The intricate engineering process began with graduate student Soumya Kannan and postdoc Shiyou Zhu, who screened nearly 400 different IscB enzymes, identifying ten capable of DNA editing in human cells. The formidable challenge lay in enhancing the enzyme’s activity while simultaneously ensuring its specificity, preventing unintended cuts elsewhere in the genome. “The key is to balance the improvement of both activity and specificity at the same time,” explained Zhu.
A crucial insight emerged from graduate student Han Altae-Tran’s studies on IscB diversity and evolution. The team observed that IscBs effective in human cells possessed a unique segment, dubbed ‘REC,’ absent in other IscBs. Structural modeling suggested that this REC segment could facilitate interaction with human DNA and potentially allow IscBs to accommodate longer, more specific RNA guides, which are vital for precise targeting.
Leveraging this knowledge, the team embarked on a rational engineering approach, a strategic methodology that dramatically accelerated their progress compared to traditional random mutation and screening methods. By swapping in parts of REC domains from various IscBs and Cas9s, and making additional guided changes based on their understanding of DNA and RNA guide interactions, they meticulously optimized the enzyme. The result was NovaIscB, a protein over 100 times more active in human cells than its bacterial progenitor, exhibiting impressive specificity for its intended targets.
The versatility of NovaIscB as a scaffold for various genome editing tools has been demonstrated. Kannan noted its biochemical similarity to Cas9, making it easy to adapt existing Cas9-optimized tools. With modifications, NovaIscB has been used to precisely alter DNA letters and modulate the activity of targeted genes in human cells. Its compact nature allows it to be readily packaged within a single adeno-associated virus (AAV), the most common and safest vector for delivering gene therapy to patients – a significant advantage over bulkier Cas9-based tools that often require more complex delivery strategies.
Further demonstrating its therapeutic promise, Zhang’s team developed OMEGAoff, a NovaIscB-based tool designed to add chemical markers to DNA, thereby dialing down the activity of specific genes. When programmed to repress a gene involved in cholesterol regulation and delivered via AAV to the livers of mice, OMEGAoff led to lasting reductions in cholesterol levels in the animals’ blood. This paves the way for potential in vivo applications for a range of genetic conditions.
The team anticipates that NovaIscB can be adapted to target most human genes and hopes that other research groups will embrace this new technology and their evolution-guided approach to rational protein engineering. “Nature has such diversity, and its systems have different advantages and disadvantages,” Zhu remarked. “By learning about that natural diversity, we can make the systems we are trying to engineer better and better.”
