New SMArT platform improves safety of CRISPR gene editing

A team of researchers led by Luigi Naldini at the San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) has developed a new strategy to significantly improve the precision and safety of CRISPR-Cas9 gene editing in human blood stem cells, potentially overcoming one of the major barriers limiting broader clinical application of genome editing therapies.

The study, published in Nature Biotechnology, introduces SMArT ("Selection by Means of Artificial Transactivators"), an innovative platform that achieves targeted integration of a gene-sized cassette and verifies the outcome of the procedure. This strategy can be used to enrich edited hematopoietic stem and progenitor cells (HSPCs) to near purity while selectively removing cells carrying unintended and potentially harmful genomic alterations generated during editing.

The work was led by Luigi Naldini, director of SR-Tiget, and Samuele Ferrari, with Daniele Canarutto and Martina Fiumara as first authors.

CRISPR-Cas9 editing has transformed the field of genetic medicine by enabling targeted modification of disease-causing genes. The first CRISPR-based therapies - including exagamglogene autotemcel (Casgevy) for sickle cell disease and transfusion-dependent beta-thalassemia - have already received regulatory approval in multiple countries, marking a historic milestone for genome editing therapies. However, despite these advances, safety concerns remain. When CRISPR-Cas9 cuts DNA, cells can repair the break in unintended ways, sometimes generating chromosomal aberrations and rearrangements that may carry an unknown risk in terms of safety.

Moreover, Casgevy is a drug based upon knock-out of a target gene by the delivered DNA break. Conversely, targeted integration of DNA cassettes into the break has been so far out of reach due to limited efficiency as the outcome of targeted integration is at odds with other editing outcomes. SMArT solves this problem by verifying that the intended outcome has occurred, hereby enabling enrichment of cells with targeted integration to 100% purity. Importantly, this increased efficiency also results in an ameliorated safety profile.

These unintended outcomes, such as large deletions of DNA sequences, have emerged as one of the most important limitations to the broader application of gene editing, especially in stem cells intended for transplantation. With SMArT, we aimed to create an intelligent selection system capable of identifying and enriching only those cells that achieved the desired genetic correction while excluding cells carrying potentially dangerous alterations."

Luigi Naldini, San Raffaele Telethon Institute for Gene Therapy

The researchers developed three increasingly sophisticated SMArT configurations that act as transient synthetic "AND-gate" systems. Only cells simultaneously carrying the intended on-target integration and preserving the integrity of the targeted locus transiently activate a selectable marker, enabling purification of the correctly edited population.

In preclinical models, SMArT enrichment generated highly pure populations of edited blood stem cells while markedly reducing the presence of large deletions and other unwanted editing outcomes. After transplantation into immunodeficient mice, the selected cells successfully engrafted and generated long-term human hematopoiesis. Importantly, the selector used to isolate correctly edited cells was only transiently expressed and became undetectable after engraftment, leaving behind a "clean" edited graft.

"Our goal was not simply to improve editing efficiency, but to fundamentally rethink how to control the quality of edited cell products," said Samuele Ferrari, co-senior author of the study. "SMArT introduces a programmable framework that can simultaneously increase precision, reduce genotoxic burden, and preserve the functional potential of stem cells."

The study focused on therapeutic gene editing strategies for severe inherited immune disorders, including X-linked severe combined immunodeficiency (SCID-X1) and Hyper-IgM 1 syndrome, but the authors believe the approach could be broadly applicable across multiple gene editing platforms and disease areas.

One of the most advanced versions of the technology, SMArT-3, exploits a single polyfunctional programmable CRISPR-based regulatory systems to transiently detect correct genomic integration and transiently activate endogenous genes that may improve stem cell engraftment.

"Gene editing has often been described as precise genome surgery, but biology is more complex than initially anticipated," said Daniele Canarutto, co-first author of the study. "SMArT helps distinguish cells that truly achieved the intended therapeutic outcome from those that underwent alternative repair processes."

"Precision medicine requires precision editing," added Martina Fiumara, co-first author. "We believe approaches like SMArT could help unlock the full therapeutic potential of gene-sized editing while addressing some of the most pressing safety concerns in the field."

The authors suggest that SMArT strategies could be integrated not only with current CRISPR-Cas9 editing approaches, but also with other emerging genome engineering technologies.

The study was supported by Fondazione Telethon, the European Union Horizon Europe Programme, the Italian Ministry of Health, the Italian Ministry of University and Research, and other international funding bodies.