Researchers at the San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) in Italy have developed a gene editing platform they say improves the precision and safety of CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells (HSPCs), a development that addresses the long-standing difficulty of ensuring that edited stem cells carry only the intended genetic change without unintended genomic alterations. Details of the new platform, called SMArT (Selection by Means of Artificial Transactivators), are published in Nature Biotechnology.
“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,” said senior author Luigi Naldini, MD, PhD, director of SR-Tiget. “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.”
CRISPR-based therapies such as exagamglogene autotemcel (Casgevy) have already reached regulatory approval for hemoglobinopathies, the vanguard of the expected wave of CRISPR-based gene therapies. But drugs like Casgevy use CRISPR to knock out the disease causing gene, while approaches aimed at creating homology-directed repair (HDR), whereby a gene is inserted have been plagued by unintended genetic changes at the editing site. To date, HDR has been constrained by cell cycle limitations and competing DNA repair pathways that can generate deletions, rearrangements, and other structural changes at the target location.
SMArT was developed to address both low HDR efficiency and the unintended heterogeneity of the gene editing approaches. To do this the investigators created a method whereby transient synthetic “AND-gate” circuits are activated only when two conditions are met: successful targeted integration of a gene-sized cassette and preservation of correct genomic architecture at the edited locus. Only these cells transiently express a selectable marker, enabling their enrichment while removing cells carrying aberrant edits.
“HDR-edited HSPCs were enriched to 80–100% purity through transient selector expression, whereas cells carrying undesired and potentially genotoxic on-target edits were preferentially depleted,” the researchers wrote. They also showed that after transplantation into immunodeficient mice, the enriched cells sustained long-term human hematopoiesis, and the selector system was no longer detectable.
SMArT was developed in three configurations SMArT-1, SMArT-2, and SMArT-3. The most advanced version, SMArT-3, employs a CRISPR-based regulatory architecture that can detect correct integration of the edit at the intended site while also transiently activating genes to support stem cell engraftment. One version of this system uses a single programmable CRISPR-based regulatory system to reduce the need for multiple components.
In preclinical models, SMArT was tested in human HSPCs targeting loci relevant to inherited immune disorders, including X-linked severe combined immunodeficiency (SCID-X1) and Hyper-IgM syndrome. The system successfully enriched for correctly edited cells while reducing large deletions and other structural abnormalities associated with CRISPR-induced DNA repair. The selected cells demonstrated multilineage differentiation capacity after engraftment in mice, exhibiting preserved stem cell function.
“Our goal was not simply to improve editing efficiency, but to fundamentally rethink how to control the quality of edited cell products,” said co-senior author Samuele Ferrari, PhD, a project leader at SR-Tiget. “SMArT introduces a programmable framework that can simultaneously increase precision, reduce genotoxic burden, and preserve the functional potential of stem cells.”
The researcher noted that the SMArT platform is designed to be usable across loci and compatible with other genome engineering technologies beyond CRISPR-Cas9, including emerging targeted integration systems. The also believe it can be used to address multiple genetic diseases, particularly those requiring durable correction of blood or immune cell lineages.
Next steps include improving manufacturing efficiency, preserving clonal diversity during selection, and optimizing delivery methods for HDR templates. SMArT will also need evaluation to determine how it performs across different disease contexts and stem cell sources, as well as how it may integrate with non-myeloablative conditioning approaches to therapeutic development.
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