New insights into long-term dysfunction of edited blood stem cells and how to overcome it

Scientists at the San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Milan, have found that gene editing using CRISPR-Cas9 in combination with AAV6 vectors can trigger inflammatory and senescence-like responses in blood stem cells, compromising their long-term ability to regenerate the blood system. The study, published in Cell Reports Medicine, outlines new strategies to overcome this hurdle, improving both the safety and efficacy of gene-editing-based therapies for inherited blood disorders.

The research was led by Dr. Raffaella Di Micco, group leader at SR-Tiget, New York Stem Cell Foundation Robertson Investigator and Associate Professor at the School for Advanced Studies (IUSS) of Pavia, in collaboration with Professor Luigi Naldini, Director of SR-Tiget, and several European research partners.

Unmasking senescence as an hidden challenge in gene editing.

Homology-directed repair (HDR)-based gene editing in hematopoietic stem and progenitor cells (HSPCs) holds great promise for the treatment of genetic blood diseases. Despite significant advances in HDR-based gene editing, translating these approaches into safe and effective clinical therapies for patients remains a major challenge. While the technology shows strong potential in laboratory settings, achieving reliable, durable outcomes in human hematopoietic stem cells - without compromising their function - has indeed proven difficult. The team of Di Micco at SR-Tiget discovered that CRISPR-Cas9 editing, especially when using AAV6 vectors to deliver the repair template, activates a strong DNA damage response (DDR) and inflammatory signals driven by p53 and IL-1/NF-κB pathways.This leads to inflammation and a senescence-like state that reduces the the regenerative capacity of the edited cells post-transplantation.

“We found that a fraction of gene-edited hematopoietic stem cells shows signs of premature aging,” explains Dr. Di Micco. “This reduces their ability to regenerate blood cells after transplantation, which can limit the long-term success and therapeutic benefit of gene therapy.”

We were surprised by the persistence of senescence-like features even months after transplantation,” says Dr. Anastasia Conti, first author of the study and Project Leader in Dr. Di Micco’s lab. “Our findings suggest that stem cells retain a ‘memory’ of the genetic engineering process. These adverse effects are not just transient stress responses—they can lead to lasting impairments in stem cell function.

Mitigating senescence for superior performance of genetically-engineered cells:

To counteract this adverse effect, the researchers tested two complementary strategies:transient p53 inhibition and the use of anti-inflammatory agents, particularly Anakinra - a clinically approved IL-1 receptor antagonist.

“Both approaches significantly reduced senescence markers in edited HSPCs and improved their ability to regenerate a healthy, diverse blood system in preclinical models. Importantly, Anakinra also reduced potential genotoxic events, such as large deletions and translocations, suggesting a safer profile compared to p53 inhibition alone” says Dr. Conti.

“Our study shows that modulating the inflammatory and senescence-related response during gene editing can preserve the fitness of edited hematopoietic stem cells and enable more stable and polyclonal hematopoietic reconstitution in the long-term. These findings help explain setbacks observed in recent clinical trials involving HDR-based gene editing in blood stem cells and offer a concrete path to enhance outcomes. The strategies proposed could be especially valuable for diseases requiring long-term correction and stem cell engraftment, such as immunodeficiencies or bone marrow failure syndromes” concludes Dr. Di Micco.

This groundbreaking research was conducted in the Di Micco Lab with support from the European Research Council (ERC), the European Innovation Council (X-PAND), and the New York Stem Cell Foundation. With a longstanding history of pioneering gene therapy technologies, SR-Tiget continues to lead international efforts to optimize CRISPR-based treatments for safe clinical translation.

 

END