CRISPR gene-editing shows promise for treating inherited blood disorders such as sickle-cell anemia and thalassemia. But in a process not fully understood, stem cells respond to CRISPR editing by working to counteract the very DNA changes designed to treat the disease.
Now, a team led by scientists in Italy has managed to contain that problem by bypassing the well-known anti-cancer gene p53.
Known as “the guardian of the genome,” the p53 gene helps stabilize DNA and prevent cancer formation. Inactivation of p53 has been linked to many cancer types, and the gene's cancer-suppressing effect has attracted much interest in the scientific world.
A University of Wisconsin-Madison team recently identified an enzyme that helps mutant p53 accumulate and therefore promote aggressive cancers. New Jersey biotech PMV Pharma, a 2017 FierceBiotech Fierce 15 company, is also working on small molecules that could correct mutant p53.
As it turns out, the DNA double-stranded breaks induced by CRISPR “scissors” such as the Cas9 enzyme could activate p53, which then prevents the edited hematopoietic stem and progenitor cells (HSPCs) from proliferating. The team, led by the San Raffaele Telethon Institute for Gene Therapy, described the findings in the journal Cell Stem Cell.
Stem cell therapy only works when the edited—and therefore normal—cells populate. But shutting down p53's natural response to DNA damage, which would make way for that HSPC growth, might allow tumors to form.
The Italian researchers found a way to avoid that. Cas9 isn’t perfect: It can snip DNA even at unintended locations. That off-target effect can in turn increase p53 activity and ultimately kill the edited cells. What’s more, the adeno-associated viral vector scientists usually use to deliver a functional DNA sequence can also induce prolonged p53 response.
So the researchers used nuclease that is highly specific to make the Cas9 scissors more precise and thus limit p53 activation. And with that technique, the p53 response “appears to be fully reversible and compatible with maintenance of the important biological properties of the hematopoietic stem cells,” Luigi Naldini, a study co-senior author, said in a statement.
The brief p53 activation only delayed HSPC proliferation, rather than stopping it, and that delay could be overcome by transiently inactivating the p53 response during gene editing, the researchers found. Short p53 inhibition improved the yield of edited cells without hurting genome stability or increasing mutations, they reported.
Exploring strategies to improve CRISPR precision is also a hot topic. In a recent Cell study co-authored by CRISPR pioneer Jennifer Doudna, a UC Berkeley team rearranged the Cas9 sequence to create variants known as ProCas9s, which can figure out what cells they’re in based on proteases, and therefore allow CRISPR to be turned on only in targeted sites.
More specific gene-editing technology that introduces fewer breaks in DNA could keep HSPC’s p53 response under control, the Italian team figures. As the findings provide molecular evidence for the potential use of CRISPR in HSPC, such precision therapies to treat blood disorders such as sickle cell anemia, thalassemia and primary immunodeficiency syndromes could be tested in humans, they said.