New research from the labs of David Liu, PhD, at the Broad Institute, and Jonathan Yen, PhD, at St. Jude Children’s Research Hospital shows that prime editing can not only fix sickle cell disease (SCD) mutations in a patient’s hematopoietic stem and progenitor cells (HSPCs) but that these prime edited cells can also engraft and help treat genetic blood disorders in mice. These findings are among the first to establish therapeutic prime editing of HSPCs, implying that prime editing and transplanting patient HSPCs may represent a promising therapeutic strategy as a one-time autologous treatment for SCD and other blood disorders.

“These results show efficient prime editing of blood stem cells and that the prime-edited cells maintain their full ability to engraft and repopulate the bone marrow of an animal,” said senior and co-corresponding author Liu, whose lab invented prime editing in 2019. “Bringing the ‘search-and-replace’ versatility of prime editing to blood stem cells raises the possibility of applying this technology to treat a wide range of diseases involving blood cells.”

The study, “Ex vivo prime editing of patient hematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice,” was published in Nature Biomedical Engineering.

While allogeneic transplantation of HSPCs is the only FDA-approved treatment for SCD, most patients lack suitable donors, and the procedure is associated with serious side effects such as graft-versus-host disease and graft rejection. The use of the patient’s own HSPCs avoids immune complications and the need for a tissue-compatible donor. 

There are several strategies for therapeutic manipulation of autologous SCD HSPCs currently being examined in clinical trials, and it is not yet known which strategy will be the safest and most effective for patients. BlueBird Bio’s approach is based on the lentiviral expression of a β-like globin that reduces the SCD variant (HbS), and other approaches include using genome editing nucleases or base editors to activate γ-globin gene transcription for induction of fetal hemoglobin (HbF). With this approach, Vertex and CRISPR Therapeutics submitted the first CRISPR treatment for sickle cell disease to the FDA in early April, and a regulatory decision is expected in 8 to 12 months.

An ideal treatment for SCD would permanently revert the SCD allele to wild type with few deleterious genomic alterations or cell state changes. Because prime editing replaces a target segment of DNA with a specified new sequence up to hundreds of base pairs in length, it enables the installation of targeted insertions, deletions, and any base-to-base substitutions directly into the genome of living cells and animals without requiring double-stranded breaks (DSBs).

An additional feature of the prime editing strategy is that it does not require DNA delivery, viral transduction, or drug selection to enrich edited cells. DNA delivery is required for HDR and gene therapy but can also lead to increased toxicity, lower engraftment frequency, or insertional mutagenesis. This is important in light of the news from Graphite Bio pertaining to the CEDAR trial—a clinical trial using Cas9 nuclease-initiated homology-directed repair (HDR) and an adeno-associated virus type 6 (AAV6)-delivered DNA template to correct the SCD mutation was stopped due to a patient developing transfusion-dependent pancytopenia.

Here, researchers show that a one-time prime edit corrects the SCD allele to wild type in HPSCs from SCD patients, does not require any viral or non-viral DNA template, and minimizes the undesired consequences of DNA double-strand breaks. With a single electroporation, cells could be efficiently edited and cryopreserved.

After being injected upon thawing to minimize loss of multipotency in virtro, edited cells efficiently engrafted into animal recipients with no loss of target prime editing efficiency after 17 weeks. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBBA levels and displayed engraftment frequencies, hemopoietic differentiation, and lineage maturation similar to those of unedited HSPCs from healthy donors.

The observed reduction in HbS, an increase in wild type (HbA), and a reduction in sickling propensity are suggestive of exceeding the predicted levels required for therapeutic benefit in SCD patients. “We optimized prime editing in long-term blood stem cells and showed that the prime editing cells maintain full engraftment efficiency in an animal with a clinically relevant system,” said co-corresponding author Yen.

This work also opens the door to developing cures for many hematological diseases. “We have identified what might be the next wave of therapies for genetic anemias,” said co-author Mitchell Weiss, MD, PhD, the St. Jude department of hematology chair. “We took the newest cutting-edge genetic engineering technology and showed that we could make meaningful gene edits for future therapies.”

While prime editing has the potential to cure many more genetic diseases, extensive manufacturing development, process optimization, and safety assessment will be required to get it to the clinic. But the proof of concept is there.

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