In a recent study, researchers from the Children’s Hospital of Philadelphia and the Perelman School of Medicine at the University of Pennsylvania developed a proof-of-concept model for delivering gene-editing tools to treat hematological disorders, enabling the direct modification of diseased blood cells in the body. This represents a significant advancement in gene therapy, potentially expanding the use of gene therapy for hematological disorders and reducing costs. Currently, many gene therapy treatments for hematological disorders require patients to undergo chemotherapy and stem cell transplants. The study’s findings were published in the July 28, 2023 issue of the journal Science, titled “In vivo hematopoietic stem cell modification by mRNA delivery”.

Dr. Stefano Rivella, a co-corresponding author of the study and a professor of pediatrics at Children’s Hospital of Philadelphia, stated, “Currently, if you want to use gene therapy to treat blood disorders like sickle cell disease and beta-thalassemia, patients must undergo conditioning treatments to make space for newly gene-corrected blood cells, which is expensive and risky. In our study, we demonstrated that we can replace diseased blood cells with gene-corrected blood cells directly in the body through a ‘one-and-done’ therapy without the need for myeloablative conditioning treatments, simplifying the delivery process of these potentially life-changing therapies. This represents a significant step forward in our approach to treating genetic diseases, with the potential to expand the use of gene therapy to the patients who need it most.”

Dr. Hamideh Parhiz, a co-corresponding author of the study and an assistant professor of infectious diseases at the Perelman School of Medicine at the University of Pennsylvania, said, “Targeted delivery of mRNA-encoded therapeutics to specific tissues and cell types will have a profound impact on the way nucleic acid-based therapies for diseases are delivered in the future. In our study, we provided a cell-specific targeting lipid nanoparticle (LNP) platform decorated with an antibody recognizing the CD117 receptor on hematopoietic stem cells. We combined this targeting platform with advances in mRNA therapy and RNA-based genome editing tools, offering a new approach to controlling hematopoietic stem cell fate and correcting genetic defects in vivo. mRNA-encoded targeted genome editing approaches offer controlled expression, high editing efficiency, and potentially safer in vivo genome modifications compared to currently available techniques.”

Hematopoietic stem cells (HSCs) reside in the bone marrow and continuously divide throughout life to generate all the cells in the blood and immune system. In non-malignant hematological diseases such as sickle cell disease and immunodeficiency disorders, these blood cells cannot function properly due to genetic mutations.

For these patients, there are currently two potentially curative treatment approaches, both involving bone marrow transplantation: one is to transplant HSCs from healthy donors, and the other is gene therapy, which involves genetically modifying the patient’s own HSCs outside the body and then transplanting them back in (often referred to as ex vivo gene therapy).

Both methods have risks, such as graft-versus-host disease in the case of donor transplantation, and both require conditioning regimens involving chemotherapy or radiation to eliminate the patient’s diseased HSCs, preparing them for the new cells. These conditioning regimens can have severe toxic side effects, making it necessary to explore less toxic approaches.

One approach that eliminates the need for both of the above methods is in vivo gene editing: directly injecting gene-editing tools into the patient’s body to edit and correct HSCs without the need for conditioning treatments.

To validate this approach, Dr. Laura Breda and Dr. Michael P. Triebwasser from Children’s Hospital of Philadelphia, along with Dr. Tyler E. Papp and mRNA vaccine pioneer Dr. Drew Weissman from the Perelman School of Medicine at the University of Pennsylvania, led a research team that used liquid nanoparticle (LNP) delivery to deliver mRNA gene-editing tools. LNPs are highly effective in packaging and delivering mRNA and were widely used for two COVID-19 vaccines in 2020.

However, for COVID-19 vaccines, LNP-mRNA constructs are not specifically targeted to particular cells or organs within the body. Since the authors wanted to target hematopoietic stem cells specifically, they decorated the surface of their experimental LNPs with antibodies recognizing the CD117 receptor on the surface of these stem cells. They then tested the efficacy of the CD117/LNP formulation through three methods.

First, they tested CD117/LNP-encapsulated reporter gene mRNA, showing successful in vivo mRNA expression and gene editing. Next, they investigated whether this approach could be used to treat hematologic diseases. They tested CD117/LNP-encapsulated mRNA encoding a Cas9 gene editor targeting the genetic mutation responsible for sickle cell disease. This gene-editing technique converts the disease-causing hemoglobin mutation into a non-disease-causing variant.

The authors tested their LNP-mRNA construct on hematopoietic stem cells from donors with sickle cell disease and found that CD117/LNP facilitated highly efficient base editing in vitro, resulting in a 91.7% increase in functional hemoglobin. They also demonstrated that sickle cells were almost entirely eliminated.

Finally, the authors explored whether LNPs could be used for in vivo conditioning, allowing marrow depletion without the use of chemotherapy or radiation. To do this, they used CD117/LNP to encapsulate mRNA encoding PUMA, a protein that promotes cell death.

In a series of in vitro and in vivo experiments, the authors found that CD117/LNP-mediated in vivo targeting effectively depleted diseased hematopoietic stem cells, allowing successful engraftment of new marrow cells without the need for chemotherapy or radiation. The engraftment rates observed in animal models were consistent with reported engraftment rates sufficient to cure severe combined immunodeficiency (SCID) using healthy donor marrow cells, suggesting that this technique may be used to treat severe immunodeficiency disorders.

Dr. Breda concluded, “These findings could revolutionize gene therapy by enabling not only cell-specific gene modifications in vivo with minimal risk but also by making previously impossible manipulations of hematopoietic stem cell physiology possible, offering a platform that, if adjusted correctly, could correct many different single-gene diseases. Such innovative delivery systems may help fulfill the promise of decades of collaboration between genetics and biomedical research to eradicate a range of human diseases.”