RNA Based Gene Editing Model Identifies Blood Disorders

Researchers at the University of Pennsylvania’s Perelman School of Medicine and Children’s Hospital of Philadelphia (CHOP) have developed a proof-of-concept model for delivering gene editing tools to treat blood disorders, allowing for the modification of diseased blood cells directly within the body. If this strategy is carried into the clinic, it has the potential to increase access and lower the cost of gene therapies for blood disorders, many of which presently require patients to have chemotherapy and a stem cell transplant. The findings were published in the journal Science.

“Right now, if you want to treat hematologic diseases like sickle cell disease and beta thalassemia with gene therapy, patients must receive conditioning treatments like chemotherapy to make space for the new, corrected blood cells, which is both expensive and comes with risks,” said co-senior author Stefano Rivella, PhD, Kwame Ohene-Frempong Chair on Sickle Cell Anemia and Professor of Pediatrics at Children’s Hospital of Philadelphia. “In our paper, we have shown that it is possible to replace diseased blood cells with corrected ones directly within the body in a ‘one-and-done’ therapy, eliminating the need for myeloablative conditioning treatments and streamlining the delivery of these potentially life-changing treatments. This is a big step forward in how we think about treating genetic diseases and could expand the access of gene therapies to patients who need them most.”

“Targeted delivery of mRNA-encoded therapeutics to specific tissues and cell types will have an immense impact on the way diseases will be treated with nucleic acids in the future,” said senior author Hamideh Parhiz, PharmD, PhD, a research assistant professor of Infectious Diseases at Penn. “In our study, we are providing a cell-specific targeted lipid nanoparticle encapsulating

mRNA therapeutics/editors as a platform technology that can be used for in vivo cellular reprogramming in many diseases in need of a precisely targeted gene therapy modality. Here, we combined the targeted platform with advances in mRNA therapeutics and RNA-based genomic editing tools to provide a new way of controlling hematopoietic stem cell fate and correcting genetic defects. A targeted mRNA-encoded genomic editing methodology could lead to controlled expression, high editing efficacy, and potentially safer in vivo genomic modification compared to currently available technologies.”

Hematopoietic stem cells (HSCs) live in the bone marrow and divide throughout life to produce all blood and immune system cells. Because of a genetic abnormality, these blood cells do not function properly in patients with non-malignant hematological illnesses such as sickle cell disease and immunodeficiency disorders.

There are currently two potentially curative treatments available for these patients, both of which involve a bone marrow transplant: a stem cell transplant with HSCs from a healthy donor, or gene therapy in which the patient’s own HSCs are modified outside of the body and transplanted back in (also known as ex vivo gene therapy). Because the HSCs come from a donor, the former procedure carries the danger of graft versus host illness, and both processes entail a conditioning regimen of chemotherapy or radiation to destroy the patient’s sick HSCs and prepare them to absorb the new cells. These conditioning treatments have significant toxic side effects, emphasizing the need for less-toxic approaches.

In vivo gene editing, in which gene editing tools are administered directly into the patient, would eliminate the requirement for the previous procedures, allowing HSCs to be edited and repaired without the need for conditioning regimens.

A research team led by Laura Breda, PhD, and Michael P. Triebwasser, MD, PhD at CHOP (now at the University of Michigan), Tyler E. Papp, BS at Penn, and Drew Weissman, MD, PhD, the Roberts Family Professor in Vaccine Research, director of the Penn Institute for RNA Innovation, and a pioneer of mRNA-vaccine research, used liquid nanoparticles (LNP) to deliver mRNA gene editing tools. Because of the LNP-mRNA platform for two major COVID-19 vaccines, LNP are particularly effective in packaging and delivering mRNA to cells and will be widely used in 2020.

The LNP-mRNA construct, however, did not target specific cells or organs within the body in the case of the COVID-19 vaccinations. Because the researchers sought to precisely target HSCs, they coated the surface of their experimental LNPs with antibodies that recognized CD117, a receptor on the surface of HSCs. They then tested the efficacy of their CD117/LNP combination using three different ways.

The researchers first tested CD117/LNP encapsulating reporter mRNA in vivo to demonstrate successful in vivo mRNA expression and gene editing.

The researchers next looked at whether this method may be utilized to treat hematologic diseases. They tried CD117/LNP encapsulating mRNA encoding a cas9 gene editor targeting the sickle cell disease mutation. This method of gene editing transforms the

A disease-causing hemoglobin mutation has been converted into a non-disease-causing variation. Using cells from sickle cell disease donors, the researchers discovered that CD117/LNP promoted effective base editing in vitro, resulting in a 91.7% increase in functional hemoglobin. They also showed a nearly complete lack of sickled cells, the crescent-shaped blood cells that cause the disease’s symptoms.

Finally, the researchers investigated whether LNPs could be utilized for in vivo conditioning, allowing bone marrow to be depleted without the use of chemotherapy or radiation. They did this by encapsulating mRNA for PUMA, a protein that induces cell death, in CD117/LNP. The researchers demonstrated that in vivo targeting using CD117/LNP-PUMA successfully decreased HSC, allowing for successful infusion and uptake of new bone marrow cells, a process known as engraftment, without the use of chemotherapy or radiation. The engraftment rates found in animal models were commensurate with those previously reported to be sufficient for the cure of severe combined immunodeficiency (SCID) using healthy donor bone marrow cells, implying that this procedure could be employed for other severe immunodeficiencies.

“These findings may potentially transform gene therapy, not only by allowing cell-type specific gene modification in vivo with minimal risk, which could allow for previously impossibly manipulations of blood stem cell physiology but also by providing a platform that, if properly tuned, can correct many different monogenic disorders,” said Dr. Breda, a research assistant professor with the Division of Hematology at Children’s Hospital of Philadelphia. “Such novel delivery systems may help translate the promise of decades of concerted genetic and biomedical research to ablate a wide array of human diseases.”

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