Researchers develop 'in vivo' RNA-based gene editing model for blood disorders

In a step forward in the development of genetic medicines, researchers at Children's Hospital of Philadelphia (CHOP) and the Perelman School of Medicine at the University of Pennsylvania 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 translated into the clinic, this approach could expand access and reduce the cost of gene therapies for blood disorders, many of which currently require patients receive chemotherapy and a stem cell transplant. The findings were published today 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) reside in the bone marrow, where they divide throughout life to produce all cells within the blood and immune system. In patients with non-malignant hematopoietic disorders like sickle cell disease and immunodeficiency disorders, these blood cells don't function correctly because they carry a genetic mutation.

For these patients, there are currently two avenues for potentially curative treatments, 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 (often referred to as ex vivo gene therapy). The former approach comes with the risk of graft versus host disease, given that the HSCs come from a donor, and both processes involve a conditioning regimen of chemotherapy or radiation to eliminate the patient's diseased HSCs and prepare them to receive the new cells. These conditioning procedures come with significant toxic side effects, underscoring the need to investigate less-toxic approaches.

One option that would eliminate the need for the above methods would be in vivo gene editing, in which gene editing tools are infused directly into the patient, allowing HSCs to be edited and corrected without the need for conditioning regimens.

To validate this approach, a research team led by Laura Breda, PhD, and Michael P. Triebwasser, MD, PhD at CHOP (presently at the University of Michigan), Tyler E. Papp, BS at Penn, and Drew Weissman, MD, PhD, the Roberts Family Professor in Vaccine Research, the director of the Penn Institute for RNA Innovation, and a pioneer of mRNA-vaccine research, used liquid nanoparticle (LNP) to deliver mRNA gene editing tools. LNP are highly effective at packaging and delivering mRNA to cells and became widely utilized in 2020, due to the LNP-mRNA platform for two leading COVID-19 vaccines.

However, in the case of the COVID-19 vaccines, the LNP-mRNA construct did not target specific cells or organs within the body. Given that the researchers wanted to target HSCs specifically, they decorated the surface of their experimental LNPs with antibodies that would recognize CD117, a receptor on the surface of HSCs. They then pursued three approaches to test the efficacy of their CD117/LNP formulation.

First, the researchers tested CD117/LNP encapsulating reporter mRNA to show successful in vivo mRNA expression and gene editing.

Next, the researchers investigated whether this approach could be used as a therapy for hematologic disease. They tested CD117/LNP encapsulating mRNA encoding a cas9 gene editor targeting the mutation that causes sickle cell disease. This type of gene editing converts the disease-causing hemoglobin mutation into a non-disease-causing variant. Testing their construct on cells from donors with sickle cell disease, the researchers showed that CD117/LNP facilitated efficient base editing in vitro, leading to a corresponding increase in functional hemoglobin of up to 91.7%. They also demonstrated a nearly complete absence of sickled cells, the crescent-shaped blood cells that cause the symptoms of the disease.

Finally, the researchers explored whether LNPs could be used for in vivo conditioning, which would allow bone marrow to be depleted without chemotherapy or radiation. To do so, they used CD117/LNP encapsulating mRNA for PUMA, a protein that promotes cell death. In a series of in vitro, ex vivo, and in vivo experiments, the researchers showed that in vivo targeting with CD117/LNP-PUMA effectively depleted HSC, allowing for successful infusion and uptake of new bone marrow cells, a process known as engraftment, without need of chemotherapy or radiation. The engraftment rates observed in animal models were consistent with those reported to be sufficient for the cure of severe combined immunodeficiency (SCID) using healthy donor bone marrow cells, suggesting this technique could be used for severe immunodeficiences.

"These findings may potentially transform gene therapy, not only by allowing cell-type specific gene modification in vivowith 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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CRISPR gene editing reveals biological mechanism behind common blood disorder

UNSW researchers have used CRISPR gene editing -- a type of 'molecular scissors' -- to understand how deletions in one area of the genome can affect the expression of nearby genes. The work, led by UNSW Associate Professor Kate Quinlan and Professor Merlin Crossley, together with collaborators from the US, will help researchers investigate new therapeutic approaches for one of the world's most devastating genetic blood disorders -- sickle cell disease.

Asymptomatic sickle cell disease patients actually lack a tiny part of the genome, scientists have shown.

The team's findings are published today in academic journal Blood. (Just last week, A/Prof. Quinlan and Prof. Crossley received a $412,919 ARC linkage grant to fund a collaboration between UNSW Sydney and CSL that follows on from the work described in this paper.)

"Sickle cell disease and beta thalassemia, a closely related disease, are inherited genetic conditions that affect red blood cells. They are fairly common worldwide -- over 318,000 infants with these conditions are born every year, and haemoglobin disorders cause 3 per cent of deaths in children aged under five years worldwide," says co-lead author A/Prof. Quinlan.

Genetic mutations -- specifically, a defect in the adult globin gene -- are responsible for the disorders. The mutant genes affect the production of haemoglobin, the protein in red blood cells that carries oxygen around our bodies.

"Interestingly, when children are born, they don't show disease symptoms at first, even if they have the mutations, because at that stage, they're still expressing foetal globin and not yet adult globin. That's because we have different haemoglobin genes that we express at different stages of development," says A/Prof. Quinlan.

"As the foetal globin gets turned off, and adult globin gets turned on -- which happens within about the first year of life -- the symptoms start to manifest."

When that happens, the red blood cells take on unusual, sickled shapes and block small blood vessels, causing pain, organ damage, and premature death. The disease is particularly common in tropical countries, and in people from places where malaria is endemic.

"The goal of our research is finding out how we can reverse the foetal to adult globin switch, so that patients continue to express foetal globin throughout life, rather than the mutant adult globin genes that cause blood cells to become stiff and block vessels," says A/Prof. Quinlan.

Interestingly, this already happens in some people with sickle cell disease: thanks to another, beneficial genetic mutation, a rare subset of patients keeps the foetal globin gene 'on' throughout their life and are protected from sickle cell symptoms.

"In these patients, the persistent expression of foetal globin effectively compensates for the defective adult globin -- but up until this piece of research, we didn't really understand the process that led to this incredible advantage," A/Prof. Quinlan says.

'Deleting' genes with CRISPR

To get to the bottom of what's going on in these lucky people's genome, UNSW PhD student Sarah Topfer compiled data on the rare families that express foetal globin throughout life.

"As a first step, Sarah compared deletions in lots of different patients' genomes -- essentially, she looked to see if any shared element was missing in all of them. What do these patients have in common? She found one very small region was deleted in all these patients' genomes."

Sarah then used CRISPR gene editing to replicate some of these big patient deletions -- and the small deleted bit they all had in common -- in cell lines in the lab.

"CRISPR allows us to 'cut' bits of DNA out of cells grown in the lab, to modify genes and see what happens as a result -- it's essentially a tool to figure out what genes do inside living cells," A/Prof. Quinlan says.

"We found that deleting just that one little bit was sufficient to make foetal globin go up and adult globin down -- which suggests that we have found the key mechanism that can explain why foetal globin levels remains high in these asymptomatic patients," A/Prof. Quinlan says.

"Effectively, by deleting the adult globin 'on switch', we made the foetal globin 'on switch' active."

Prof. Quinlan says the results were unexpected.

"It was surprising to see the findings -- many people have studied these mutations for many years, so the idea that there'd be one unifying hypothesis that could explain them rather than them all working through different mechanisms will be surprising for the field.

"While we went in with the hypothesis that there might be one mechanism, we didn't expect it to come out so cleanly -- we thought that perhaps it would be more complicated than what we'd initially thought."

The CRISPR revolution and potential therapies

Co-lead author Prof. Crossley, who is also UNSW's Deputy Vice-Chancellor, Academic & Student Life, says it was impossible to test this model prior to the advent of CRISPR gene editing.

"Our group has specialised in using this new technology to understand globin gene switching," Prof. Crossley says. "Australia now has a significant number of people with either sickle cell disease or thalassemia.

"The work, supported by the National Health and Medical Research Council, is an important example of how the CRISPR gene editing revolution is accelerating scientific understanding and will deliver new therapies to the clinic."

The scientists say the work revealed today is improving our fundamental understanding of the mechanism behind sickle cell disease.

"What this really helps us to do is understand this process of turning off foetal globin and turning on adult globin and how we could reverse that, so that we can use this understanding of the mechanism to help us look for new therapeutic approaches -- it's a key piece of the puzzle," A/Prof. Quinlan says.

Some of Prof. Crossley's team's previous discoveries in the field are informing clinical trials already -- by using beneficial mutations they've discovered in the past that could lead to therapies for these disorders.

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