Part of the job as a pediatric hematologist for Michael P. Triebwasser, MD, PhD, is to take care of patients during their bone marrow transplantation. Just last week, he took care of a patient with a severe combined immunodeficiency (SCID) disorder and another with a bone marrow disorder.

Even though there has been progress in the use of autologous cells for ex vivo gene therapies for hematopoietic disorders like sickle cell disease and beta thalassemia, Triebwasser warns that it still requires the invasive procedure of taking cells out of the body, putting electrophoresis on these cells, and “conditioning” the patients to get rid of their own hematopoietic stem cells (HSCs) to make room for the gene-edited ones. That’s why Triebwasser said that he and others have been on the hunt for “the holy grail of HSC gene therapy”—in vivo genome editing of HSCs.

“In theory, we can replace that gene or correct it in some way and offer them a curative therapy that doesn’t have some of the same issues that hematopoietic stem cell transplantation (HSCT) does, namely graft versus host disease,” Triebwasser told GEN. “Some patients just don’t even have a donor available. When we look at large numbers, like large registries, some patients just don’t have a good option. There are some alternative transplantation methods using partially related donors, but those have some challenges and can have a lot of comorbidities.”

Nobel Laureate Jennifer Doudna, PhD, goes one step further, telling GEN that arguably the biggest challenge in realizing the full potential of CRISPR is improved in vivo delivery. Doudna said that this is important for developing therapies that are affordable and accessible, making it an active part of her lab’s research, publishing a preprint in April demonstrating cell-specific molecular delivery with enveloped delivery vehicles (EDVs) that enable genome editing ex vivo and in vivo. “To reach the point where CRISPR is the standard of care for all the types of diseases we know it can address, we need to be able to target more cell and tissue types precisely.”

In a study published today in Science, Triebwasser and co-first authors Laura Breda, PhD, and Tyler E. Papp demonstrated genome editing of HSCs in vivo (and ex vivo) through mRNA delivered by lipid nanoparticles (LNPs) decorated with targeting moieties. With the support of co-senior authors Stefano Rivella, PhD, from the Children’s Hospital of Philadelphia, and Hamideh Parhiz, PhD, from the Perelman School of Medicine at the University of Pennsylvania, they used LNPs targeting a stem cell factor on HSCs (CD117) for delivery of mRNA to correct human sickle cells ex vivo and to target HSCs in mice in vivo.

Stuart Orkin, MD, professor of pediatrics at Harvard Medical School and a Howard Hughes Medical Institute investigator, told GEN, “If the goal is to reduce the burden of sickle cell disease (or thalassemia) at a population level, it cannot be done with existing approaches to gene/editing therapy; an in vivo approach, if efficient enough in the clinical setting, can solve this problem. This is an excellent starting point.

This work builds off research published last January in Science, in which Papp and Parhiz were part of a research team that included Jonathan A. Epstein, PhD, and Carl June, MD, that demonstrated in vivo targeted LNP-based delivery of modified mRNA to differentiated T cells in vivo for treating cardiac injury.

“The beauty of this mRNA-LNP approach is that it’s a highly modular platform where we are able to decorate the surface of these LNPs, which were the foundation for the most efficacious COVID-19 vaccines for Moderna and Pfizer, to target certain cell types, and we can modify the mRNA cargo to express (or not express) in certain cell types in the body,” said Papp, a research scientist in the Parhiz lab. “I think we’re on the verge of designing personalized therapeutics that can have a higher regulation of unintended side effects.”

This research follows the publication of a few studies that share a great deal of overlap. In March 2023, Dennis Shi, Sho Toyonaga, and Daniel G. Anderson from the Massachusetts Institute of Technology (MIT) published an article in Nano Letters in which they used a similar approach with targeted LNPs to deliver mRNA to HSCs in vivo; however, they did not provide any functional studies. Also in April 2023, a study published in Blood demonstrated the use of adeno-associated virus (AAV) to deliver base editors for in vivo gene editing of HSCs from the labs of Hans-Peter Kiem, David R. Liu, Evangelia Yannaki, and André Lieber.

Targeted LNPs to HSCs

One limitation with LNP-based delivery is that there’s a pretty strong tropism for the liver. In some cases, this has been a boon for a number of clinical trials and companies. Intellia Therapeutics has an ongoing clinical trial for the in vivo editing of liver cells to treat hereditary transthyretin amyloidosis. Also, Verve Therapeutics has a single-course in vivo liver base editing medicine targeting PCSK9 is currently being evaluated in our Phase Ib heart-1 clinical trial in patients with high-risk heterozygous familial hypercholesterolemia (HeFH), established atherosclerotic cardiovascular disease (ASCVD), and uncontrolled LDL-C levels on oral standard-of-care therapy.

But for delivery beyond the liver, there is a specificity issue, which is why in this study the researchers sought to improve the targeting efficiency of HSCs by conjugating an antibody for a CD117 onto LNPs with diverse mRNA cargos.

“[The LNP] was very effective for the COVID vaccine that many people received from either Pfizer or Moderna,” said Triebwasser. “Here we’ve put an antibody on the outside to take the LNPs out of the bloodstream and into the bone marrow where the stem cells are, and we can actually get delivery of the mRNA to HSCs and gene edit those cells while they’re in their niche.”

They demonstrated that this targeted-LNP platform can deliver all sorts of advanced genome editing systems, such as adenine base editors (ABEs), to HSCs ex vivo. With the delivery of an anti-human CD117/LNP-based editing system, the researchers saw a near-complete correction of hematopoietic sickle cells.

“We showed that [the delivered advanced genome editing systems] are very effective— potentially the most effective reported strategy or modality for delivering ABEs for the correction of the sickle cell disease mutation with higher rates of correction than the gold standard for electroporation,” said Triebwasser. “The next step would be to try that same kind of genome editing of a disease allele in a model organism.”

The potential of ex vivo editing is in itself quite promising because it presents an alternative to the electroporation that’s being done currently for sickle cell disease and beta thalassemia by, for instance, Vertex Pharmaceuticals and CRISPR Therapeutics. These patients are having very good outcomes, showing the safety and efficacy of exa-cel, which is currently being evaluated by the FDA for approval to treat sickle cell disease and beta thalassemia. Nevertheless, a head-to-head comparison of viability and editing efficiency between electroporation and the LNP-based approach could prove valuable.

“Our data shows that there’s almost no effect on the viability of the primary [HSCs] with the LNP-based gene editing approach,” said Triebwasser. “So, as a start, I think it could be used to modify those cells ex vivo in place of electroporation, and this would increase the number of CD34+ HSCs or true stem cells that patients receive as part of that therapy and hopefully improve their clinical course.”

In vivo genome editing of HSCs

For in vivo genome editing, the researchers delivered CD117-targeted LNPs with pro-apoptotic PUMA (p53 upregulated modulator of apoptosis) mRNA that affected HSC function in the bone marrow niche in vivo, which permitted nongenotoxic conditioning for HSCT. With cargoes like PUMA that can kill off targeted cells, Papp said that he sees this technology as eventually replacing the chemotherapy necessary to ablate malignant hemopathies that require HSCT.

“Conventionally, CAR T-cell therapy is done through retroviral-based approaches that have a more permanent T-cell population, with patients showing continued success 10 years after they’ve been administered the T-cell,” said Papp. “One of the applications of this technology is for preconditioning for HSCTs or cancer chemotherapy. Instead of going through all of that, you just have to get one injection of these LNP-mRNA therapeutics, which, keep in mind, are acute—the mRNA expresses, degrades, and is gone from the body.”

There are many advantages to the in vivo gene editing approach, including cost, resources, and the need for chemotherapy or conditioning. But Papp, who worked a lot on the chemistry of conjugation, the targeting of the LNPs, and the design of the mRNA, said that there are worries about the off-targeting of cell types with their strategy because there are also cells in the lung that express CD117. Additionally, Triebwasser noted that the significance of this is currently unknown in the case of the sickle cell disease mutation because the targeted gene is pretty specifically expressed in hematopoietic cells and not in the lung or liver.

While companies are already using nucleoside-modified mRNA in GMP settings, IND-enabling studies may not be just around the corner. Given that targeted LNP-mRNA could be a game-changing technology for HSC gene therapy, further research into platform and process improvements should most likely be pursued at the preclinical level. 

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