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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Novel ultrasound uses microbubbles to open blood-brain barrier to treat glioblastoma in humans

A major impediment to treating the deadly brain cancer glioblastoma has been that the most potent chemotherapy can't permeate the blood-brain barrier to reach the aggressive brain tumor.

But now Northwestern Medicine scientists report results of the first in-human clinical trial in which they used a novel, skull-implantable ultrasound device to open the blood-brain barrier and repeatedly permeate large, critical regions of the human brain to deliver chemotherapy that was injected intravenously.

The four-minute procedure to open the blood-brain barrier is performed with the patient awake, and patients go home after a few hours. The results show the treatment is safe and well tolerated, with some patients getting up to six cycles of treatment.

This is the first study to successfully quantify the effect of ultrasound-based blood-brain barrier opening on the concentrations of chemotherapy in the human brain. Opening the blood-brain barrier led to an approximately four- to six-fold increase in drug concentrations in the human brain, the results showed.

Scientists observed this increase with two different powerful chemotherapy drugs, paclitaxel and carboplatin. The drugs are not used to treat these patients because they do not cross blood-brain barrier in normal circumstances.

In addition, this is the first study to describe how quickly the blood-brain barrier closes after sonication. Most of the blood-brain barrier restoration happens in the first 30 to 60 minutes after sonication, the scientists discovered. The findings will allow optimization of the sequence of drug delivery and ultrasound activation to maximize the drug penetration into the human brain, the authors said.

"This is potentially a huge advance for glioblastoma patients," said lead investigator Dr. Adam Sonabend, an associate professor of neurological surgery at Northwestern University Feinberg School of Medicine and a Northwestern Medicine neurosurgeon.

Temozolomide, the current chemotherapy used for glioblastoma, does cross the blood-brain barrier, but is a weak drug, Sonabend said.

The paper will be published May 2 in The Lancet Oncology.

The blood-brain barrier is a microscopic structure that shields the brain from the vast majority of circulating drugs. As a result, the repertoire of drugs that can be used to treat brain diseases is very limited. Patients with brain cancer cannot be treated with most drugs that are otherwise effective for cancer elsewhere in the body, as these do not cross the blood-brain barrier. Effective repurposing of drugs to treat brain pathology and cancer require their delivery to the brain.

In the past, studies that injected paclitaxel directly into the brain of patients with these tumors observed promising signs of efficacy, but the direct injection was associated with toxicity such as brain irritation and meningitis, Sonabend said.

Blood-brain barrier recloses after an hour

The scientists discovered that the use of ultrasound and microbubble-based opening of the blood-brain barrier is transient, and most of the blood-brain barrier integrity is restored within one hour after this procedure in humans.

"There is a critical time window after sonification when the brain is permeable to drugs circulating in the bloodstream," Sonabend said.

Previous human studies showed that the blood-brain barrier is completely restored 24 hours after brain sonication, and based on some animal studies, the field assumed that the blood-brain barrier is open for the first six hours or so. The Northwestern study shows that this time window might be shorter.

In another first, the study reports that using a novel skull-implantable grid of nine ultrasound emitters designed by French biotech company Carthera opens the blood-brain barrier in a volume of brain that is nine times larger than the initial device (a small single-ultrasound emitter implant). This is important because to be effective, this approach requires coverage of a large region of the brain adjacent to the cavity that remains in the brain after removal of glioblastoma tumors.

Clinical trial for patients with recurrent glioblastoma

The findings of the study are the basis for an ongoing phase 2 clinical trial the scientists are conducting for patients with recurrent glioblastoma. The objective of the trial -- in which participants receive a combination of paclitaxel and carboplatin delivered to their brain with the ultrasound technique -- is to investigate whether this treatment prolongs survival of these patients. A combination of these two drugs is used in other cancers, which is the basis for combining them in the phase 2 trial.

In the phase 1 clinical trial reported in this paper, patients underwent surgery for resection of their tumors and implantation of the ultrasound device. They started treatment within a few weeks after the implantation.

Scientists escalated the dose of paclitaxel delivered every three weeks with the accompanying ultrasound-based blood-brain barrier opening. In subsets of patients, studies were performed during surgery to investigate the effect of this ultrasound device on drug concentrations. The blood-brain barrier was visualized and mapped in the operating room using a fluorescent die called fluorescein and by MRI obtained after ultrasound therapy.

"While we have focused on brain cancer (for which there are approximately 30,000 gliomas in the U.S.), this opens the door to investigate novel drug-based treatments for millions of patients who suffer from various brain diseases," Sonabend said.

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Gel-like, radioactive tumor implant obliterates pancreatic cancer in mice

Biomedical engineers at Duke University have demonstrated the most effective treatment for pancreatic cancer ever recorded in mouse models. While most mouse trials consider simply halting growth a success, the new treatment completely eliminated tumors in 80% of mice across several model types, including those considered the most difficult to treat.

The approach combines traditional chemotherapy drugs with a new method for irradiating the tumor. Rather than delivering radiation from an external beam that travels through healthy tissue, the treatment implants radioactive iodine-131 directly into the tumor within a gel-like depot that protects healthy tissue and is absorbed by the body after the radiation fades away.

The results appear online October 19 in the journal Nature Biomedical Engineering.

"We did a deep dive through over 1100 treatments across preclinical models and never found results where the tumors shrank away and disappeared like ours did," said Jeff Schaal, who conducted the research during his PhD in the laboratory of Ashutosh Chilkoti, the Alan L. Kaganov Distinguished Professor of Biomedical Engineering at Duke. "When the rest of the literature is saying that what we're seeing doesn't happen, that's when we knew we had something extremely interesting."

Despite accounting for only 3.2% of all cancer cases, pancreatic cancer is the third leading cause of cancer-related death. It is a very difficult to treat because its tumors tend to develop aggressive genetic mutations that make it resistant to many drugs, and it is typically diagnosed very late, when it has already spread to other sites in the body.

The current leading treatment combines chemotherapy, which keeps cells in a stage of reproduction vulnerable to radiation for longer periods of time, with a beam of radiation targeted at the tumor. This approach, however, is ineffective unless a certain threshold of radiation reaches the tumor. And despite recent advances in shaping and targeting radiation beams, that threshold is very difficult to reach without risking severe side effects.

Another method researchers have tried involves implanting a radioactive sample encased in titanium directly within the tumor. But because titanium blocks all radiation other than gamma rays, which travel far outside the tumor, it can only remain within the body for a short period of time before damage to surrounding tissue begins to defeat the purpose.

"There's just no good way to treat pancreatic cancer right now," said Schaal, who is now director of research at Cereius, Inc., a Durham, North Carolina biotechnology startup working to commercialize a targeted radionuclide therapy through a different technology scheme.

To skirt these issues, Schaal decided to try a similar implantation method using a substance made of elastin-like polypeptides (ELPs), which are synthetic chains of amino acids bonded together to form a gel-like substance with tailored properties. Because ELPs are a focus of the Chilkoti lab, he was able to work with colleagues to design a delivery system well suited for the task.

The ELPs exist in a liquid state at room temperature but form a stable gel-like substance within the warmer human body. When injected into a tumor along with a radioactive element, the ELPs form a small depot encasing radioactive atoms. In this case, the researchers decided to use iodine-131, a radioactive isotope of iodine, because doctors have used it widely in medical treatments for decades and its biological effects are well understood.

The ELP depot encases the iodine-131 and prevents it from leaking out into the body. The iodine-131 emits beta radiation, which penetrates the biogel and deposits almost all its energy into the tumor without reaching the surrounding tissue. Over time, the ELP depot degrades into its constituent amino acids and is absorbed by the body -- but not before the iodine-131 has decayed into a harmless form of xenon.

"The beta radiation also improves the stability of the ELP biogel," Schaal said. "That helps the depot last longer and only break down after the radiation is spent."

In the new paper, Schaal and his collaborators in the Chilkoti laboratory tested the new treatment in concert with paclitaxel, a commonly used chemotherapy drug, to treat various mouse models of pancreatic cancer. They chose pancreatic cancer because of its infamy for being difficult to treat, hoping to show that their radioactive tumor implant creates synergistic effects with chemotherapy that relatively short-lived radiation beam therapy does not.

The researchers tested their approach on mice with cancers just under their skin created by several different mutations known to occur in pancreatic cancer. They also tested it on mice that had tumors within the pancreas, which is much more difficult to treat.

Overall, the tests saw a 100% response rate across all models, with the tumors being completely eliminated in three-quarters of the models about 80% of the time. The tests also revealed no immediately obvious side effects beyond what is caused by chemotherapy alone.

"We think the constant radiation allows the drugs to interact with its effects more strongly than external beam therapy allows," Schaal said. "That makes us think that this approach might actually work better than external beam therapy for many other cancers, too."

The approach, however, is still in its early preclinical stages and will not be available for human use anytime soon. The researchers say their next step is large animal trials, where they will need to show that the technique can be accurately done with the existing clinical tools and endoscopy techniques that doctors are already trained on. If successful, they look toward a Phase 1 clinical trial in humans.

"My lab has been working on developing new cancer treatments for close to 20 years, and this work is perhaps the most exciting we have done in terms of its potential impact, as late-stage pancreatic cancer is impossible to treat and is invariably fatal," Chilkoti said. "Pancreatic cancer patients deserve better treatment options than are currently available, and I am deeply committed to taking this all the way into the clinic."

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Stem cell discoveries hold potential to improve cancer treatment

Two recent discoveries by stem cell scientists at Cedars-Sinai may help make cancer treatment more efficient and shorten the time it takes for people to recover from radiation and chemotherapy.

In the first study, published in the journal Blood, investigators discovered a protein that is expressed by blood stem cells that could aid in identifying, studying and deploying the cells for treatments.

"We show that this protein, syndecan-2, identifies primitive blood stem cells and it regulates stem cell function," said John Chute, MD, director of the Division of Hematology and Cellular Therapy at Cedars-Sinai and senior author of the study.

Blood stem cells are found in small quantities in the bone marrow and in peripheral blood -- the type that travels through the heart, arteries, capillaries and veins. These stem cells are of interest to scientists because they produce all blood cells and immune cells in the body. They are used in the curative treatment of people with leukemia and lymphoma.

This approach faces a major challenge: Hematopoietic stem cells make up less than 0.01% of cells in the bone marrow and peripheral blood, and there is not yet a good way to separate them from other cells. This means that when people receive infusions of bone marrow and peripheral blood cells, they get a tiny number of stem cells that are therapeutic along with a lot of other cells that are not.

To study this phenomenon, investigators at the Chute laboratory led by first author Christina M. Termini, PhD, extracted bone marrow cells from adult mice and ran the samples through a device that can detect hundreds of different types of cells based on the proteins that live on their surfaces.This process revealed that hematopoietic stem cells have a high concentration of syndecan-2, which is part of a family of proteins called heparan sulfate proteoglycans, on the cell surface.

The researchers found this protein plays an important role in how hematopoietic stem cells reproduce. When stem cells that express syndecan-2 were transplanted into mice following irradiation, their cells repopulated. But when stem cells that lacked syndecan-2 were transplanted, the cells stopped replicating.

By transplanting only cells that express syndecan-2, it may be possible to make blood stem cell transplants more efficient and less toxic.

Second Discovery

The second discovery by Chute and his team -- published in the journal Nature Communications -- revealed a mechanism through which the blood vessels in the bone marrow respond to injury, such as from chemotherapy or radiation.

When people receive radiation or chemotherapy as part of their cancer treatment, their blood counts plummet. It typically takes several weeks for these counts to return to normal levels.

Chute and colleages found that when mice receive radiation treatment, the cells that line the inner walls of the blood vessels in the bone marrow produce a protein called semaphorin 3A. This protein tells another protein, called neuropilin 1, to kill damaged blood vessels in the bone marrow.

When the investigators blocked the ability of these blood vessel cells to produce neuropilin 1 or semaphorin 3A, or injected an antibody that blocks semaphorin 3A communication with neuropilin 1, the bone marrow vasculature regenerated following irradiation. In addition, blood counts increased dramatically after one week.

"We've discovered a mechanism that appears to control how blood vessels regenerate following injury," said Chute, senior author of the paper. "Inhibiting this mechanism causes rapid recovery of the blood vessels and blood cells in bone marrow following chemotherapy or irradiation. In principle, targeting this mechanism could allow patients to recover following chemotherapy in one to two weeks, instead of three or four weeks as currently experienced."

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Half the amount of chemo prevents testicular cancer from coming back, new trial shows

Testicular cancer can be prevented from coming back using half the amount of chemotherapy that is currently used, a new clinical trial has shown.

In many men who have had surgery for an aggressive form of testicular cancer, the disease can come back elsewhere in their bodies and need intensive treatment, often within two years after initial diagnosis.

The new trial showed that giving men one cycle of chemotherapy was as effective at preventing men's testicular cancer from coming back as the two cycles used as standard.

Crucially, lowering the overall exposure to chemotherapy reduced the debilitating side effects which can have a lifelong impact on patients' health.

The 111 trial has already begun to change clinical practice, reducing the number of hospital admissions, and lowering the costs of treatment.

The trial, led by The Institute of Cancer Research, London, and University Hospitals Birmingham NHS Foundation Trust, involved nearly 250 men with early-stage testicular cancer at high risk of their cancer returning after surgery.

The research was published in the journal European Urology today (Thursday), and was funded by Cancer Research UK and the Queen Elizabeth Hospital Birmingham Charity.

Testicular cancer is the most common cancer affecting young men, with many patients being diagnosed in their twenties or thirties.

After surgery, patients are currently offered two cycles of chemotherapy to destroy any cancer cells that may have already spread, or a watch-and-wait approach -- where they receive no treatment unless their cancer comes back, at which point they are given three cycles of chemo.

Survival rates are very high, but as men are diagnosed young, if they choose to have chemotherapy they may have to live with long-term side effects for many decades.

In the new study, patients were given one three-week cycle of a chemotherapy known as BEP -- a combination of the drugs bleomycin, etoposide and the platinum agent cisplatin.

The researchers looked at the percentage of men whose testicular cancer returned within two years of being treated with one cycle of chemotherapy, and compared these relapse rates with established data from previous studies in patients who were given two cycles.

The researchers found that only three men -- 1.3 per cent -- saw their testicular cancer return after finishing treatment -- a nearly identical rate to previous studies using two cycles of BEP chemotherapy.

In the new study, 41 per cent of men receiving one cycle of chemotherapy experienced one or more serious side effects while receiving treatment, such as an increased risk of infection, sepsis or vomiting. But only a small number -- six patients, or 2.6 per cent -- experienced long-term side effects such as damage to their hearing.

It is well established that lower chemotherapy doses are related to reduced rates of side effects, and the researchers are confident that the rates found in this study are substantially lower than those currently seen in the clinic.

Professor Robert Huddart, Professor of Urological Cancer at The Institute of Cancer Research, London, and Consultant in Urological Oncology at The Royal Marsden NHS Foundation Trust, said:

"Men with testicular cancer who are at high risk of recurrence have generally been treated with two cycles of chemotherapy -- but our new study found that one cycle was enough to stop their tumour from coming back.

"Reducing the overall dose of chemotherapy could spare young men who have their whole lives ahead of them from long-term side effects, and also means they will need fewer hospital visits for their treatment.

"This new trial is already changing clinical practice on a global scale, and is set to improve patients' quality of life as well as reducing the cost of testicular cancer treatment.

"Reducing the number of cycles and the dosage of chemotherapy for testicular cancer could save the NHS money, and free up valuable hospital time and resources."

Kris Taylor, 35, from the West-Midlands, was treated as part of the 111 trial at the Queen Elizabeth Hospital Birmingham after having surgery for his testicular cancer. He said:

"I was playing football semi-professionally at the time I was diagnosed. Even though my prognosis was good, knowing that you have cancer is really scary, and the key thing for me was to get back to normality as soon as possible. I'd already had to have time off for surgery, so, when I was offered the chance to have less chemo but with no greater risk the cancer would return, I jumped at it.

"The side effects of the treatment were really difficult, but I was straight back on the pitch as soon as it finished -- five years on, and I'm still fighting fit. It's great to know that others may now be able to benefit from the trial's findings. Being able to reduce the amount of chemotherapy a person receives can make such a big difference to their quality of life in both the short-term and the long-term."

Professor Emma Hall, Deputy Director of the Clinical Trials and Statistics Unit at The Institute of Cancer Research, London, said:

"We tend to be focused on whether we can cure a cancer or not, but for a disease like testicular cancer which affects young people, it is also crucial to ensure treatment does not leave patients with a lifetime of adverse effects.

"There is an important balance to be struck in giving men enough chemotherapy to stop their testicular cancer from coming back, without giving them so much that they suffer unnecessary side effects.

"Our study has found strong evidence to suggest that testicular cancer chemotherapy can be safely reduced from two cycles to just one -- making their treatment shorter, kinder and cheaper."

Martin Ledwick, Cancer Research UK's head information nurse, said:

"Thanks to advances in treatments, survival for testicular cancer is very high, but the chemotherapy can cause unpleasant, sometimes lasting side effects. That's why it's such good news to see that we can cut down the amount of treatment we give.

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