A cure inside the womb

The lifelong impact of cystic fibrosis (CF), a chronic—and sometimes fatal—multi-organ disease, begins before a baby is even born. As early as 16 weeks of gestation, babies with CF already show signs of damage to their pancreas, and upon birth, experience reduced birth weight, meconium ileus (a bowel obstruction), and other major issues. It results in a lifelong chronic inflammatory lung disease that leads to a shortened life expectancy.

A multidisciplinary team at Yale is uniting an array of cutting-edge technologies to make the leap to treating this disease in utero, before much of the irreparable organ damage begins. Yale medical students have been at the heart of this effort, working across four labs alongside some of Yale’s best-known innovators. Their insights have helped the strategy’s building blocks—in utero gene editing, delivered via nanoparticles—fall into place.

One of those students is ninth-year MD/PhD student Adele Ricciardi, who arrived at Yale aiming to apply her keen interest in biomedical engineering and surgery to translational research. She began her Yale career splitting time between the labs of W. Mark Saltzman, PhD, chair and Goizueta Foundation Professor of Biomedical Engineering and Peter Glazer, MD ‘87, PhD ‘87, chair and Robert E. Hunter Professor of Therapeutic Radiology and professor of Genetics. Saltzman is widely known for his transformative work engineering nanoparticles: tiny—fewer than a thousand nanometers in size—biodegradable polymer droplets that can act as powerful and versatile drug delivery vehicles. Glazer is a pioneer of gene editing via PNA, or peptide nucleic acid. PNA can be devised to bind to a particular gene containing a mutation in a strand of DNA, causing a lesion that the cell itself is compelled to remove. The gene is then corrected when a strand of DNA without the mutation is inserted in the mutation’s place.

These are two powerful advances on their own. Together, their potential is exponentially greater. Enter the students: according to Ricciardi, another MD-PhD student, Nicole McNeer, who also worked in Saltzman’s lab, attended an MD-PhD retreat. There, Joanna Chin, a researcher from Peter Glazer’s lab, Joanna Chin, was presenting work on delivering PNA into cells via electroporation, in which electricity temporarily punctures a cell membrane. There were limitations to this technology: “You can do that in a dish of cells, but not in a living organism,” said Ricciardi. At the seminar, McNeer approached Chin and said that she had an idea for how they might deliver these molecules in a safe and gentle way. Doctors could load nanoparticles with PNA and corrected DNA, producing synthetic vehicles that carried their contents into a target cell. “This was a huge advance,” Ricciardi said. McNeer is now a hematology/oncology fellow at Memorial Sloan Kettering Cancer Center in New York.

Armed with this powerful combination, Glazer’s and Saltzman’s labs began to examine how this strategy would work, particularly in blood diseases such as sickle cell anemia and thalassemia. However, near the end of McNeer’s PhD, Ricciardi said, the team had another idea: because it is caused by a single mutation, they believed CF would make a challenging but excellent candidate for gene editing therapy. McNeer began working in the lab of Marie Egan, MD, professor of pediatrics (respiratory), and of cellular and molecular physiology, and director of Yale’s Cystic Fibrosis Center.

“CF is a real challenge for gene editing because there are so many organs and tissues that are involved,” said Egan. There was also the problem of how to best get the PNA-loaded nanoparticles to the right cells to address the organs impacted by CF. In 2010, Egan applied for a grant from The Hartwell Foundation, which funds pediatric research at top medical institutions, to investigate how this could be done. “They support projects that are very innovative and high risk, which often are not funded by other organizations,” said Egan. The risk taken by Hartwell and the Yale team proved fruitful: in a Nature Communications paper, the team demonstrated that by delivering PNA-loaded nanoparticles to mice via inhalation, they were able to correct the most common mutation in CF.

The team then had another idea: in utero delivery. According to Ricciardi, “There is already so much disease present at birth in CF patients, I thought, if we can do systemic correction, can we correct all of the organs where the patient is experiencing disease? It seemed possible we could do that in utero.” Prenatal testing can now diagnose CF in fetuses at a very early stage of development, in time to intervene to prevent some of this damage before birth.

McNeer, Ricciardi said, working with another MD/PhD student, Elias Quijano, along with post-doctoral fellow, Raman Bahal, in Glazer’s lab, discovered that stem cells can be edited at a higher frequency than already differentiated cells, and they’re more abundant in utero, when organs are developing, than after birth. For her own PhD work, Ricciardi built on that idea. “Can we edit an organism when it is mainly stem progenitor cells?” Ricciardi asked. “And can we try to deliver nanoparticles directly to the fetus intravenously or into the amniotic fluid?”

Serendipitously, she said, David Stitelman, MD, assistant professor of surgery (pediatrics) arrived at Yale close to this time. He applied his experience with fetal therapy and viral vectors to achieve gene editing therapy in utero. Stitelman’s lab then became the fourth to join the collaboration.

“If you are going to correct a disease long-term, you want to correct the stem cells that make the tissue,” said Stitelman. Another compelling reason for gene editing in utero is cost. Because of the size of the dose required in comparison to the size of the patient, he said, the treatment would be much more expensive in an adult than in a fetus.

“Adele has done a masterful job leading a collaborative effort among the Saltzman, Glazer, Stitelman, and Egan labs at Yale to advance PNA/nanoparticle technology for in utero gene editing in mouse models of both thalassemia and cystic fibrosis,” said Glazer. “Her cystic fibrosis work, although in early stages, has established the possibility that in utero intervention could provide for amelioration of both pulmonary and GI manifestations of the disease.”

Right now, “We have really convincing data that we can correct this gene mutation in utero and that this results in sustained activity of that protein into adulthood of these mice,” Ricciardi said. If continued examination and development of this strategy is successful, the promise to patients is great. “We can make the quality of life better for a child and their family,” said Ricciardi. As a Yale medical student, she was paired with a CF patient through the Yale Postdoc Buddy program, and that patient, who is now 15, and her family, have become an inspiration to her. “Seeing what it is like to be a patient with CF, and what this can mean for someone is really motivating. That is why translational medicine is so powerful.”