In June 2012, Kirstie Ennis was six months into her second deployment to Afghanistan and recently promoted to sergeant. The helicopter gunner and seven others were three hours into a routine mission of combat resupplies and troop transport when their CH-53D helicopter went down hard.
Miraculously, all eight people onboard survived, but Ennis' injuries were many and severe. She had a torn rotator cuff, torn labrum, crushed cervical discs, facial fractures, deep lacerations and traumatic brain injury. Despite a severely fractured ankle, doctors managed to save her foot, for a while at least.
In November 2015, after three years of constant pain and too many surgeries to count, Ennis relented. She elected to undergo a lower leg amputation but only after she completed the 1,000-mile, 72-day Walking with the Wounded journey across the UK.
On Veteran's Day of that year, on the other side of the country, orthopedic surgeon Cato Laurencin announced a moonshot challenge he was setting out to achieve on behalf of wounded warriors like Ennis: the Hartford Engineering A Limb (HEAL) Project.
Laurencin, who is a University of Connecticut professor of chemical, materials and biomedical engineering, teamed up with experts in tissue bioengineering and regenerative medicine from Harvard, Columbia, UC Irvine and SASTRA University in India. Laurencin and his colleagues at the Connecticut Convergence Institute for Translation in Regenerative Engineering made a bold commitment to regenerate an entire limb within 15 years – by the year 2030.
Dr. Cato Laurencin pictured in his office at UConn.
Photo Credit: UConn
Regenerative Engineering -- A Whole New Field
Limb regeneration in humans has been a medical and scientific fascination for decades, with little to show for the effort. However, Laurencin believes that if we are to reach the next level of 21st century medical advances, this puzzle must be solved.
An estimated 185,000 people undergo upper or lower limb amputation every year. Despite the significant advances in electromechanical prosthetics, these individuals still lack the ability to perform complex functions such as sensation for tactile input, normal gait and movement feedback. As far as Laurencin is concerned, the only clinical answer that makes sense is to regenerate a whole functional limb.
Laurencin feels other regeneration efforts were hampered by their siloed research methods with chemists, surgeons, engineers all working separately. Success, he argues, requires a paradigm shift to a trans-disciplinary approach that brings together cutting-edge technologies from disparate fields such as biology, material sciences, physical, chemical and engineering sciences.
As the only surgeon ever inducted into the academies of Science, Medicine and Innovation, Laurencin is uniquely suited for the challenge. He is regarded as the founder of Regenerative Engineering, defined as the convergence of advanced materials sciences, stem cell sciences, physics, developmental biology and clinical translation for the regeneration of complex tissues and organ systems.
But none of this is achievable without early clinician participation across scientific fields to develop new technologies and a deeper understanding of how to harness the body's innate regenerative capabilities. "When I perform a surgical procedure or something is torn or needs to be repaired, I count on the body being involved in regenerating tissue," he says. "So, understanding how the body works to regenerate itself and harnessing that ability is an important factor for the regeneration process."
The Birth of the Vision
Laurencin's passion for regeneration began when he was a sports medicine fellow at Cornell University Medical Center in the early 1990s. There he saw a significant number of injuries to the anterior cruciate ligament (ACL), the major ligament that stabilizes the knee. He believed he could develop a better way to address those injuries using biomaterials to regenerate the ligament. He sketched out a preliminary drawing on a napkin one night over dinner. He has spent the next 30 years regenerating tissues, including the patented L-C ligament.
As chair of Orthopaedic Surgery at the University of Virginia during the peak of the wars in Iraq and Afghanistan, Laurencin treated military personnel who survived because of improved helmets, body armor and battlefield medicine but were left with more devastating injuries, including traumatic brain injuries and limb loss.
"I was so honored to care for them and I so admired their steadfast courage that I became determined to do something big for them," says Laurencin.
When he tells people about his plans to regrow a limb, he gets a lot of eye rolls, which he finds amusing but not discouraging. Growing bone cells was relatively new when he was first focused on regenerating bone in 1987 at MIT; in 2007 he was well on his way to regenerating ligaments at UVA when many still doubted that ligaments could even be reconstructed. He and his team have already regenerated torn rotator cuff tendons and ACL ligaments using a nano-textured fabric seeded with stem cells.
Even as a finalist for the $4 million NIH Pioneer Award for high-risk/high-reward research, he faced a skeptical scientific audience in 2014. "They said, 'Well what do you plan to do?' I said 'I plan to regenerate a whole limb in people.' There was a lot of incredulousness. They stared at me and asked a lot of questions. About three days later, I received probably the best score I've ever gotten on an NIH grant."
In the Thick of the Science
Humans are born with regenerative abilities--two-year-olds have regrown fingertips--but lose that ability with age. Salamanders are the only vertebrates that can regenerate lost body parts as adults; axolotl, the rare Mexican salamander, can grow extra limbs.
The axolotl is important as a model organism because it is a four-footed vertebrate with a similar body plan to humans. Mapping the axolotl genome in 2018 enhanced scientists' genetic understanding of their evolution, development, and regeneration. Being easy to breed in captivity allowed the HEAL team to closely study these amphibians and discover a new cell type they believe may shed light on how to mimic the process in humans.
"Whenever limb regeneration takes place in the salamander, there is a huge amount of something called heparan sulfate around that area," explains Laurencin. "We thought, 'What if this heparan sulfate is the key ingredient to allowing regeneration to take place?' We found these groups of cells that were interspersed in tissues during the time of regeneration that seemed to have connections to each other that expressed this heparan sulfate."
Called GRID (Groups that are Regenerative, Interspersed and Dendritic), these cells were also recently discovered in mice. While GRID cells don't regenerate as well in mice as in salamanders, finding them in mammals was significant.
"If they're found in mice. we might be able to find these in humans in some form," Laurencin says. "We think maybe it will help us figure out regeneration or we can create cells that mimic what grid cells do and create an artificial grid cell."
What Comes Next?
Laurencin and his team have individually engineered and made every single tissue in the lower limb, including bone, cartilage, ligament, skin, nerve, blood vessels. Regenerating joints and joint tissue is the next big mile marker, which Laurencin sees as essential to regenerating a limb that functions and performs in the way he envisions.
"Using stem cells and amnion tissue, we can regenerate joints that are damaged, and have severe arthritis," he says. "We're making progress on all fronts, and making discoveries we believe are going to be helping people along the way."
That focus and advancement is vital to Ennis. After laboring over the decision to have her leg amputated below the knee, she contracted MRSA two weeks post-surgery. In less than a month, she went from a below-the-knee-amputee to a through-the-knee amputee to an above-the-knee amputee.
"A below-the-knee amputation is night-and-day from above-the-knee," she said. "You have to relearn everything. You're basically a toddler."
Kirstie Ennis pictured in July 2020.
Photo Credit: Ennis' Instagram
The clock is ticking on the timeline Laurencin set for himself. Nine years might seem like forever if you're doing time but it might appear fleeting when you're trying to create something that's never been done before. But Laurencin isn't worried. He's convinced time is on his side.
"Every week, I receive an email or a call from someone, maybe a mother whose child has lost a finger or I'm in communication with a disabled American veteran who wants to know how the progress is going. That energizes me to continue to work hard to try to create these sorts of solutions because we're talking about people and their lives."
He devotes about 60 hours a week to the project and the roughly 100 students, faculty and staff who make up the HEAL team at the Convergence Institute seem acutely aware of what's at stake and appear equally dedicated.
"We're in the thick of the science in terms of making this happen," says Laurencin. "We've moved from making the impossible possible to making the possible a reality. That's what science is all about."
In November 2020, messenger RNA catapulted into the public consciousness when the first COVID-19 vaccines were authorized for emergency use. Around the same time, an equally groundbreaking yet relatively unheralded application of mRNA technology was taking place at a London hospital.
Over the past two decades, there's been increasing interest in harnessing mRNA — molecules present in all of our cells that act like digital tape recorders, copying instructions from DNA in the cell nucleus and carrying them to the protein-making structures — to create a whole new class of therapeutics.
Scientists realized that artificial mRNA, designed in the lab, could be used to instruct our cells to produce certain antibodies, turning our bodies into vaccine-making factories, or to recognize and attack tumors. More recently, researchers recognized that mRNA could also be used to make another groundbreaking technology far more accessible to more patients: gene editing. The gene-editing tool CRISPR has generated plenty of hype for its potential to cure inherited diseases. But delivering CRISPR to the body is complicated and costly.
"Most gene editing involves taking cells out of the patient, treating them and then giving them back, which is an extremely expensive process," explains Drew Weissman, professor of medicine at the University of Pennsylvania, who was involved in developing the mRNA technology behind the COVID-19 vaccines.
But last November, a Massachusetts-based biotech company called Intellia Therapeutics showed it was possible to use mRNA to make the CRISPR system inside the body, eliminating the need to extract cells out of the body and edit them in a lab. Just as mRNA can instruct our cells to produce antibodies against a viral infection, it can also teach them to produce the two molecular components that make up CRISPR — a guide molecule and a cutting protein — to snip out a problem gene.
"The pandemic has really shown that not only are mRNA approaches viable, they could in certain circumstances be vastly superior to more traditional technologies."
In Intellia's London-based clinical trial, the company applied this for the first time in a patient with a rare inherited liver disease known as hereditary transthyretin amyloidosis with polyneuropathy. The disease causes a toxic protein to build up in a person's organs and is typically fatal. In a company press release, Intellia's president and CEO John Leonard swiftly declared that its mRNA-based CRISPR therapy could usher in a "new era of potential genome editing cures."
Weissman predicts that turning CRISPR into an affordable therapy will become the next major frontier for mRNA over the coming decade. His lab is currently working on an mRNA-based CRISPR treatment for sickle cell disease. More than 300,000 babies are born with sickle cell every year, mainly in lower income nations.
"There is a FDA-approved cure, but it involves taking the bone marrow out of the person, and then giving it back which is prohibitively expensive," he says. It also requires a patient to have a matched bone marrow done. "We give an intravenous injection of mRNA lipid nanoparticles that target CRISPR to the bone marrow stem cells in the patient, which is easy, and much less expensive."
Meanwhile, the overwhelming success of the COVID-19 vaccines has focused attention on other ways of using mRNA to bolster the immune system against threats ranging from other infectious diseases to cancer.
The practicality of mRNA vaccines – relatively small quantities are required to induce an antibody response – coupled with their adaptable design, mean companies like Moderna are now targeting pathogens like Zika, chikungunya and cytomegalovirus, or CMV, which previously considered commercially unviable for vaccine developers. This is because outbreaks have been relatively sporadic, and these viruses mainly affect people in low-income nations who can't afford to pay premium prices for a vaccine. But mRNA technology means that jabs could be produced on a flexible basis, when required, at relatively low cost.
Other scientists suggest that mRNA could even provide a means of developing a universal influenza vaccine, a goal that's long been the Holy Grail for vaccinologists around the world.
"The mRNA technology allows you to pick out bits of the virus that you want to induce immunity to," says Michael Mulqueen, vice president of business development at eTheRNA, a Belgium-based biotech that's developing mRNA-based vaccines for malaria and HIV, as well as various forms of cancer. "This means you can get the immune system primed to the bits of the virus that don't vary so much between strains. So you could actually have a single vaccine that protects against a whole raft of different variants of the same virus, offering more universal coverage."
Before mRNA became synonymous with vaccines, its biggest potential was for cancer treatments. BioNTech, the German biotech company that collaborated with Pfizer to develop the first authorized COVID-19 vaccine, was initially founded to utilize mRNA for personalized cancer treatments, and the company remains interested in cancers ranging from melanoma to breast cancer.
One of the major hurdles in treating cancer has been the fact that tumors can look very different from one person to the next. It's why conventional approaches, such as chemotherapy or radiation, don't work for every patient. But weaponizing mRNA against cancer primes the immune cells with the tumor's specific genetic sequence, training the patient's body to attack their own unique type of cancer.
"It means you're able to think about personalizing cancer treatments down to specific subgroups of patients," says Mulqueen. "For example, eTheRNA are developing a renal cell carcinoma treatment which will be targeted at around 20% of these patients, who have specific tumor types. We're hoping to take that to human trials next year, but the challenge is trying to identify the right patients for the treatment at an early stage."
Repairing Damaged mRNA
While hopes are high that mRNA could usher in new cancer treatments and make CRISPR more accessible, a growing number of companies are also exploring an alternative to gene editing, known as RNA editing.
In genetic disorders, the mRNA in certain cells is impaired due to a rogue gene defect, and so the body ceases to produce a particular vital protein. Instead of permanently deleting the problem gene with CRISPR, the idea behind RNA editing is to inject small pieces of synthetic mRNA to repair the existing mRNA. Scientists think this approach will allow normal protein production to resume.
Over the past few years, this approach has gathered momentum, as some researchers have recognized that it holds certain key advantages over CRISPR. Companies from Belgium to Japan are now looking at RNA editing to treat all kinds of disorders, from Huntingdon's disease, to amyotrophic lateral sclerosis, or ALS, and certain types of cancer.
"With RNA editing, you don't need to make any changes to the DNA," explains Daniel de Boer, CEO of Dutch biotech ProQR, which is looking to treat rare genetic disorders that cause blindness. "Changes to the DNA are permanent, so if something goes wrong, that may not be desirable. With RNA editing, it's a temporary change, so we dose patients with our drugs once or twice a year."
Last month, ProQR reported a landmark case study, in which a patient with a rare form of blindness called Leber congenital amaurosis, which affects the retina at the back of the eye, recovered vision after three months of treatment.
"We have seen that this RNA therapy restores vision in people that were completely blind for a year or so," says de Boer. "They were able to see again, to read again. We think there are a large number of other genetic diseases we could go after with this technology. There are thousands of different mutations that can lead to blindness, and we think this technology can target approximately 25% of them."
Ultimately, there's likely to be a role for both RNA editing and CRISPR, depending on the disease. "I think CRISPR is ideally suited for illnesses where you would like to permanently correct a genetic defect," says Joshua Rosenthal of the Marine Biology Laboratory in Chicago. "Whereas RNA editing could be used to treat things like pain, where you might want to reset a neural circuit temporarily over a shorter period of time."
Much of this research has been accelerated by the COVID-19 pandemic, which has played a major role in bringing mRNA to the forefront of people's minds as a therapeutic.
"The pandemic has really shown that not only are mRNA approaches viable, they could in certain circumstances be vastly superior to more traditional technologies," says Mulqueen. "In the future, I would not be surprised if many of the top pharma products are mRNA derived."
"Making Sense of Science" is a monthly podcast that features interviews with leading medical and scientific experts about the latest developments and the big ethical and societal questions they raise. This episode is hosted by science and biotech journalist Emily Mullin, summer editor of the award-winning science outlet Leaps.org.