In the landscape of modern medicine, early-onset rare genetic disorders do not receive much attention, mainly because they occur so infrequently. Yet, in the few unfortunate cases, the ordeal is lifelong and extremely difficult to manage. Take hereditary tyrosinemia Type I (HT1), which affects one in 100,000 individuals worldwide and is characterized by elevated blood levels of tyrosine, a key protein building block. Newborns with HT1 experience diarrhea, vomiting, and jaundice, then quickly progress to liver failure. Medication taken twice daily from infancy and severe diet restrictions can control the disease, but even so, some patients eventually advance to liver cancer. Gene editing represents one way to correct HT1 at the molecular level. However, because it manifests itself early in life, the opportunity for treatment may be limited after birth.
A recent collaboration between researchers from the Children’s Hospital of Philadelphia (CHOP) and the Perelman School of Medicine at the University of Pennsylvania has produced a proof-of-concept for therapeutic gene editing before birth. The study demonstrated that CRISPR-based gene editing tools, delivered using viral vectors into developing mouse fetuses, successfully edited two target genes and resolved HT1 long after birth. This points to a new therapeutic approach for congenital diseases that currently have no effective treatments.
This study, codirected by Kiran Musunuru, Associate Professor of Medicine at Penn Med, and William Peranteau, attending surgeon in the Division of General, Thoracic and Fetal Surgery at CHOP, represents a partnership between two complementary specialties—Musunuru, a cardiovascular geneticist, is a gene editing expert; Peranteau is an expert on using viral vectors to deliver therapeutic genes and stem cells to the developing fetus for the potential treatment of congenital disorders. They were working across the street from each other, and quickly recognized the opportunity for an effective collaboration to explore prenatal, or in utero, gene editing. If successfully proven, this approach would be exceptionally useful in the context of therapy, primarily because fetuses are smaller than newborns, so high treatment doses per body weight can be administered. “Also, the immune system of the fetus is immature, and thus unlikely to reject the therapy,” Peranteau said. Moreover, unlike embryonic editing, which results in genetic changes that are heritable, treating at the fetal stage allows genetic corrections to be limited to target organs. Finally, and most compelling, in utero gene editing could treat a disease before irreversible effects set in.
In this study, the researchers first demonstrated the feasibility of using viral vectors to deliver gene-editing tools to the fetus. When a normal virus infects a cell, it releases its own genetic material that is usually integrated into the host genome. Viral vectors are “hollowed out” versions of viruses, instead filled with gene-editing molecular machinery. These vectors retain their ability to zero in on and infect target cells to deliver their payloads. For the first test run, the payload was a general-purpose CRISPR-Cas9 gene-editing toolkit, configured to target the liver. The vectors were injected into mouse fetuses via a blood vessel that drains directly into the fetal liver. As expected, on the first day after birth, the researchers observed editing in the newborns’ heart and liver.
With this positive result, the researchers attempted base-specific gene editing in utero. This is much more challenging since the machinery has to identify and modify a single base out of billions in the genetic sequence. Base-specific editing may be useful for treating congenital disorders; however, for many such disorders, there is only one base that must be targeted. To this end, the researchers turned to a recent technology called Base Editor 3 (BE3), a safer and less error-prone refinement of CRISPR-Cas9. But they also had to use a different type of vector, called an adenovirus, to accommodate the large cargo size of BE3.
For base-specific editing, the researchers decided to first target a cholesterol gene called Pcsk9. Musunuru had previously worked on deactivating Pcsk9 in adult mice, leading to reduced cholesterol levels. The researchers configured the BE3 toolkit to target Pcsk9, packaged it in adenoviral vectors, and delivered it to mouse fetuses. Upon birth, they observed correct editing only in the liver and not in any other organ, just as desired. To be sure, they analyzed maternal DNA and found no evidence of editing. “[This] was a highlight in the project. I distinctly remember the evening when we got data showing that mice showed signs of editing after birth,” Musunuru said. The researchers also monitored cholesterol levels, which remained low even after three months. For reference, the same editing technique was applied to mice five months after birth, but the effects of editing were significantly reduced after two to three months. Moreover, only the postnatally treated mice developed immune responses against the treatment.
The success of the Pcsk9 demonstration prompted the researchers to move on to target early-onset congenital disorders, as they initially planned. “These patients have the highest unmet need; they have the most to gain and the least to lose,” Musunuru said. Hereditary tyrosinemia type 1 (HT1) fits the bill, as it can be treated by altering a single base in the Hpd gene in the liver. The same method was used with the BE3 toolkit reconfigured to target and alter the Hpd gene. For comparison, they used diseased mice that survived after birth on medication. The in utero-treated mice survived past three months without medication, essentially cured of the disease. The real highlight is how the mice treated in utero thrived—they gained weight even faster than their counterparts on medication. The researchers also observed normal liver function after three months, which corresponds to 25 human years. This shows that CRISPR-based prenatal gene editing is indeed viable for permanent treatment of such congenital disorders as HT1.
To Musunuru and Peranteau, this study is simply proof-of-concept—more fine-tuning is required for clinical application. For one, the adenoviral vectors used to transport the large BE3 toolkit are not safe. “Adenoviral vectors have known issues when applied to human subjects. Lipid nanoparticles and other viral vectors are more clinically relevant delivery approaches,” Peranteau said. Lipid nanoparticles involve encasing the gene editing toolkit in a nanoscale globule of fat. This delivery method is safe, but the packaging aspect is highly challenging. Separately, the researchers still have to prove that the effects of editing persist for a lifetime. Since the publication of the study, they are still monitoring the mice, which have lived for one year at this point. No unusual effects have been observed so far. Another foreseeable goal is to target other organs besides the liver, the obvious strategy being to coat the vectors with molecules that can be specifically recognized by the target organ. This will expand the repertoire of diseases amenable to the new treatment approach. Finally, the clinical application of this work depends on advancement in prenatal gene testing, to identify individuals who need treatment in the first place.
Looking back, the collaboration has been beneficial for both researchers. “The beauty of this collaboration is that now, Kiran is equally knowledgeable of the benefits of prenatal gene delivery, while I have learned a lot from Kiran and am well versed in gene editing approaches,” Peranteau said. Both agree that their experience practicing medicine has greatly informed their research. Musunuru studies cardiovascular diseases, and has identified genes such as Pcsk9 that confer protection against heart disease. “We want to extend this genetic advantage to those who did not win the genetic lottery,” Musunuru said. To him, therapeutic gene editing, which corrects the genetic cause of diseases, is no different from vaccinations. For Peranteau’s part, his expertise in fetal surgery, a specialty developed barely 15 years ago, gives him perspective on how technologies can progress and significantly impact future medicine. “My hope is that in utero gene editing, just like fetal surgery years ago, will be the future of prenatal interventions,” Peranteau said.
In any case, this successful study gives us reason to look forward to a future in which children can be cured of debilitating or even fatal genetic disorders before they are born, if only because treatment after birth will be too late.