Children born with deleterious mutations in both copies of the CLN3 gene typically show no symptoms until age four or five, when their vision begins to deteriorate. This first sign of trouble launches a diagnostic odyssey that eventually leads to Batten disease, a family of fatal neurological disorders that arise when intracellular lysosomes fail to break down waste molecules. Any delay in diagnosis is costly for patients, who only survive through childhood or possibly into young adulthood (1). But often health care providers don’t have experience with Batten disease because of its rarity: It’s estimated to affect one out of every 100,000 people worldwide (2). With such a small patient pool, developing, testing, and investing in therapeutics is difficult, and there are none currently on the market for CLN3 Batten disease, the most common form. 

To create a high fidelity animal model for evaluating therapeutics, Sanford Health neuroscientist Jill Weimer and a team of researchers recently reported a new porcine model of CLN3 Batten disease in a study published in Disease Models & Mechanisms (3). They characterized the pigs’ potential to recapitulate the cellular trademarks of the disease and symptoms including vision loss, cognitive impairment, and motor decline. “These genetically modified models are extremely helpful,” said Weimer. “In the grand scheme of things for drug development, they’re going to be absolutely essential.”

Previously, the only animal models for CLN3 Batten disease were mice where the CLN3 gene is knocked out. “They have the pathology of the disease, so you can see the storage material accumulation. You can see neuronal death,” said Weimer. “But they have no phenotype.” Without displaying the symptoms of Batten disease, these mice cannot report on the efficacy of therapeutics aimed at arresting or reversing those symptoms.

Hoping that pigs would prove to be an upgrade, Weimer’s research group partnered with Precigen Exemplar, a subsidiary of the larger biopharmaceutical company Precigen. Precigen Exemplar specializes in developing models of human disease in Yucatan miniswine, and Weimer’s group had already worked with the company to develop a porcine model of CLN2 Batten disease (4). While the term miniswine might conjure images of tiny pigs, the animals actually weigh 180 pounds at maturity, mimicking the size and metabolism of an adult male human. 

In the present study, the researchers knocked out CLN3 genes in Yucatan miniswine fetuses and then monitored 29 animals for three years after birth and five animals for four years after birth. To measure visual decline, the researchers checked the electrical response of the pigs’ retinas to light every six months. By four years of age, pigs with CLN3 mutations had impaired vision relative to unmodified pigs.

Monitoring deterioration of motor and cognitive function was less straightforward because pigs move through and sense the world differently than humans. “You have to be creative in how you’re demonstrating that [animal models] have a phenotype because they’re not human,” said Weimer. 

To check for cognitive changes, the researchers put pigs in a simple, T-shaped maze with a food reward consistently located at the end of one arm. Pigs with CLN3 mutations didn’t struggle to locate the food any more than their healthy peers, but that doesn’t necessarily mean that they were cognitively well. “Pigs also have a hyper sense of smell,” said Weimer. “They may also be able to compensate for some of the learning and memory by smelling.” Future experiments would need to account for the pigs’ powerful noses.

These genetically modified models are extremely helpful. ...In the grand scheme of things for drug development, they’re going to be absolutely essential. 
- Jill Weimer, Sanford Health

Similarly, assessing motor function required Weimer’s team to consider the fact that pigs walk on four legs. “They actually are able to compensate for gait defects much better than a human would,” said Weimer. To pick up subtle changes in the animals’ movements, the researchers recorded measurements as the pigs walked across pressure-sensitive platforms. They extracted data about the pigs’ footfall patterns, stride characteristics, and balance, which revealed discernable differences in the movement features of pigs with CLN3 mutations relative to unmodified pigs. 

Finally, Weimer’s team extracted the brains of pigs at different ages and checked to see how CLN3 mutation affected their neurons and glial cells. The researchers looked at regions of the brain typically affected by CLN3 Batten disease, including the somatosensory cortex, motor cortex, and specific areas of the thalamus and hippocampus. Pigs with CLN3 mutations showed some unique patterns of glial cell activation that may be species-specific, but they displayed the expected accumulation of intracellular waste and loss of motor neurons. 

Jonathon Cooper, a neuropathologist at the Washington University School of Medicine in St. Louis who was not involved in this study, said that Weimer’s team laid important groundwork. “They’ve done the very sensible thing, which is to look in the places that we know are more affected within the brain of a mouse or within the brain of a person,” Cooper said. But as the researchers begin to use the porcine model to test therapeutics, he’d like to see a more global analysis of the disease pathology in the pigs to determine if CLN3 mutation affects additional, unexpected areas of the brain and body in these animals.

Pigs won’t replace existing models of diseases, but rather complement them. Successfully integrating porcine models into the current research and regulatory landscape could accelerate development of therapeutics for Batten disease and other conditions that drastically shorten patients’ lives. “We’re building the plane as we’re flying it,” Weimer said. 

References

1. Goebel, H. & Wisniewski, K. Current state of clinical and morphological features in human NCL. Brain Pathol  14, 61-69 (2004).
2. Johnson, T. et al. Therapeutic landscape for Batten disease: current treatments and future prospects. Nat Rev Neurol  15, 161-178 (2019).
3. Swier, V. et al. A novel porcine model of CLN3 Batten disease recapitulates clinical phenotypes. Dis Models Mech  16, dmm050038 (2023).
4. Swier, V. et al. A novel porcine model of CLN2 Batten disease that recapitulates patient phenotypes. Neurother  19, 1905-1919 (2022).