Collaborating researchers at Aarhus University’s department of biomedicine and department of clinical medicine have harnessed CRISPR-Cas9 gene editing technology to generate a herd of Göttingen minipigs that could help to advance Alzheimer’s disease (AD) research and potentially unlock future treatment strategies.

The cloned pigs were born with a mutation in the gene SORL1 (sortilin-related receptor 1), which is altered in up to 2–3% of all early-onset cases of Alzheimer’s in humans. Studying the animals, the researchers confirmed that pigs with the SORL1 mutation develop signs of Alzheimer’s disease at a young age, providing an opportunity to follow the early stages and signs of the disease. The cloned animals also showed changes in the same biomarkers that are used to diagnose Alzheimer’s disease in people.

“By following the changes over time in the pigs, we can better understand the earliest changes in the cells,” said associate professor Olav Michael Andersen, PhD, who is first and co-corresponding author of the team’s published report in Cell Reports Medicine. “Later, these changes lead to the irreversible alterations in the brain that are the cause of dementia. But now we can follow the pigs before they lose their memory, change their behavior, etc., which will make it possible to test new drugs that can be used at an early stage to prevent SORL1-associated Alzheimer’s disease … Pigs resemble human beings in many ways, which is why this increases the possibilities of producing drugs that will work to counteract Alzheimer’s. It is important to have a workable animal model to bridge the gap between research and drug development,” he explained.

Anderson and the teams at Aarhus University, together with international collaborators, described their studies in a paper titled, “A genetically modified minipig model for Alzheimer’s disease with SORL1 haploinsufficiency.”

Since the 1990s, researchers have known of three genes which, if they mutate, can directly cause Alzheimer’s disease. “The established causal genes in Alzheimer’s disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease’s initial preclinical phase,” the authors wrote.

Through intense research over the past 20 years, it has now been established that a mutation in a fourth gene, SORL1—which encodes the endosome recycling receptor SORLA—can also directly cause Alzheimer’s disease. SORL1 was first identified at Aarhus University in 1996, and was established as a cause of Alzheimer’s disease in 2021. Noting that previous studies have demonstrated a genetic link between SORL1 and AD, the investigators stated, “Most recently, genetic epidemiology studies have identified rare truncation mutations in SORL1 almost exclusively in individuals with AD, suggesting that SORL1 functions as a causal gene.” If this gene is defective, the person carrying the genetic defect will develop Alzheimer’s.

Many people have contributed to the development of the pigs, which has taken place in a collaboration between the department of biomedicine and the department of clinical medicine at Aarhus University. Associate professor Olav Michael Andersen, PhD, and associate professor Charlotte Brandt Sørensen, PhD, are first and last authors, respectively, of the study. [Simon Byrial Fischel]

The Aarhus University researchers wanted to further investigate the causality of SORL1 in relation to AD. They developed a Göttingen minipig model that was genetically engineered to be SORL1 haploinsufficient, by heterozygous knockout (KO) of the porcine SORL1 gene. This mimicked the genetic status of humans with AD who carried heterozygous SORL1 truncating mutations.

“We have created an animal model for Alzheimer’s in minipigs by changing one of just four genes that are currently known to be directly responsible for the disease,” said Anderson. “The pigs can be used in the pharmaceutical industry to develop new drugs—and at the same time, this can provide researchers with better possibilities to understand the early changes in the brains of people who will later develop Alzheimer’s.”

Researchers have previously developed pig models for Alzheimer’s and other diseases by means of cloning. The technique, somatic cell nuclear transfer (SCNT) is done by removing the hereditary material from an unfertilized egg cell taken from one pig, and then fusing that cell with a skin cell from another pig. The researchers had previously used CRISPR-Cas9-based gene editing to destroy the SORL1 gene in a skin cell taken from a minipig of the Göttingen breed.

Using this technique, the reconstructed embryo, i.e., a cloned egg, develops into a new individual with the same genetic characteristics as the gene-edited skin cell, so the cloned minipigs are born with a damaged SORL1 gene. As the mutation is inherited, researchers can now breed these SORL1 het pigs that show the first signs of Alzheimer’s before they reach the age of three.

“The pigs resemble Alzheimer’s patients who have the SORL1 gene defects—in contrast to previous pig models for Alzheimer’s, which have had one or more mutated human genes inserted in the hope of accelerating the disease,” said associate professor Charlotte Brandt Sørensen, PhD, who was responsible for the development of the genetically modified, cloned pigs. As the authors wrote, “SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of ß-amyloid (Aß) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans.”

The study could help researchers better understand human AD. “We know from human genetics that when the SORL1 gene is destroyed, we develop Alzheimer’s,” Anderson stated. We have shown that if we destroy this gene in pigs, precisely the early changes occur in the animals’ brain cells that we had dared to hope for. This makes it possible to find biomarkers that reflect the initial, preclinical phase of the disease.”

The authors concluded in their paper, “Besides providing evidence that SORL1 is an AD-causing gene, the SORL1 het Göttingen minipigs allow future longitudinal studies for biochemical and neuroimaging biomarker discovery and may provide important clues regarding the sequence of events that occurs in the pharmacologically valuable treatment window between the very early preclinical stage of the disease, with raised Aß and tau CSF levels, and the time point of amyloid brain deposition, irreversible neurodegeneration, and subsequent cognitive impairment.”

Anderson further suggests, “The best thing would be to develop new medicines on the basis of this pig model, and we are already well advanced with the preparations. The group of patients who carry mutations of SORL1 is far larger than the group of patients who have errors in the three other known genes.”

Danish company Ellegaard Göttingen Minipigs owns the rights to, and breeds the Göttingen pig variety.

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