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Two CRISPR-based Gene Therapies Focus on Amyloid, APOE4 Allele

​Two experimental CRISPR-based gene therapies for Alzheimer's disease (AD) target amyloid and APOE4, the strongest genetic risk factor for the condition, according to findings presented in July at the Alzheimer's Association International Conference.

Brent Aulston, PhD—working in the lab of Subhojit Roy, MD, PhD, professor of pathology at the University of California at San Diego—targeted the gene for amyloid precursor protein (APP), which, when cleaved by beta-secretase, produces amyloid and leads to the development of plaques.

“We know from APP knockout mouse studies that if you delete the entire gene, you have abnormal brain development, decreased cognition, and neuroinflammation," Dr. Aulston said, so deletion of the gene is not an ideal strategy. Instead, he and his colleagues used CRISPR to cleave a six amino acid sequence from the gene's terminus, which maintains gene expression but protects the now-truncated protein from internalization and beta-secretase cleavage; it remains at the membrane, where alpha-secretases can cleave it to generate neuroprotective cleavage products.

The APP-targeting machinery was packaged in an AAV vector and injected into brains of mice carrying APP with three causative mutations at 1.5 months of age. Evaluations from 6 months to 12 months indicated a significant reduction in amyloid plaques compared to injections with control non-targeted CRISPRs, reduced inflammation, and improved performance on the novel recognition test of cognition. To investigate safety of the gene-editing approach, the team deleted the same six amino acid sequence in wild-type mice and found no loss of neurons, no signs of inflammation, and no cognitive impairments.

“These data demonstrate that deletion of the APP C terminus is safe and efficacious," Dr. Aulston said, and it supports further development of this approach for translation to humans.

Boris Kantor, PhD, associate professor of neurobiology at Duke University; Ornit Chiba-Falek, PhD, professor in neurology and division chief of translational brain sciences at Duke; and colleagues took a different approach, using CRISPR to target the APOE4 allele. Rather than using the CRISPR system to disrupt the gene, they took advantage of its exquisitely precise targeting capability to deliver a gene repressor to the APOE gene regions, reducing expression of the resulting protein. Unlike gene editing, this approach does not introduce breaks and nicks in the DNA.

They tested their approach in human-based cell models, including cerebral organoids and microglia-like cells, as well as in mice carrying a human APOE gene. They showed that treatment could reduce APOE4 mRNA expression in all cell systems, without affecting expression of the e3 allele. Unilateral hippocampal injection of 4-month-old mice led to a significant reduction in APOE4 production compared to control injection of the opposite-side hippocampus within the same mice.

“This study is a proof of principle that the expression of the e4 allele can be specifically reduced by this method," Dr. Kantor said, which may reduce the risk of developing AD.

Dr. Chiba-Falek added that “the technology offers opportunities for precision medicine in AD," and “this epigenome editing system is also useful for investigating the biological role of APOE and the mechanism by which the e4 allele exerts its pathogenic effect."

“The effects of targeting APP's C-terminus region on long-term potentiation and behavior are promising," said Li-Huei Tsai, PhD, director of the Picower Institute for Learning and Memory and the Picower Professor of Neuroscience at Massachusetts Institute of Technology, who was not involved in the study. “A major side effect of many A-beta targeting antibodies has been vascular damage and ARIA [amyloid-related imaging abnormalities]. If this gene therapy approach can avoid those side effects, it may be a promising strategy in reducing brain amyloid."

The potential benefit of gene therapy targeting APOE4 is less clear, he noted. “The mechanisms by which APOE4 predisposes individuals to AD remains to be explored, and APOE4's effects are likely due to both toxic gain of function and loss of function" Dr. Tsai said. “Moreover, APOE knockout mice are predisposed to developing a plethora of metabolic disorders, including atherosclerosis. Finally, it's important to note that many individuals with APOE4 never develop AD, and APOE4-associated risk of developing AD varies widely by ethnic background and sex of the individual. Therefore, how to deploy APOE4 gene therapy and to which particular demographic of people would have to be carefully thought through."


Dr. Roy is co-founder of the company CRISPRAlz.

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