13 Mar 2019

Researchers have enlisted CRISPR technology to edit Alzheimer’s disease-related genes in isolated cells and in wild-type mice. The March 11 Nature Neuroscience now offers a glimpse of what CRISPR can do in mouse models of adult amyloidosis. A group led by Jongpil Kim at Dongguk University in Seoul injected nanocomplexes carrying CRISPR-Cas9 and guide RNAs targeting β-secretase (BACE1) into the hippocampi of AD mice. The complexes suppressed BACE1 expression, Aβ deposits waned, and the mice fared better in tests of learning and memory.

Researchers injected CRISPR nanocomplexes into the hippocampi of AD mouse models.

This knocked down BACE1, amyloid plaques, and improved memory.

CRISPR nanocomplex application in Alzheimer’s disease remains a ways off.

“The idea of using CRISPR/Cas9 nanocomplexes for Alzheimer’s disease is pretty exciting,” said Subhojit Roy, University of Wisconsin in Madison, who has tested CRISPR in cells and wild-type mice. Nanoparticle-based approaches to deliver Cas9 into the brain have mostly proven inefficient, so the new findings are important if they stand up to confirmation, he noted.

Recently, researchers in the AD field have used CRISPR/Cas gene editing to suppress mutant or normal Aβ precursor protein (APP) in adult wild-type mice and to generate mutant APP knock-in mice in utero (May 2018 news; Sun et al., 2019; Nagata et al., 2018). In the new study, first author Hanseul Park tasked CRISPR with eliminating BACE1, the enzyme that makes the first of two cuts that release Aβ from APP. Park used the amphiphilic peptide R7L10, which self-assembles into micelles in aqueous solution, to package Cas9 and guide RNAs (gRNAs) into nanocomplexes. The gRNAs were chosen to generate premature stop codons. The scientists first tested the nanocomplexes in cultured cells, including human neurons differentiated from neural stem cells, and found low toxicity and high levels of on-target BACE disruption.

Less BACE, Fewer Plaques. 5xFAD mice injected with nanocomplexes loaded with CRISPR-Cas9 and a guide RNA targeting BACE1 accumulate fewer amyloid deposits (green dots, right) than untreated controls (left). [Courtesy of Park et al., Nature Neurosci, 2019.]

With this, they next injected the nanocomplexes into the hippocampi of wild-type mice. The 10 μl injection reduced the number of hippocampal BACE1-positive cells by approximately 75 percent over four weeks. Markers for inflammation and reactive gliosis (GFAP, Iba1, Itgam, and CD86) remained at similar levels as in controls one to14 weeks post-injection, although levels of Il-12 and CD68 seemed to rise temporarily the first week. No signs of toxicity were detected, as assessed by blood urea nitrogen levels, an indicator of kidney and liver health, and cleaved caspase-3, a marker of apoptosis.

“The advantage of our Cas9 nanocomplex system is that it can lead to efficient gene targeting in post-mitotic neurons of adult mouse brain without genomic integration, and with very low cytotoxicity,” noted Kim.

Does this work in an AD mouse? Park injected BACE1sgRNA-Cas9 nanocomplexes into the CA3 hippocampal region of six-month-old 5xFAD transgenic AD mice. Western blots revealed a 70 percent drop in BACE1 in the hippocampus, and nearly equal reduction in the APP β-cleavage product C99. Giving the mice a second dose of nanocomplexes reduced BACE1 even more, to about 10 percent of wild-type levels.

Park then used thioflavin T to light up Aβ plaques in hippocampal brain sections four weeks post-injection. Mice receiving a single injection had 30 percent less plaque burden than sham-injected controls; mice injected twice had half as much as controls. In both cases, Aβ42 levels dropped 10-15 percent in the hippocampus as assessed by ELISA.

Treated mice reportedly performed better on behavioral tests than their untreated peers. In a contextual fear conditioning task, mice injected once with BACE1sgRNA-Cas9 nanocomplexes froze in place three times longer than did controls after hearing a tone previously paired with an electric shock. They also better remembered which arms of a Y-maze they had previously visited, and found the hidden platform in the Morris water maze approximately twice as fast on the fourth day of training.

These differences persisted for eight and 12 weeks after injection. Four-month-old APP knock-in mice, APPNL-G-F/NL-G-F, similarly improved on learning and memory tasks, and had less plaque, after they were injected.

Jochen Herms of the DZNE in Munich found the results impressive but raised concerns about targeting BACE1. “This approach does not overcome the fundamental problem with BACE inhibition in AD treatment—the inhibition of Sez6 cleavage, which we have shown is responsible for alterations in spine plasticity,” he wrote (Filser et al., 2015; Zhu et al., 2018). Herms believes Sez6 cleavage may explain recent setbacks in BACE1 inhibitor clinical trials (see Nov 2018 news).

Still, the findings represent proof of principle that CRISPR could be used to edit or modulate expression of genes related to AD. How might the strategy likely be used to treat or study other neurodegenerative diseases? Roy noted that nanocomplexes have some advantages over viral delivery systems, including temporary exposure to Cas9, larger cargo capacity, and lower immunogenicity. However, getting the particles into neurons in the brain has proven challenging (for example, see Staahl et al., 2017). “We have tried using nanoparticles, but just can’t get them in efficiently,” Roy said.

Another challenge will be achieving the brain-wide effects that will likely be required to treat AD. “Eventually, we need to develop a Cas9 nanocomplex capable of systemic intravenous delivery,” Kim wrote. He noted that refining the system to target specific cells would be helpful.—Marina Chicurel