What will it take before CRISPR/Cas gene-editing technology might treat Alzheimer’s disease? Much more work, to be sure, but researchers are devising approaches, and grappling with the challenges of translating the technology from cells to animals, and some day to people. The prospect of correcting disease-causing mutations seems most straightforward for early onset, autosomal-dominant forms of AD (ADAD), and toward that goal, Martin Ingelsson, Uppsala University, Sweden, and colleagues published the first instance of using CRISPR/Cas9 to neutralize the Swedish APP mutant in patient cells and in mice. In the April 18 in Molecular Therapy: Nucleic Acids, the scientists describe how they knocked out the mutant allele, diminishing the production of Aβ in patient cells, while leaving the wild-type allele intact. Using the same strategy they also modified the genome in transgenic mice bearing the human APPswe allele, but don’t know yet if that will affect AD pathology.

  • CRISPR knockout of  Swedish mutation in patient cells reduces Aβ.
  • A separate CRISPR approach to blunt Aβ production could be tried in sporadic AD.

“This manuscript elegantly demonstrates the potential for correcting genetic defects both in cell models and in an animal model of AD based on the Swedish mutation. These findings move the field one step closer to being able to genetically correct harmful mutations, such as dominantly inherited AD,” said Randall Bateman, Washington University, St. Louis. “If and how genetic modification could treat AD caused by mutations, and even sporadic AD, will be closely watched,” he said.

In another new study, Subhojit Roy’s group at the University of Wisconsin, Madison, took on the challenge of sporadic AD. In a paper posted April 28 on the bioRχiv preprint server, the group report how they used CRISPR to frameshift the C-terminus of APP. This introduced an early stop codon, truncated the protein, and dramatically decreased Aβ production. This edit worked in wild-type mice, too, but whether it affects amyloid production in vivo remains to be seen.

CRISPR/Cas9 technology allows researchers to rewrite the genome practically at will. The technique has been used to create cell models of ADAD by introducing presenilin and APP mutations into human induced pluripotent stem cells (iPSCs); neurons derived from those cells showed disease-related changes in Aβ production (May 2016 news). Other groups have used CRISPR to correct familial AD mutations in the presenilin gene in patient-derived iPSCs (Pires et al., 2016Poon et al., 2016). In another study, researchers corrected a presenilin 2 mutation in iPSC-derived basal forebrain cholinergic neurons from patients with autosomal-dominant AD, and showed this normalized the cells’ electrophysiological function and Aβ secretion (Ortiz-Virumbrales et al., 2017).

In contrast, Ingelsson’s idea was not to correct, but to destroy APP harboring the Swedish mutation. This double point mutation in exon 16 just upstream of the BACE cleavage site promotes the amyloidogenic cleavage of APP, increasing production and secretion of Aβ40 and Aβ42 in brain and peripheral tissues. Simply knocking out the mutant allele, Ingelsson reasoned, should forestall Aβ accumulation and disease. While on sabbatical in co-author Xandra Breakefield’s lab at Massachusetts General Hospital in Boston, Ingelsson worked with co-first authors Bence György and Camilla Lööv to devise a way to slay the foe with CRISPR.

The Swedish mutation is an attractive target. Two mismatches between mutant and wild-type sequence should ensure better specificity of guide RNAs used in CRISPR than the one mismatch found in other APP and PS FAD mutations. Besides, a TGG trinucleotide right next to the mutated bases is a preferred binding motif for the Cas9 nuclease. “It was a fortunate combination that made it straightforward to design a system to target the mutation with high precision,” Ingelsson told Alzforum.

First, the researchers tested guide RNAs, transfecting them along with Cas9 into patient–derived fibroblasts from three Swedish mutation carriers and two non-affected family members. Guide RNAs complementary to the mutated allele caused Cas9 to make small deletions, resulting in frame shifts in APP that wiped out the BACE cleavage site in the mutant, but not in the wild-type, allele. The most effective guide, dubbed SW1, left APP protein levels in the cells unchanged but reduced Aβ40 and Aβ42 production by 50–60 percent in three independently generated cell lines.

Therapeutic targeting. A guide RNA (blue) binds exon 16 of human APP, spanning the two-base Swedish mutation (yellow) directly adjacent to a TGG Cas9-binding protospacer adjacent motif (pink) and near the β-secretase cleavage site (arrow). [Courtesy of György et al., 2018.]

To see if this would work in vivo, the authors used Tg2576 mice, which carry multiple copies of the human APP Swe transgene. In a preliminary study, they packaged DNA encoding both Cas9 and guide RNAs in adeno-associated virus 9 (AAV) vectors, and infected cultured primary cortical neurons from Tg2576 embryos. After three weeks, they detected disrupted APP Swe alleles, mainly in the form of one base pair insertions. Then they tested the vectors in adult mice, injecting them into the hippocampus. All the mice showed some disruption of the APP Swe gene, again mostly from single base pair insertions.

However, the approach hit only a small fraction of transgenes. In cultured and in vivo Tg2576 neurons, 2.3 and 1.3 percent of transgenes, respectively, were disrupted. That’s low, but unsurprising, Ingelsson said. “This is a model with roughly 100 copies of the transgene per neuron, so it seems the CRISPR system got overwhelmed,” he said. The AAV delivery and CRISPR activity appeared efficient, as a guide RNA targeting a control endogenous gene modified 36 percent of target alleles in cultured Tg2576 neurons.

Because of the low modification rate in the overexpression model, it made no sense to look at plaque or other downstream effects in the mice, Ingelsson said. He plans to do that using a knock-in model. Ingelsson does not know what percentage of genes would need to be modified to reduce plaque formation in vivo, but thinks effects could add up over time.

In the second study, first author Jichao Sun in Roy’s group wanted a CRISPR therapy that would work on any case of AD, familial or sporadic. Previously, the group found that clipping off the C-terminal, cytoplasmic portion of APP prevented the protein from trafficking into endosomes, where it meets up with BACE and undergoes its first cleavage on the road to Aβ (Aug 2013 newsDec 2015 news). In the new work, Sun took advantage of a protospacer adjacent motif (PAM) trinucleotide in the far C-terminal sequence of APP to target CRISPR cleavage, and truncated the gene. Whether in mouse cells or in human iPSC-derived neurons, this strategy led to a dramatic drop in Aβ40/42 secretion, and an increase in non-amyloidogenic α-secretase products.

Roy’s group also tried to disrupt APP in mice. When they injected AAV9 expressing a guide RNA and Cas9 into the hippocampus, they detected less binding of the APP C-terminal antibody Y188, indicating the gene had been truncated. They were able to induce brain-wide expression of truncated APP after injecting AAV into the ventricles of newborn mice.

One problem with CRISPR gene editing is that if the guide RNAs are not 100 percent faithful, they can induce mutations in other genes, too. To look for off-target effects, Sun analyzed the top five most likely non-APP targets, based on the RNA guide sequence, and the two APP homologs APLP1 and 2, finding no evidence of disruption in them. The researchers detected no obvious side effects of CRISPR in primary hippocampal neurons. In other words, truncating APP did not alter neurite outgrowth, spine number, synapse density, or electrophysiological activity.

“The work from Roy’s lab represents another interesting approach of making therapeutic use of CRISPRs in the AD setting,” said Ingelsson. “It is more generally applicable compared to our approach, which has to be tailored to a specific disease-causing mutation,” he said. “The CRISPR/Cas technology certainly offers us a fascinating repertoire of strategies to disrupt or correct alleles that are involved in AD and other neurodegenerative diseases.”

What Lies Ahead
CRISPR has shown promise in animal models of Huntington’s and amyotrophic lateral sclerosis (Yang et al., 2017; Monteys et al., 2017; Dec 2017 news) even as new tools are expanding editing capabilities from DNA to RNA. Recently, one group directed CRISPR to RNA to correct aberrant tau splicing in neurons derived from people with frontotemporal dementia (Mar 2018 news). The next goal for AD researchers will be to show that CRISPR can curb amyloid deposition, and improve function, in an AD mouse model.

Even after crossing this hurdle, obstacles remain before CRISPR will be ready to use in the clinic. It’s likely that any therapeutic agent would need to be expressed widely throughout the brain, which rules out local injection as a means of delivery. Ingelsson thinks that brain-wide delivery is achievable with the latest AAV9-based vectors, which broadly transduce cells in the CNS after peripheral injection (Dec 2008 newsSep 2014 news; Dashkoff et al., 2016), However, whether they will drive sufficiently robust expression remains to be determined.

Off-target effects also cause concern. “The main challenge with CRISPR is that while you can watch and control as it repairs the mutation you want to eliminate, the enzyme can create new mutations elsewhere. To some extent you can find those with whole-genome sequencing, but if you miss one, that could be disaster,” Sam Gandy, Mount Sinai School of Medicine in New York, wrote in an email to Alzforum. Gandy’s group has used the technology to correct PS2 mutations in patient cells.

Ingelsson agrees. “We don't have a clear picture yet of what off-target effects we may get. People are trying to come up with ways of assessing this for a particular treatment, and those systems will have to be further developed before we can run the risk of exposing patients to this type of treatment.”

Human trials of CRISPR anti-cancer therapies started in China in 2015, and have been approved to begin at the University of Pennsylvania. CRISPR Therapeutics of Cambridge, Massachusetts, is pursuing trials of gene editing to treat hemoglobin disorders, including sickle cell anemia and β-thalassemia. All these efforts involve the removal of blood cells, ex vivo gene knockout, and reinfusion. Genetic editing in situ will be more complicated, say researchers. “I would like to see it perfected in blood diseases before jumping into the brain,” said Gandy.—Pat McCaffrey


  1. I think the György et al. paper is quite exciting. They clearly show that they can target specifically the mutant allele in fibroblasts and that these express lower amounts of Aβ40 and Aβ42. So the in vitro part is quite convincing. The in vivo part is not so clear, since they state that it is difficult for them to measure delivery of the CRISPR constructs by the AAV virus. It would be interesting to see more systematic analyses of the targeted cells in the hippocampus. How far can the virus spread and alter the mutant allele?

    They also claim that it was advantageous to target the Swedish APP mutation since it is a mutation affecting two nucleotides. This keeps me wondering how effective the approach is when only a single nucleotide is affected. I also did not see any off-target screening in the paper and I was wondering if that was addressed. Obviously this is a concern, even though the latest research has shown that there are few, if any, off-target effects it would have been important to check. Also, one needs to keep in mind that AD is rarely caused by such familial mutations, but for the few affected individuals this could be an appropriate therapy. 

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News Citations

  1. CRISPR Verifies Risk Alleles, Improves Gene Editing
  2. Neural Activity Tips Endosomal Balance, Hastens Amyloid Pathology
  3. Close Encounters: A New Look at Where APP and BACE1 Meet
  4. Gene Editing Delays Disease Onset in ALS Model Mice
  5. New RNA CRISPR Tool Normalizes Tau Splicing
  6. Virus Slips Through Blood-Brain Barrier to Deliver the Gene Goods
  7. Neuroscientists Probe CRISPR Transgenics and Treatment Paradigms

Research Models Citations

  1. Tg2576

Antibody Citations

  1. AβPP (Y188)

Paper Citations

  1. . Generation of a gene-corrected isogenic control cell line from an Alzheimer's disease patient iPSC line carrying a A79V mutation in PSEN1. Stem Cell Res. 2016 Sep;17(2):285-288. Epub 2016 Aug 7 PubMed.
  2. . Generation of a gene-corrected isogenic control hiPSC line derived from a familial Alzheimer's disease patient carrying a L150P mutation in presenilin 1. Stem Cell Res. 2016 Nov;17(3):466-469. Epub 2016 Sep 24 PubMed.
  3. . CRISPR/Cas9-Correctable mutation-related molecular and physiological phenotypes in iPSC-derived Alzheimer's PSEN2 N141I neurons. Acta Neuropathol Commun. 2017 Oct 27;5(1):77. PubMed.
  4. . CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington's disease. J Clin Invest. 2017 Jun 30;127(7):2719-2724. Epub 2017 Jun 19 PubMed.
  5. . CRISPR/Cas9 Editing of the Mutant Huntingtin Allele In Vitro and In Vivo. Mol Ther. 2017 Jan 4;25(1):12-23. PubMed.
  6. . Tailored transgene expression to specific cell types in the central nervous system after peripheral injection with AAV9. Mol Ther Methods Clin Dev. 2016;3:16081. Epub 2016 Dec 7 PubMed.

Other Citations

  1. Swedish mutation

Further Reading

No Available Further Reading

Primary Papers

  1. . CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease. Mol Ther Nucleic Acids. Volume 11, p429–440, 1 June 2018
  2. . A CRISPR/Cas9 based strategy to manipulate the Alzheimer's amyloid pathway. bioRχiv 310193; Apri 28, 2018