Brain-wide genome editing via a single systemic dose of modified adeno-associated virus (AAV) variants that cross the blood-brain barrier (BBB) may represent a promising new approach for the development of disease-modifying treatments for familial Alzheimer's disease (FAD).

This strategy could also be applicable to other central nervous system (CNS) disorders, according to a proof-of-concept (PoC) study led by researchers at The Hong Kong University of Science and Technology (HKUST).

"We believe this is the first demonstration of brain-wide gene editing via modified AAV variants, with this PoC study providing experimental evidence for developing a gene-editing approach for FAD treatment," said study leader Nancy Ip, Morningside professor of life science and director of the State Key Laboratory of Molecular Neuroscience at HKUST.

"This strategy can be applied to monogenetic CNS disorders affecting multiple regions, including FAD, amyotrophic lateral sclerosis and Parkinson's disease," Ip, the vice-president for research and development at HKUST, told BioWorld Science.

AD is a common progressive neurodegenerative disease characterized by amyloid-beta (Abeta) peptide deposition and neurofibrillary tau protein tangles in the brain.

FAD is caused by mutations in genes encoding amyloid precursor protein (APP) and the presenilin 1 and 2 (PS1 & 2) catalytic components of the gamma-secretase complex, which leads to Abeta peptide accumulation.

However, no effective treatments are available for FAD, despite improved understanding of its genetic causes and the advent of CRISPR-Cas9-mediated genome editing.


CRISPR-Cas9 can powerfully target specific mutations, but FAD affects multiple brain regions, which has made delivery challenging.

"Genome editing in vivo requires a vehicle with which to deliver Cas9 protein into cells, but most Cas9 vehicles cannot be systemically and efficiently delivered into the brain, as they cannot pass the BBB," said Ip. "Hence, intra-parenchymal injection is required to deliver Cas9 into the brain region, but since it can only affect the brain within that region, its beneficial effects are restricted."

Therefore, efficient and global genome editing that can be used throughout the adult brain is urgently needed, such as using modified AAV variants that can cross the BBB.

Modified AAV variants

Modified AAV variants have enabled widespread CNS transduction after intravenous delivery, having been shown to promote brain-wide post-stroke recovery in mice.

Therefore, modifying expression of CNS disease-modifier gene(s) using a BBB-crossing virus could possibly be developed as a therapeutic strategy for FAD.

However, the ability of such a virus to deliver CRISPR-Cas9 genome-editing components to disease phenotypes remains untested in vivo.

FAD cases are mostly caused by monogenic mutations, with CRISPR-Cas9-mediated genome editing having been used to correct these mutations and decrease Abeta in vitro.

Most FAD patients have a heterozygous pathogenic mutation, while those carrying one copy of APP, or PSEN1 or PSEN2 encoding PS1 or PS2, do not show neurologic symptoms.

The Swedish patient

In the present study, the team targeted the so-called APP Swedish mutation, which is located at the beta-secretase cleavage site of APP and results in significantly higher Abeta production in affected patients.

"We used this mutation with a strong disease phenotypic outcome to demonstrate the beneficial effects of the new therapeutic approach, which can be applied to other FAD mutations using specific single-guide RNAs (sgRNAs)," Ip explained.

However, while genome editing had previously been used to disrupt APPswe in mutated transgenic mice, low genome-editing efficiency prevented its therapeutic evaluation.

Reported in the July 26, 2021, edition of Nature Biomedical Engineering, the HKUST-led study therefore investigated AAV-mediated delivery of Cas9 and sgRNA specifically targeting the APPswe mutation.

Injected directly into adult hippocampus, a single administration of a BBB-crossing AAV-Cas9 vector targeting the APPswe mutation was shown to achieve efficient genome editing and alleviated multiple Abeta-associated pathologies in transgenic mouse models of Abeta deposition.

"We demonstrated the genome editing efficiency of this approach using T7 endonuclease I assay and a targeted deep sequencing method," explained Ip.

"This approach achieved a genome editing efficiency of 27%, which is very high compared to other studies using genome editing for AD," she said.

Using both immunolabeling and behavioral tests, the team showed that "genome editing significantly alleviated pathologies including Abeta deposition, gliosis, neurite dystrophy and impaired cognitive performance," Ip said. The effects persisted for at least 6 months.

In AD, "microgliosis is a major contributor to disease pathogenesis, as impaired microglia cannot clear Abeta, while activated microglia release pro- and anti-inflammatory cytokines affecting brain functions."

Consequently, "decreasing microgliosis can restore microglial functions and reduce neuroinflammation, subsequently restoring normal brain function," she noted.

"Neurite dystrophy also contributes to AD pathogenesis, as it alters axonal transport and synaptic release properties, disrupting synaptic communication, so reducing neurite dystrophy can also help protect synaptic function."

Brain-wide disease-modifying genome editing could therefore represent a viable strategy for treating familial AD and other monogenic diseases affecting multiple brain regions.

"This PoC study demonstrates the feasibility of using this genome editing approach for efficient brain-wide gene editing and provides experimental evidence that it can reduce disease pathology and improve cognitive functions.

"It further serves as an important step in promoting use of genome editing in brain disease treatment, specifically in developing precision medicine for inherited forms of neurodegenerative disorders," said Ip.

"However, much more work is needed, especially safety evaluation, before this research can be translated into the clinic. Therefore, we will be further evaluating the safety of this systemic Cas9 delivery system and analyzing the long-term beneficial effects of genome editing in transgenic mice."