Adeno-Associated Virus-Mediated Central Nervous System Gene Transfer to Suppress Alzheimer’s Disease High-Risk APOE4 Variant and Replace with Protective APOE2

Common genetic variants of APOE are major risk factors for sporadic late-onset Alzheimer’s disease (AD). APOE has three common variants: APOE3, APOE4, and APOE2. Epidemiological, clinical, and experimental evidence demonstrate that APOE3 is associated with an average risk for AD, APOE4 is pathogenic and conveys a high risk, and APOE2 is protective and reduces risk. In prior mouse studies, we have demonstrated that (1) adeno-associated virus (AAV)-mediated central nervous system (CNS) gene transfer of APOE2 is highly protective against the toxic effects of APOE4 and (2) AAV-mediated CNS transfer of anti-APOE4 microRNA (miRNA) significantly suppresses the expression of APOE4. The ideal therapy for APOE4 homozygotes would both reduce CNS levels of APOE4 and substitute APOE4 with the addition of APOE2. We have developed a “silence-and-replace” therapy, where suppression of CNS APOE4 is achieved through AAV-mediated expression of miRNAs designed to silence the endogenous human APOE4 messenger RNA (mRNA), together with simultaneous replacement by an APOE2 coding sequence [APOE2(R)] resistant to those miRNAs. AAV vectors expressing APOE2(R) with and without the miRNAs were administered to the hippocampus of human APOE4 mice, and APOE levels at the mRNA and protein levels were the same for both vectors. Dideoxy Sanger sequencing and allele-specific real-time quantitative PCR were used to assess the CNS APOE2 to APOE4 ratio. As expected, both vectors with APOE2(R) increased the E2/E4 ratio relative to controls by 3.5 ± 0.6-fold for APOE2(R) and 6.3 ± 0.4-fold for APOE2(R) + miRNAs. We conclude that a single AAV vector can deliver a cassette with dual functionality: suppression of the toxic APOE4 variant and simultaneous delivery of the protective APOE2. This dual approach may provide a more potent gene therapy for APOE4-dependent AD than either strategy.