Neurotherapeutics. 2021 Nov 11
Noelia Lopez-Sanchez, Alberto Garrido-Garc, Morgan Ramon-Landreau, Vanesa Cano-Daganzo, and Jose M. Frade
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AAV vector packaging The human E2F4 coding sequence with Thr248Ala/ Thr250Ala mutations and containing the Kozak sequence (ACC ATG G) and either a final stop codon (hE2F4DN) or the c-Myc tag sequence plus a stop codon (hE2F4DNmyc) was synthesized (GeneScript, Piscataway, NJ) and cloned into the pcDNA 3.1( +) vector (Invitrogen). After confirming the sequences, they were PCR amplified and seamless cloned [37] into the KD10 plasmid (Packgene, Worcester, MA), flanked by the human synapsin 1 (hSyn1) promoter [38] and the mutant woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence described by [39] (WPRE3SL), which lacks tumorigenic capacity [39]. The WPRE3SL cassette has the SV40 late polyadenylation signal sequence plus the upstream polyadenylation enhancer element repeated in tandem described by Schambach et al. [40]. The expected sequence was confirmed by Sanger sequencing. Research-grade, single-stranded AAV.PHP.B.hSyn1. hE2F4DN-myc.WPRE3SL (AAV-hE2F4DN-myc), AAV.PHP.B.hSyn1.hE2F4DN.WPRE3SL (AAV-hE2F4DN), and AAV.PHP.B.hSyn1.EGFP.WPRE3SL (AAV-EGFP) vectors for preclinical testing were manufactured by Packgene by co-transfecting the respective KD10 plasmids together with the pAdDeltaF6 helper vector containing essential adenoviral elements (E4 transcription unit, viralassociated RNA, and adenoviral DNA-binding protein) and the pRep2-Cap-PHP.B vector expressing both Repand PHP.B-specific Cap proteins [33]. Request Quote

Research Field: CNS

Keywords: E2F4 phosphorylation, Aβ deposits, Neuroinflammation, Neuronal tetraploidy, Y-maze

AAV Serotype: AAV.PHP.B

Dose: AAV.PHP.B vectors were intravenously (i.v.) administered through the tail vein at 3.12 × 10^13 vg/kg, 6.25 × 10^13 vg/kg, or 1.25 × 10^14 vg/kg.

Routes of Administration: intravenously (i.v.) administered through the tail vein


After decades of unfruitful work, no effective therapies are available for Alzheimer’s disease (AD), likely due to its complex etiology that requires a multifactorial therapeutic approach. We have recently shown using transgenic mice that E2 factor 4 (E2F4), a transcription factor that regulates cell quiescence and tissue homeostasis, and controls gene networks affected in AD, represents a good candidate for a multifactorial targeting of AD. Here we show that the expression of a dominant negative form of human E2F4 (hE2F4DN), unable to become phosphorylated in a Thr-conserved motif known to modulate E2F4 activity, is an effective and safe AD multifactorial therapeutic agent. Neuronal expression of hE2F4DN in homozygous 5xFAD (h5xFAD) mice after systemic administration of an AAV.PHP.B-hSyn1.hE2F4DN vector reduced the production and accumulation of Aβ in the hippocampus, attenuated reactive astrocytosis and microgliosis, abolished neuronal tetraploidization, and prevented cognitive impairment evaluated by Y-maze and Morris water maze, without triggering side effects. This treatment also reversed other alterations observed in h5xFAD mice such as paw-clasping behavior and body weight loss. Our results indicate that E2F4DN-based gene therapy is a promising therapeutic approach against AD.

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