Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder which lacks effective disease-modifying therapies. We have investigated the therapeutic potential of pDNA encoding apolipoprotein E2 (ApoE2), or nerve growth factor (NGF) by transporting pDNA across the blood brain barrier (BBB) and expressing the ApoE2 or NGF into brain, using brain-targeted liposomal nanoparticles for treatment of AD. We explored the neuroprotective functions of ApoE2 and survival-promoting properties of NGF through gene therapy as potential disease-modifying therapies for AD. We designed brain-targeted gene delivery systems with prolonged systemic circulation and enhanced cellular penetration by conjugating transferrin (Tf) ligand and cell-penetrating peptide (CPP) to liposome via DSPE-PEG phospholipid. In vitro characterization studies showed that the nanoparticles had homogeneous particle size, positive zeta potential and protected plasmid DNA against enzymatic degradation. Additionally, they exhibited low hemolytic potential and low cytotoxicity. Cellular uptake occurred in a time-dependent manner through multiple endocytosis pathways. Reporter gene transfection and consequent protein expression in different cell lines were significantly higher using CPP-Tf-liposomes compared to single modified liposomes. The ability of these liposomes to escape from endosomes can be an important factor which may have likely contributed to the high transfection efficiency observed. In vivo brain targeting efficiency of designed liposomes was evaluated using in vitro triple co-culture BBB model. Dual-modified liposomes efficiently crossed in vitro BBB and, subsequently, transfected primary neuronal cells. Increasing NGF expression in primary neuronal cells following treatment with liposomes increased the levels of pre-synaptic marker synaptophysin in vitro. PenTf-liposomes containing pDNA efficiently induced protein expression in the brain of mice. A dose response study was performed in order to select the appropriate dose of pNGF to induce significant NGF expression and, consequently, a therapeutic effect. Administration of PenTf-liposomes containing pNGF to APP/PS1 mice (aged 3 months) for four weeks (one injection per week) decreased the levels of toxic soluble and insoluble Aβ peptides. Additionally, the treatment stimulated cell proliferation and increased the levels of synaptic markers, synaptophysin and PSD-95. These data suggest the therapeutic potential of PenTf-liposome-mediated NGF gene therapy which can be considered as a candidate for treatment of AD.