Highlights • Amino acid residue at the junction of fusion glycoprotein segments was optimized. • A novel vector showed the highest efficacy of retrograde transfer in mouse brains. • The vector resulted in efficient retrograde gene transfer in marmoset brains. • This vector system is useful for basic neuroscience studies and clinical treatment. Abstract Background Pseudotyping of a lentiviral vector with fusion glycoproteins composed of rabies virus glycoprotein (RVG) and vesicular stomatitis virus glycoprotein (VSVG) segments achieves high gene transfer efficiency through retrograde transport in the nervous system. In our previous study, we determined the junction of RVG/VSVG segments of glycoproteins that enhances the transduction efficiency of the neuron-specific retrograde gene transfer (NeuRet) vector (termed fusion glycoprotein type E or FuG-E). New Method We aimed to optimize the amino acid residue at position 440 in the membrane–proximal region of FuG-E to improve the efficiency of retrograde gene transfer in the brain. Results We constructed variants of FuG-E with 18 kinds of single amino acid substitutions at residue 440 to generate lentiviral vectors pseudotyped with these variants, and tested in vivo gene transfer of the vectors in the mouse brain. The FuG-E (P440E) variant, in which proline was substituted by glutamate at residue 440 in FuG-E, showed the greatest retrograde gene transfer efficiency in the brain, bearing the property of the NeuRet vector. The FuG-E (P440E) pseudotype also displayed efficient retrograde gene transfer in the common marmoset brain. Comparison with Existing Methods The NeuRet vector with the FuG-E (P440E) variant demonstrated higher retrograde gene transfer efficiency into different neural pathways compared with the parental FuG-E vector. Conclusions The FuG-E (P440E) pseudotype provides a powerful tool to investigate neural circuit mechanisms underlying various brain functions and for gene therapy trials of neurological and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]