A note on retrograde gene transfer efficiency and inflammatory response of lentiviral vectors pseudotyped with FuG-E vs. FuG-B2 glycoproteins

Abstract

Pseudotyped lentiviral vectors give access to pathway-selective gene manipulation via retrograde transfer. Two types of such lentiviral vectors have been developed. One is the so-called NeuRet vector pseudotyped with fusion glycoprotein type E, which preferentially transduces neurons. The other is the so-called HiRet vector pseudotyped with fusion glycoprotein type B2, which permits gene transfer into both neurons and glial cells at the injection site. Although these vectors have been applied in many studies investigating neural network functions, it remains unclear which vector is more appropriate for retrograde gene delivery in the brain. To compare the gene transfer efficiency and inflammatory response of the NeuRet vs. HiRet vectors, each vector was injected into the striatum in macaque monkeys, common marmosets, and rats. It was revealed that retrograde gene delivery of the NeuRet vector was equal to or greater than that of the HiRet vector. Furthermore, inflammation characterized by microglial and lymphocytic infiltration occurred when the HiRet vector, but not the NeuRet vector, was injected into the primate brain. The present results indicate that the NeuRet vector is more suitable than the HiRet vector for retrograde gene transfer in the primate and rodent brains.

Figure 1

Production efficiency of pseudotyped lentiviral vectors. HEK293T cells in 72 10-cm tissue culture dishes were transfected with the envelope plasmid encoding FuG-E or FuG-B2, together with the transfer plasmid encoding RFP and packaging plasmids. Vector particles were concentrated by centrifugation, purified with ion-exchange chromatography and finally prepared in 110 μl of PBS. (a) The yield in the vector stock solutions measured by quantitative reverse transcription-PCR analysis (open bar for FuG-B2, gray bar for FuG-E). Each value was obtained from five individual experiments and expressed as the mean ± SEM. *P < 0.05, significant difference from the FuG-B2 pseudotype (Student’s t test). (b) Fluorescent images of SDS-PAGE of the viral protein (p24 and p17) from vectors pseudotyped with FuG-B2 (4.0 × 10⁶ genome copies) and FuG-E (4.0 × 10⁶ and 1.6 × 10⁷ genome copies). A full-length gel image is presented in Supplementary Fig. S3. (c) Fluorescent images of Hoechst-stained HEK293T and Neuro2A (NA) cells after transduction of the vectors pseudotyped with FuG-B2 and FuG-E (100 genome copies/cell). Scale bar, 50 μm. (d) Ratio of the RNA genome copies to the transduction units (open bars for FuG-B2, gray bars for FuG-E). Each value was obtained from five individual experiments and expressed as the mean ± SEM. **P < 0.01, significant difference from the FuG-B2 vector (Student’s t test).

Figure 2

Pattern of gene transfer of the HiRet and NeuRet vectors around injection sites in macaque monkeys. The HiRet and NeuRet lentiviral vectors (each 2.5 × 10¹⁰ genome copies/ml) were injected into the putamen (Put) of macaque monkeys, and histological analyses were performed in the brains fixed at four weeks postinjection. (a) Nissl staining and RFP immunostaining at the injection sites of the HiRet (left) and NeuRet (right) vectors with the same titer. ac, anterior commissure; Cd, caudate nucleus; GPe, external segment of the globus pallidus; GPi, internal segment of the globus pallidus; ic, internal capsule. Scale bar, 2 mm. (b) Double immunostaining for RFP (red) and NeuN (upper; green) or GFAP (lower; green) at injection sites of the HiRet (left) and NeuRet (right) vectors. Shown in yellow are double-immunostained cells. Scale bars, 50 μm.

Figure 3

Transgene expression after intrastriatal injections of the HiRet and NeuRet vectors in macaque monkeys. Intrastriatal injections of HiRet (2.5 × 10¹⁰ genome copies/ml) and NeuRet (2.5 × 10¹⁰ and 7.5 × 10¹⁰ genome copies/ml) pseudotyped lentiviral vectors were made in macaque monkeys, and histological analyses were performed in the brains fixed at four weeks postinjection. (a) RFP immunostaining in the substantia nigra pars compacta (SNc), centromedian nucleus (CM), and supplementary motor area (SMA) of the ipsilateral and contralateral hemispheres for the HiRet (top), lower-titer NeuRet (middle), and higher-titer NeuRet (bottom) vectors. cp, cerebral peduncle; MD, mediodorsal nucleus; SNr, substantia nigra pars reticulate; III, layer III; V, layer V. Scale bars, 1 mm for SNc and CM, 500 μm for SMA. (b) Number of RFP-positive neurons in the SNc and the centromedian-parafascicular complex (CM-Pf). Eleven or seven sections were used for cell counts in the SNc or CM-Pf, respectively. (c) Density of RFP-positive neurons in the SMA of the ipsilateral and contralateral hemispheres. Ten sections were analyzed. Each symbol (open or filled circle, triangle, or square) indicates data for each animal, and the same symbol represents data for the same animal. Averaged data (n = 2) are denoted by open (for the HiRet vector), filled (for the NeuRet vector with lower titer), and gray (for the NeuRet vector with higher titer) bars.

Figure 4

Transgene expression after intrastriatal injections of the HiRet and NeuRet vectors in marmosets. The HiRet and NeuRet vectors (each 1.0 × 10¹⁰ genome copies/ml) were injected into the Cd and Put of marmosets, and histological analyses were performed in the brains fixed at four weeks postinjection. (a) Injection sites of the HiRet (upper; gray) and NeuRet (lower; gray) vectors and cells double-immunostained for RFP (red) and GFAP (green) at the injection sites. Shown in yellow are double-immunostained cells. Scale bars, 1 mm for injection sites, 50 μm for double-immunostained cells. (b) RFP immunostaining in the SNc, CM-Pf, and Brodmann’s area 6 m (A6m) of the ipsilateral and contralateral hemispheres for the HiRet (upper) and NeuRet (lower) vectors. The roman numerals in each A6m panel indicate the cortical layers. fr, fasciculus retroflexus; Pf, parafascicular nucleus. Scale bars, 500 μm. (c) Number of RFP-positive neurons in the SNc and CM-Pf. Seven or four sections were used for cell counts in the SNc or CM-Pf, respectively. (d) Density of RFP-positive neurons in the A6m of the ipsilateral and contralateral hemispheres. Eight sections were analyzed. Each symbol (open or filled circle or triangle) indicates data for each animal, and the same symbol represents data for the same animal. Averaged data (n = 2) are denoted by open (for the HiRet vector) and filled (for the NeuRet vector) bars. Other conventions are as in Figs 2 and 3.

Figure 5

Transgene expression after intrastriatal injections of the HiRet and NeuRet vectors in rats. The HiRet and NeuRet vectors (each 1.0 × 10¹⁰ genome copies/ml) were injected into the striatum (Str) of rats, and histological analyses were performed in the brains fixed at four weeks postinjection. (a) Nissl staining and RFP immunostaining at the injection sites of the HiRet (left) and NeuRet (right) vectors. LV, lateral ventricle. Scale bar, 1 mm. (b) RFP immunostaining in the SNc, Pf, and primary motor cortex (M1) of the ipsilateral and contralateral hemispheres for the HiRet (upper) and NeuRet (lower) vectors. In each M1 panel, a Nissl-stained image of the corresponding region is also depicted. Scale bars, 500 μm for SNc, Pf, and M1. (c) Number of RFP-positive neurons in the SNc and Pf. Six or four sections were used for cell counts in the SNc or Pf, respectively. (d) Density of RFP-positive neurons in the M1 of the ipsilateral and contralateral hemispheres. Four sections were analyzed. Averaged data (n = 4 for each group; error bar, SEM) are denoted by open (for the HiRet vector) and filled (for the NeuRet vector) bars. **P < 0.01, ***P < 0.001, significant difference from the HiRet vector (Student’s t test). Other conventions are as in Figs 2–4.

Figure 6

Inflammatory responses after intrastriatal injections of the HiRet and NeuRet vectors in macaque monkeys and rats. (a) Nissl staining, RFP-native fluorescence (red), and immunofluorescent staining for Iba1 (green) and CD8 (magenta) at the injection sites of the HiRet (top), lower-titer NeuRet (middle), and higher-titer NeuRet (bottom) vectors in macaque monkeys. Put, putamen, Scale bar, 500 μm. (b) Nissl staining, RFP-native fluorescence (red), and immunofluorescent staining for Iba1 (green) and CD8 (magenta) at the injection sites of the HiRet (upper) and NeuRet (lower) vectors in rats. Str, striatum. Scale bar, 500 μm.