Enhanced Pseudotyping Efficiency of HIV-1 Lentiviral Vectors by A

ORIGINAL ARTICLE Enhanced pseudotyping efﬁciency of HIV-1 lentiviral vectors by a rabies/vesicular stomatitis virus chimeric envelope glycoprotein

DCJ Carpentier, K Vevis1, A Trabalza, C Georgiadis, SM Ellison, RI Asfahani2 and ND Mazarakis

Rabies virus glycoprotein (RVG) can pseudotype lentiviral vectors, although at a lower efficiency to that of vesicular stomatitis virus glycoprotein (VSVG). Transduction with VSVG-pseudotyped vectors of rodent central nervous system (CNS) leads to local neurotropic gene transfer, whereas with RVG-pseudotyped vectors additional disperse transduction of neurons located at distal efferent sites occurs via axonal retrograde transport. Attempts to produce high-titre RVG-pseudotyped lentiviral vectors for preclinical and clinical trials has to date been problematic. We have constructed several chimeric RVG/VSVG glycoproteins and found that a construct bearing the external/transmembrane domain of RVG and the cytoplasmic domain of VSVG shows increased incorporation onto HIV-1 lentiviral particles and has increased infectivity in vitro in 293T cells and in differentiated neuronal cell lines of human, rat and murine origin. Stereotactic application of vector pseudotyped with this RVG/VSVG chimera in the rat striatum resulted in efficient gene transfer at the site of injection showing both neuronal and glial tropism. Distal neuronal transduction in the substantia nigra, thalamus and olfactory bulb via retrograde axonal transport also occurs after intrastriatal administration of chimera-pseudotyped vectors at similar levels to that observed with a RVG-pseudotyped vector. This is the first report of distal transduction in the olfactory bulb. The enhanced pseudotyping with this envelope should enable easier production of higher-titre pseudotyped lentiviral vectors that exhibit efficient local and dispersed neuronal transduction in the CNS. Gene Therapy (2012) 19, 761–774; doi:10.1038/gt.2011.124; published online 8 September 2011

Keywords: lentivirus vectors; pseudotyping; rabies virus; vesicular stomatitis virus; glycoprotein

INTRODUCTION neuronal axons.8–14 RVG is the most promising GP of Lyssavirus HIV particles produced by cells superinfected with other viruses, such (rabies virus (RV) and rabies-related virus) origin for the targeting of as Moloney murine leukaemia virus, herpes simplex virus and neuronal cells in vitro and in vivo.13 This presents the possibility of vesicular stomatitis virus (VSV), display an expanded cell tropism targeting gene therapy for neurodegenerative diseases specifically to because of the presence of heterologous envelope-incorporated surface neuronal cells. Additionally, it opens up the possibility of a non- glycoproteins (GPs).1–5 This process of pseudotyping, reviewed in invasive administration of the vector by targeting the peripheral sites Cronin et al.,6 has been extensively harnessed to alter, improve and of neuromuscular synapses to target cells of the CNS affected by such refine the cell and tissue tropism, and transduction efficiency of diseases as amyotrophic lateral sclerosis and spinal muscular atrophy. replication-defective gene therapy vectors based on retroviruses and One barrier to the clinical use of RVG-pseudotyped lentivirus both primate and non-primate lentiviruses. vectors is the relatively low efficiency of transduction displayed by The high transduction efficiency and wide cell tropism of vesicular these vectors. HIV vectors pseudotyped with either RV racSADB19 stomatitis virus GP (VSVG)-pseudotyped lentivirus vectors has made strain or the related Mokola virus GPs showed almost fivefold lower this the standard against which other pseudotypes are compared. transduction efficiency when compared with the equivalent number of VSVG-pseudotyped lentiviral vectors are stable and capable of with- VSVG-pseudotyped vector particles on the same cell types.14 Different standing purification and concentration protocols required for their strains of RV and related GPs appear to pseudotype lentivirus vectors clinical use.7 Their broad cell tropism, however, do not make them at different efficiencies. RV ERA (Evelyn-Rokitnicki-Abelseth) and amenable for targeting gene delivery to specific disease sites, and they CVS (Challenge Virus Standard; B2c) strains have been shown to lack the ability to access difficult to reach sites (such as the central efficiently pseudotype lentivirus vectors based on equine infectious nervous system (CNS)) without invasive delivery methods. anaemia virus (EIAV), although at a lower efficiency than VSVG.8,10 Pseudotyping of lentivirus vectors with the envelope GP of the However, efficient pseudotyping of HIV-1-based vectors remains neurotrophic rabies virus (RVG) confers both neurotropism and the problematic in our hands and that of others;11 the RV CVS (B2c ability to mediate retrograde trafficking of vector particles along substrain) pseudotypes HIV-1 vector particles with the best efficiency.

Department of Gene Therapy, Centre for Neurosciences, Division of Experimental Medicine, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London, UK Correspondence: Professor ND Mazarakis, Department of Gene Therapy, Centre for Neurosciences, Division of Experimental Medicine, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Burlington Danes Building, Du Cane Road, London W12 0NN, UK. E-mail: [email protected] 1Current address: UCL Institute of Ophthalmology, London, UK. 2Current address: Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK. Received 11 May 2011; revised 20 July 2011; accepted 2 August 2011; published online 8 September 2011 Enhanced chimeric envelope pseudotyping DCJ Carpentier et al 762

In this study we aimed to generate a chimeric envelope GP bearing fusion. The VSVG protein sequence shows low but significant the RVG receptor-binding domain that will more efficiently pseudo- sequence homology with RVG25 and is thought to share several type HIV-1 vectors by replacing the cytoplasmic, transmembrane and structural and functional properties with RVG. The structure of the extracellular stalk regions of RVG with the corresponding regions from VSVG extracellular domain reviewed in Roche et al.26 has been VSVG. The pseudotyping and transduction efficiency of these new determined both in its prefusion24 and low pH23 forms by X-ray constructs was evaluated. We show that replacing the cytoplasmic crystallography after proteolytic treatment to remove disordered region of RVG with the cytoplasmic region of VSVG resulted in higher regions. Based on these observations we defined the extracellular levels of GP incorporation on the vector particles, and that this portion of both VSVG and RVG as consisting of the receptor-binding correlated with an increase in transduction efficiency. The ability of domain (also mediating membrane fusion) and a less ordered this chimera vector to transduce the CNS was also evaluated in vivo in stalk domain (see Figure 2b) linking it to the hydrophobic transmem- the rat CNS. brane domain as defined in Rose et al.27 Using the PCR and splicing by overlap extension (SOE),28 envelope RESULTS protein expression plasmids were generated encoding RVG/VSVG Design and construction of chimeric rabies/ VSVG expression chimeric proteins in which: (1) the cytoplasmic domain of RVG was plasmids replaced with the cytoplasmic domain of VSVG (RVG/VSVG(C)), Lentiviral vectors have in the past been pseudotyped with RVG from a (2) the cytoplasmic and transmembrane domains of RVG were number of different strains of rabies viruses. The ERA strain15 replaced by the cytoplasmic and transmembrane domains of VSVG envelope GP has been used effectively to pseudotype EIAV vectors (RVG/VSVG(TC)) and (3) the cytoplasmic and transmembrane for therapeutic delivery of vascular endothelial growth factor to the domains and most of the extracellular stalk domain of RVG were SOD1G93A mouse model of amyotrophic lateral sclerosis.16–19 In our replaced by the corresponding region of VSVG (RVG/VSVG(STC)). hands, however, the ERA strain does not seem to pseudotype HIV Figure 2 gives details of the chimeric constructs generated. vectors very efficiently (P Patsali and ND Mazarakis, unpublished results). Other labs have had some success using the RVG from the Lentiviral vectors pseudotyped with a chimeric RVG possessing the rabies virus PV (Pasteur vaccines) strain.11,13 We have had very good VSVG cytoplasmic domain show increased transduction efficiency results using the CVS strain, particularly the B2c substrain, to in 293T cells pseudotype HIV-1 vectors (SM Ellison, RI Asfahani, ND Mazarakis, HIV-1 vectors, carrying a green fluorescent protein (GFP) expression unpublished results). Bioinformatic analysis and sequence alignment cassette, pseudotyped with the VSVG, RVG and the three RVG/VSVG of the C-terminus of both the ERA and CVS (B2c and N2c substrains) chimeric constructs, were produced (triplicate small-scale prepara- GP amino-acid sequences revealed a region of low sequence homol- tions) by co-transfection of 293T cells by calcium phosphate pre- ogy, mapping to the cytoplasmic tail and the transmembrane a-helix, cipitation (see Materials and Methods). Supernatants were harvested compared with the rest of the protein sequence (Figure 1). This and vector particles were concentrated and purified by ultracentrifu- suggested that the differences in these regions could play a role in gation through a 20% sucrose cushion. The biological titre of the determining the pseudotyping efficiency of the respective strains of vectors was determined by flow cytometry of eGFP expressing RVG. We were interested in generating a recombinant RVG with transduced 293T cells (Figure 3a and Table 1). The vector particle enhanced HIV-1 pseudotyping efficiency. To achieve this we decided concentration of each preparation was estimated by enzyme-linked to construct chimeric GP constructs replacing regions of the immunosorbent assay (ELISA) for the HIV-1 P24 capsid protein. The RVG (CVS-B2c) with corresponding regions from VSVG, known to values were used to calculate a vector infectivity value (transduction efficiently pseudotype HIV-1 vectors. units (TU) per ng P24). None of the RVG receptor-binding domain VSVG and RVG are members of the class III viral fusion proteins.20 containing constructs came close to matching the transduction They are both membrane-associated proteins possessing a single efficiency of VSVG (RVG showed almost a 10-fold lower infectivity transmembrane helix toward the carboxy terminus, and an amino value compared with VSVG; see Figure 3b). The TC and STC terminal signal peptide that gets cleaved off during post-translational constructs showed almost complete loss of transduction. The con- processing. Both G proteins are incorporated into virus particles as struct in which only the cytoplasmic domain had been replaced trimers21–24 and mediate receptor interaction, entry and membrane showed a significant (P¼0.0343) but small (approximately twofold)

Figure 1 Rhabdoviridae envelope glycoprotein bioinformatics sequence alignment. Alignment of the C terminal portion of RVG from the CVS-B2c, CVS-N2c and the ERA vaccine strains and VSVG from the Indiana strain. Highly conserved (shared by X75% of the sequences) residues are shaded black. Moderately conserved residues (shared by 50% of the sequences) are shaded grey. The numbering is based on the start codon being the ﬁrst residue, unlike numbering in some publications that base it on the position relative to the proteolytic cleavage site after the signal peptide. Regions as deﬁned for VSVG based on structural and biochemical studies are indicated above the sequence.

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Figure 2 Construction of RVG/VSVG chimera GPs. (a) Schematic comparison of the chimeric constructs used in this study showing how they relate to the RVG and VSVG sequences. The regions of the respective proteins used in each chimeric construct are shown in the table and the amino acids flanking the fusion site are shown in detail for each construct. (b) Schematic representation of the envelope constructs used in this study showing how the structures of the protein relate and how the recombinants have been altered. increase in vector infectivity compared with RVG-pseudotyped buffered saline (PBS), lacking bovine serum albumin, failed to dispel vectors. Assuming B2000 molecules of p24 per HIV particle (pub- this band. lished estimates range from 1500 to 2100),29–31 an equivalent of B104 To further confirm that the observed differences in RVG levels particles per pg of p24 equates to 3593 particles per TU for RVG and were not because of varying affinity of the antibody to the various 1899 particles per TU for RVG/VSVG(C) (Table 1). constructs, we repeated the western blot and probed the membrane with an alternative antibody to RVG (SNB-1, kindly provided by The VSVG cytoplasmic domain increases incorporation levels of Dr Edward Wright, Wohl Virion Centre, University College London, chimeric RVG into HIV-1 vector particles London, UK). These blots gave similar results to those done with To determine if the changes in transduction efficiency observed for the the 1C5 antibody and only detected a minor nonspecific band chimeric constructs were because of an increase in the level of present in all samples, including the control lane (Figure 4aiii). envelope protein incorporation into vector particles, vector prepara- Densitometrical analysis of three separate blots carried out on tions were analysed by sodium dodecyl sulphate-polyacrylamide gel three independent sets of preparations (Figure 4b) further supported electrophoresis (SDS-PAGE) and western blot analysis. Equal numbers that there was a significant difference in the incorporation levels of of vector particles were lysed in SDS-PAGE loading buffer, run on a both C (P¼0.0184, paired two-tailed t-test) and STC (P¼0.0325, 15% SDS-PAGE gel and blotted onto nitrocellulose membrane. paired two-tailed t-test) GPs compared with RVG, whereas the TC GPs Probing the membrane with an antibody to HIV-gag (aHIV-1 did not show a significant difference in incorporation levels compared p55+p24+p17; Ab63917; Abcam, Cambridge, UK) confirmed that with RVG. equal amounts of gag had been loaded for each sample (Figure 4ai). Stripping and reprobing the same membrane with an antibody specific Chimeric RVG-pseudotyped HIV-1 vectors more efficiently to RVG (clone 1C5) indicated that different GPs were incorporated transduce differentiated neuronal cell lines in vitro into vector particles at different efficiencies (Figure 4aii). Both C and Our results suggest that vectors pseudotyped with the chimeric RVG/ STC chimeric GPs were incorporated at high levels, whereas the TC VSVG(C) construct are more efficient at transducing cells. To verify GPs appeared to be incorporated at low levels. All showed higher whether this is true in neuronal cells, the intended target of RVG- incorporation levels than wild-type RVG. The 1C5 antibody detected a pseudotyped vectors, we compared transduction levels of lentiviral nonspecific band in all vector preparations that was also present in vectors pseudotyped with the RVG/VSVG(C) chimera and wild-type control samples taken from untransfected cells that had not been RVG CVS(B2c) in three differentiated neuronal cell lines: PC12, a rat transfected, making densitometry measurements difficult, as in several pheochromocytoma of the adrenal medulla;32 SH-SY5Y, a thrice blots the band overlapped with the RVG band (particularly the larger cloned human neuroblastoma;33 and NSC-34, a mouse hybrid cell wild-type RVG). We hypothesised that the antibody might be detect- line displaying motor neurone-like properties created from the fusion ing a component of the bovine serum albumin present in the vector of the neuroblastoma cell line N18TG2 and motor neurone-enriched resuspension buffer; however, using unsupplemented phosphate mouse spinal cord cells.34

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determined by P24 ELISA and lentivirus genome quantitative PCR (see Table 2). The biological activity of the three different pseudotypes was determined on 293T, differentiated PC12, differentiated SH-SY5Y and differentiated NSC-34 cells (see Figure 5a). Infectivity was calculated as TU per 106 genomes or TU per ng P24 for the three different pseudotypes on each of the cell lines (see Figure 5b). Cells transduced by equal genome copy numbers showed clear differences in LacZ-positive cells 72 h post transduction. Vectors pseudotyped with the chimeric RVG/VSVG(C) construct consistently transduced more cells than RVG-pseudotyped vectors and less than VSVG-pseudotyped vectors. This pattern was confirmed and shown to be statistically significant by quantitative analysis of the vector infectivity calculated as TU per 106 genomes in each of the cell lines. 293T and SH-SY5Y cells showed similar transduction patterns (Figure 5b, top panel). Infectivity levels were slightly lower in SH-SY5Y, probably because of the more disperse cell distribution caused by growth in differentiation conditions. A similar pattern was also seen in PC12 cells grown under differentiation conditions, although overall transduction levels on these cells were much lower for all three vector pseudotypes (B10-fold lower for VSVG and B1000-fold lower for RVG and the chimera). During the differentiation process of NSC-34 cells a large number of the cells die, resulting in a very sparse cell distribution. Additionally, we observed a large number of cells dying when we used the standard transduction protocol, resulting in a very patchy cellular distribution across the tissue culture dish at the time of LacZ staining. This was potentially caused by the presence of polybrene in the transductions. The experiment was repeated without using polybrene, resulting in much lower levels of cell death; however, the transduction levels were also much lower (10–20-fold lower for RVG/VSVG(C) and RVG, and 5-fold lower for VSVG). When the data were normalised to the amount of P24 present in the vector preparations, the same general pattern was observed, although with a much lower statistical significance (Figure 5b, bottom panel). In 293T cells the infectivity values (TU per ng P24) in one experiment were practically identical to those observed in the analysis of small-scale preparations. A second experiment however gave more variable results (giving the borderline significant P-value of 0.0528), potentially because of the greater variability in vector aliquots and to harsher pelleting and resuspension protocols used in the large-scale preparations compared with the small-scale preparations. This larger variability also resulted in the lack of statistical significance observed in SH-SY5Yand NSC-34 cells when data were normalised to P24 concentration. We analysed both P24 and vector genome content of our preparations as measures of physical particle content. Neither one of these is a good measure of the biological activity of vector preparations because of the high variability implicit in the production by transient transfection method.35 Our numbers suggest that only 2–5% of the particles (as defined by P24 content and assuming 104 particles per pg of P24) contain genomic RNA molecules. This low level of genome incorporation probably reflects the larger size of the LacZ encoding Figure 3 Comparison of infectivity. (a) Vector particle concentration (ng P24 protein per ml) determined by P24 ELISA and biological activity values genome, resulting in less efficient packaging within vector particles. determined by fluorescence-activated cell sorting (FACS) on 293T cells (TU ml–1) for triplicate small-scale preparations purified by sucrose cushion VSVG and RVG-CVS-B2c-pseudotyped vector transduction in vivo protocol. Values are given in Table 1. (b) Relative infectivity or transduction The transduction efficiency of eGFP expressing HIV vectors pseudo- efficiency of the RVG/VSVG chimeric constructs compared with wild-type typed with VSVG (Indiana strain) or RVG (CVS-B2c strain) was RVG and VSVG constructs. Infectivity (given as TU per ng of P24 protein) ± assessed in the brains of rats (n¼6) by intrastriatal injections with 4 ml was calculated for each prep. Error bars show s.e.m. (*P¼0.0343 two- 6 6 tailed t-test compared with RVG). of either vector (containing 510 and 2.810 transducing units, respectively). Immunohistochemical staining of sections to enhance Large-scale vector preparations encoding the lacZ reporter, pseu- the eGFP signal showed a distinctive difference in their pattern of dotyped with VSVG, RVG and chimeric RVG/VSVG(C), were pro- transduction, with VSVG showing a localised expression within the duced and concentrated 2000-fold. Physical vector particle titres were striatum alone, whereas the RVG pseudotype expression spreading

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Table 1 Characterisation of pseudotyped vector preparations

RVG/VSVG(C) RVG/VSVG(TC) RVG/VSVG(STC) RVG VSVG

(P24; ng ml–1) 5.35103±1.51103 5.64103±4.13102 6.18103±1.20103 5.82103±5.15102 5.02103±1.18103 Biological titre on 293T cells (TU ml–1) 2.59107±3.90106 2.10106±3.97105 8.33105±8.33105 1.64107±2.57106 9.31107±2.83107 Transduction efﬁciency (particles per TU) 1.90103 2.59104 9.52104 3.59103 5.56102

Abbreviations: RVG, rabies glycoprotein; TU, transduction unit; VSVG, vesicular stomatitis virus glycoprotein. Values represent the average of three independent small-scale preparations for each pseudotyped construct±s.e.m. beyond the striatum anteroposteriorly to regions distal to the injection 42.81±1.298% of striatal cells with a neuronal profile (NeuN+), site (Figures 6a and b). whereas the RVG/VSVG(C) pseudotype differs significantly The extent of transduction at the site of injection appears to span (F(1,2)¼1938.1, Po0.001) from the former, transducing only across a larger area of the striatum in the case of the RVG-pseudotyped 14.73±0.079% of neurons (Figure 8a). Although less vector as opposed to VSVG (Figure 6a). Unbiased counts of total (37.06±1.169% versus 24.31±3.982%) astrocytes (GFAP+) were eGFP+ cells over DAPI+ cells in the striatum revealed an equal to transduced by the RVG/VSVG(C) pseudotype than the RVG pseudo- slightly higher number of transduced cells for VSVG (1.74% versus type, this difference was found not to be significant (Figure 8b). In 2.62%). High-magnification images of both striatal sections confirm some animals, staining of neuronal fibres was also observed in the the counts as the VSVG-transduced cells appear tightly packed globus pallidus (Figure 7aiii, arrow), most likely resulting from compared with the more sparse appearance of RVG (Figure 6b, see anterograde migration of eGFP into cell projections from its site of insert). The higher intensity seen with RVG is mainly because of expression in the soma located in the striatum. transduced (expression of eGFP) fibres projecting to distal sites of the Retrograde transport efficiency of these pseudotyped vectors was brain such as the olfactory bulb, thalamus and substantia nigra where compared in more detail by single immunohistochemical staining on expression was also observed (Figure 6bi, v and vi). Dual staining of sections obtained from the olfactory bulb and thalamus, using anti- striatal sections with antibodies against eGFP and either NeuN or body against eGFP (Figure 7 inserts), and by dual staining, using anti- GFAP revealed the neuronal tropism of both pseudotypes with RVG GFP and anti-TH antibodies, for the sections containing substantia further transducing cells with a glial morphology/profile (astrocytes, nigra. Despite the presence of transduced cells from both pseudotyped Figure 6c). vectors in the olfactory bulb, mainly but not exclusively in the mitral cell layer, a significant difference between them was noticed Chimeric glycoprotein-pseudotyped vectors transduce neuronal (F(1,4)¼8.1883, Po0.05; Figure 9a). In this area, RVG-pseudotyped cells within the site of infection and distal areas by retrograde vector transduced 4.963±0.421% of DAPI+ cells, whereas RVG/ transport VSVG(C) chimera transduced 2.036±0.9322% of DAPI+ cells. Unlike Adult rats (n¼6) were used to compare the transduction pattern of areas reported above, both vectors showed approximately the RVG- and RVG/VSVG(C)-pseudotyped HIV-1 vectors expressing same level of gene expression for the thalamus (RVG pseudotype: eGFP from the PGK promoter following sterotaxic administration 0.545±0.116% of DAPI+; RVG/VSVG(C) pseudotype: 0.497±0.103% into the striatum and were killed 5 weeks after injection. The lentiviral of DAPI+; Figure 9b) and substantia nigra pars compacta (RVG preps used for these in vivo experiments had a biological titre of pseudotype: 3.205±0.545 eGFP+/TH+ cells per animal; RVG/ 1.66109 TU per ml in 293T cells (2000-fold concentration) for both VSVG(C) pseudotype: 3.125±0.5 eGFP+/TH+ cells per animal; wild-type and chimeric envelope, with P24 values of 129 and 89 mgml–1 Figure 9c). Thalamic nuclei in which transduced cells could be and infectivity values of 1.2104 and 1.8104 TU per ng P24, observed were the ventromedial, ventrolateral, centromedial, the respectively, indicating that there are no stability issues with scaling paracentral, the submedial (Figure 7), the mediodorsal, the posterior up the chimeric pseudotype. A selection of 40 mm thick sections was and the parafascicular (not shown) nuclei. stained with an anti-eGFP antibody to enhance the green fluorescence signal and evaluate the extent of transduction throughout the brains of DISCUSSION injected rats (Figure 7). Possible colocalisation with neurons, astro- Of the three chimeric constructs tested in this study, only one was cytes, oligodendrocytes and microglia was examined by dual staining found to produce pseudotyped vectors possessing higher biological with antibodies against eGFP in conjunction with NeuN, GFAP, Rip activity. Replacing the RVG cytoplasmic tail with that of VSVG (data not shown) and ED1 (data not shown), respectively (Figure 8). resulted in more RVG being incorporated per vector particle and in In agreement with previously published results concerning lentiviral preparations possessing higher biological activity. It is unclear whether EIAV CMV-LacZ vectors,8,10,16 eGFP immunoreactivity was seen this increased incorporation is because of an ability of the VSVG throughout the striatum for both pseudotypes (Figures 7aii and bii). cytoplasmic tail to preferentially localise in cholesterol-rich detergent- As for RVG-pseudotyped vector injections reported above, the trans- resistant microdomains believed to be the main site of HIV particle duced cells appeared sparse and a considerable part of immunoreac- budding. It is also unclear if this is because of a direct or indirect tivity came from fibres rather than somas. This is true for both the interaction with HIV capsid or matrix proteins, or because of the fact pseudotypes examined. As expected, the RVG-pseudotyped vector that the VSVG cytoplasmic tail is considerably shorter than the RVG confirmed its retrograde transport capability, and also the RVG/ cytoplasmic tail. Studies using Measles virus envelope proteins to VSVG(C) chimera showed this property. In fact, cells positive for pseudotype lentiviral vectors found that truncating the cytoplasmic eGFP were observed in the olfactory bulb, in the thalamus and in the tail increased the biological titre of preparations and increased the substantia nigra in brains injected with both pseudotypes. All these level of both heamaglutinin and Fusion protein incorporation into sites are known to send projections to the striatum. Within the vector particles.36 It is possible that the added steric hindrance of striatum the RVG pseudotype was found to transduce larger cytoplasmic tails may exclude GP molecules from being

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incorporated into budding vector particles. Interestingly, in this study replacing the transmembrane domain also resulted in loss of biological activity and a drop in RVG incorporation into vector particles, whereas extending the regions replaced to the extracellular stalk rescued incorporation levels but not biological activity. This suggests some form of interaction between the transmembrane domain of VSVG and the extracellular stalk in either maintaining the stability of the protein, mediating its membrane insertion or directing its localisation to regions of HIV budding. Although every effort was made to limit the replaced region to parts of the protein not involved in the structure of the receptor-binding domain, by referring to the structural models of VSVG,23–24 it is possible that these regions play an important role in the biological activity, structure or multimerisation of the GPs. Our initial evaluation of the infectivity of the different constructs was done using small-scale sucrose cushion purified preparations to transduce human 293T cells. We proceeded to compare the infectivity of chimera-pseudotyped vector with RVG-pseudotyped vectors by transducing a range of neuronal cell lines from a spectrum of species (PC12; rat, SH-SY5Y; human, NSC-34; mouse) grown in conditions that induce neuronal cell differentiation using large-scale preparations. Differences in infectivity values were less clearcut than with the small- scale preparations (although they still showed the same trend), probably because of the higher variability introduced in the preparations by the harsh pelleting and resuspension protocol. These pro- cesses may result in more vector particle damage and a lower purity (because of longer and more intense centrifugation without the benefit of a sucrose cushion), resulting in a higher level of non-biologically active particle-associated P24 and genomic RNA molecules. This would affect the normalisations carried out in the in vitro experiments (Figure 5). However, it is interesting to note that the TU per ng P24 was very similar between the small prep experiment and the large prep B 3 Figure 4 Comparison of GP incorporation into vector particles. (a) Western experiment ( 510 TU per ng for the chimera in 293T cells). blot of purified pseudotyped vector particles on a 15% SDS-PAGE gel. All pseudotyped vectors possessed considerably higher ‘infectivity’ Supernatant from untransfected 293T cells put through the same values on human cells compared with rodent cells (roughly 100- to purification process as the particles were loaded as a background control 1000-fold higher numbers of cells were transduced in 293T and (lane 1). Equal numbers of vector particles were loaded into all wells (lanes SH-SY5Y compared with PC12 and NSC-34 cells). CVS-24 is a 2–6) based on the values of a P24 ELISA (see Table 1). Electrophoresed mouse-adapted rabies virus strain from which two quasispecies proteins were transferred onto a nitrocellulose membrane and blotted for the presence of HIV-1 gag using an antibody that binds both capsid and matrix variants have been isolated. The N2c variant shows a stronger domains of HIV-1 gag (i), as a loading control to verify that equal levels of preference to neuronal cells in vitro and in vivo, and is more vector particles were present in all the samples. HIV-gag proteolytic processing intermediates pr55 and p41 were detected by the antibody along with the completely processed p24 capsid and p17 matrix protein. Table 2 Vector titration for in vitro transductions Membranes were blotted with two alternative antibodies detecting different epitopes of the RVG protein (1C5 (ii) and SNB-1 (iii), both specific for (P24; ng ml–1) Genome copy number (genomes per ml) the receptor-binding domain). Specific bands for the RVG protein were detected migrating at B60 kDa size. A size difference was observed VSVG 4.16104±1.78103 2.221010±7.92109 between the wild-type RVG and the chimeric proteins because of the RVG 4.01104±4.61103 6.371010±1.581010 difference in the size of their respective cytoplasmic domains. Images (i) RVG/VSVG(C) 7.20 103±6.82 102 4.52 109±5.27 108 and (ii) represent the same membrane that has been stripped and reprobed. (b) Quantification of the levels of GP incorporation by densitometry of Abbreviations: RVG, rabies glycoprotein; VSVG, vesicular stomatitis virus glycoprotein. triplicate independent vector production experiments and blotted with the Physical lentiviral particle titration of large-scale preps used for in vitro transduction experiment ± (Figure 5), by P24 enzyme-linked immunosorbent assay (ELISA; to measure the level of SNB-1 antibody. Error bars show s.e.m. (*Po0.05 compared with RVG by lentiviral protein present) and quantitative PCR (qPCR; to measure the vector genome copy paired two-tailed t-test). number). Average values are given of X3 measurements±s.e.m.

Figure 5 In vitro transduction of differentiated neuronal cell lines. (a) Phase contrast micrographs of 293T, and differentiated SH-SY5Y, PC12 and NSC-34 cells transduced with normalised genome copy numbers (1.125106 genome copies per well) of RVG/VSVG(C), RVG- and VSVG-pseudotyped lentiviral vectors encoding the LacZ reporter gene. Cells were fixed 48 h post transduction (hpt) and stained for LacZ activity with X-gal. Scale bars represent 100 mm. (b) Quantification of transduction efficiency of the different pseudotypes in SH-SY5Y, PC12 and NSC-34 (in the presence and absence of polybrene) cells. LacZ-positive foci of colonies (FOCs) were counted from triplicate wells in two independent experiments and used to calculate the biological titre of the different pseudotyped vector preparations±s.e.m. (error bars). A titration on 293T cells carried out in parallel is shown for comparison. Graphs show the number of transducing units calculated per 106 genomes (top panel) and per ng P24 (bottom panel) as determined for the individual preparations (see Table 2). Numbers are the average of two independent experiments each with triplicate samples (n¼6). For NSC-34 cells, one experiment was done in the presence of polybrene (n¼3) and one was done without polybrene (n¼3). In the graphs, the y axis is log10 scale. *, ** and *** indicate levels of statistical significance.

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Figure 7 Comparison of vector-mediated gene delivery pseudotyped with RVG or RVG/VSVG(C) after intrastriatal injection in rats. GFP expression at the site of injection and distal sites (i, olfactory bulb; ii, striatum; iii, thalamus; iv, substantia nigra) injected with P24-normalised (a) RVG or (b) RVG/VSVG(C)- pseudotyped HIV-1 lentiviral vectors (single injection of 4 ml volume carrying 4.58106 and 6.64106 TU, respectively). Brain sections, 40 mm thick, from n¼6 Wistar rats harvested 5 weeks post injection were obtained and stained with anti-eGFP antibody to enhance the eGFP signal (shown in black). High- magnification inserts corresponding to marked regions (dashed box) of the panoramic section images indicate the transduction of cell bodies and fibres. Arrow in (aiii) demonstrates expression of eGFP in globus pallidus region. Thalamus nuclei: CM, centromedial; PC, paracentral; SM, submedius; VL, ventrolateral, VM ventromedial. Panoramic section images were captured by epifluorescent microscopy with a 4 objective and inserts with a 10 objective.

Figure 6 Evaluation of pseudotyped HIV-1 vector-mediated gene delivery after single intrastriatal injection in rats. (a) GFP expression in the striatum at the site of injection transduced with either VSVG (5106 TU) or RVG (2.8106 TU) pseudotyped HIV-1 lentiviral vectors (single injection of 4 ml volume). Sections of brains, 30 mm thick, harvested 5 weeks post injection were stained with anti-eGFP antibody to enhance the eGFP signal and delineate the extent of transduction (shown in black). (b) Analysis of sites distal to the striatum showing RVG-pseudotyped HIV-1 vector-mediated transduction of both afferent and efferent projecting neurones (olfactory bulb, globus pallidus, thalamus and substantia nigra (SNc, pars compacta; SNr, pars reticulata)). Panoramic section images were captured by epifluorescent microscopy with a 2.5 objective and compiled into the final images using ImageJ. Inserts show higher magnification views of the indicated regions (dashed box). Black arrows on striatal regions (a and biii) correspond to the respective VSVG and RVG striatum inserts. (c) Tropism of pseudotyped lentiviral vectors in the striatum. GFP+ sections were stained using anti-GFP antibodies (Alexa488 secondary antibody, to enhance the GFP signal) shown in green, and antibodies to either NeuN (Alexa594 secondary antibody, to stain neuronal cells) or glial fibrillary acidic protein (GFAP, Alexa594 secondary antibody, to stain astrocytes) shown in red. Neuronal tropism (NeuN-positive eGFP-expressing cells) of the vectors at the site of injection was seen in both VSVG- and RVG- mediated transductions. The bottom panel shows confirmation that RVG also transduces astrocytes (GFAP-positive GFP-expressing cells). Top and bottom panels are confocal microscopy images of stained tissue sections. The middle panel was acquired by epifluorescence microscopy. Scale bars show 20 mm.

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Gene Therapy Enhanced chimeric envelope pseudotyping DCJ Carpentier et al 771 pathogenic in adult mice, whereas the B2c variant has been described could be defined by their morphology and relative positioning as as preferentially replicating in nonneuronal cells and being more mitral cells, not all of them appear to belong to the same category, or pathogenic in neonatal mice.37 to be tufted cells. These cells constitute the sole output of the olfactory The in vivo results show that both wild-type RVG- and chimera- bulb, but the finding of transduced cells possibly belonging to other pseudotyped vectors are able to efficiently transduce cells around the categories (granule cells) raises some fascinating consideration about site of injection as well as cells in distal sites reachable by retrograde the connectivity in this area. However, without using specific markers axonal trafficking of vectors. We observed differences in the affinity for for the various cytological classes mentioned above, it is impossible to neuronal and glial striatal subpopulations for the two pseudotypes. make further statements at present. The RVG/VSVG(C)-pseudotyped vector showed a significantly lower One of the many hurdles to advancing virus-based gene therapy rate of neuronal tropism compared with RVG, where approximately vectors into clinical trials is the relatively high cost and effort required half of the transduced cells were neurons. Taken together with the to produce enough material to administer therapeutically active doses lower, but not statistically significant, difference in the level of to any recipient organism larger than a rodent. Any advance that astrocyte transduction between the two pseudotypes, this would either increases the production levels of these vectors or makes them suggest that the increase in pseudotyping efficiency does not translate more efficient gene delivery vehicles would be a welcome contribution. into improved neuronal transduction in vivo. Several strategies have been employed to simplify and optimise Concerning the retrograde transport properties of the two pseudo- production methods, including establishing stable cell lines expressing types examined here, besides a slightly better overall efficiency of the components of the vectors,39 producing vectors in alternative expres- RVG pseudotype, especially in the olfactory bulb, they were both sion systems40 or using alternative ways to introduce vector compo- found to be able to transduce similar distal areas. The striatum nent expression constructs into producer cells.41 The VSVG represented the ideal target of intracranial injection because of both pseudotype has been used in many studies because of the wide its size and position and its extensive interconnections throughout the tropism and good transduction levels observed in most cell types brain, the former because of the virtual absence of a misplacing with these vectors.19 However, its high promiscuity (capable of occurrence, the latter because of the plethora of different structures transducing a very wide range of cell types), potential cytotoxicity,42 that send to or receive connections from it, thus allowing accurate sensitivity to inactivation by human complement43 and high immu- assay of the retrograde transport properties of pseudotyped vectors. nogenicity44 may prevent its extensive use in the clinic. For the The olfactory bulb transduction is a new observation of retrograde treatment of diseases with wide pathology in the CNS, the lack of transport of RVG-pseudotyped vectors via projections to ventral axonal retrograde transport makes delivery of VSVG-pseudotyped striatum, in accordance with published observations.38 To our knowl- vectors problematic, requiring direct delivery to multiple sites within edge, this is the first report of such evident and defined expression of the CNS to target them. Although RVG-pseudotyped lentiviral vector-carried gene in cells of the olfactory bulb after intrastriatal vectors that are capable of retrograde trafficking to the CNS injection. Although most of the cells transduced in the olfactory bulb after intramuscular delivery have shown promise as treatments for

Figure 9 Comparison of distal site efficiency of wild-type RVG vs hybrid RVG/VSVG-pseudotyped HIV-1 vector-mediated gene delivery after intrastriatal injection in rats. Efficiency of gene transfer of pseudotyped vectors in the olfactory bulb. The ratio of eGFP+ relative to DAPI+ total cell number is plotted. *Po0.05, significant difference between the two pseudotypes (analysis of variance (ANOVA)). (b) Efficiency of gene transfer of pseudotyped vectors in the thalamus. The ratio of GFP+ relative to DAPI+ total cell number is plotted. (c) Efficiency of gene transfer of pseudotyped vectors in the substantia nigra pars compacta. The number of double-positive eGFP+/TH+ is plotted.

Figure 8 Characterisation of cells transduced by RVG- and RVG/VSVG(C)-pseudotyped vectors at sites distal to the site of injection. (a) Neuronal tropism of pseudotyped lentiviral vectors in the striatum. eGFP+ sections were stained using anti-eGFP antibody (Alexa488 secondary antibody, to enhance the eGFP signal) shown in green, and antibody to NeuN (Alexa594 secondary antibody, to stain neuronal cells) shown in red. Graph shows the efficiency of gene transfer of pseudotyped vectors. The ratio of eGFP+/NeuN+ relative to total NeuN+ cell number is plotted. ***Po0.001, significant difference between the two pseudotypes (analysis of variance (ANOVA)). (b) Glial tropism of pseudotyped lentiviral vectors in the striatum. eGFP+ sections were stained using anti- GFP antibody (shown in green), and antibody to GFAP (Alexa594 secondary antibody, to stain astrocytes) shown in red. Graph shows the efficiency of gene transfer of pseudotyped vectors. The ratio of eGFP+/GFAP+ relative to total GFAP+ cell number is plotted. (c) Pseudotyped vectors gene expression in nigrostriatal dopamine neurons in substantia nigra (10 magnification: SNc, pars compacta, SNr, pars reticulata). GFP+ sections were stained using anti- eGFP antibody shown in green, and antibody to tyrosine hydroxylase (TH; Alexa594 secondary antibody, to stain dopaminergic neurons) shown in red. Scale bars show 100 mm.

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neurodegenerative diseases,17 the difficulty of producing high-titre Production and purification of lentiviral vectors preparations has hindered their advancement to clinical trials. In this Recombinant non-replicative HIV-1-based lentiviral vectors were produced study we looked at whether creating a hybrid RVG protein possessing using a modified transient calcium phosphate transfection protocol (Professor various regions of VSVG that potentially play a role in their incorpora- John Olson, University of North Carolina, personal communication) based on 46 tion into lentiviral vector particles would allow the production of more the standard four plasmid transfection of 293T cells. Briefly, 150 mm tissue culture dishes were seeded with 1.4–1.6107 293T cells per dish. The next day, efficient vectors possessing higher biological activity. We found that this the cells were transfected with 15 mg vector plasmid (pRRLsincppt-CMV-eGFP- strategy did produce enhanced pseudotyping that should enable easier WPRE, pRRLsincppt-hPGK-eGFP-WPRE or pRRLsincppt-CMV-LacZ-WPRE production of higher-titre pseudotyped lentiviral vectors that exhibit expressing either GFP or LacZ from the CMV or human PGK promoter), 15 mg efficient local and dispersed neuronal transduction in the CNS. plasmid expressing the HIV-1 gag/pol gene (pMD2-LgpRRE), 3 mgplasmid expressing HIV-1 Rev (pRSV-Rev) and 5.1 mg of the plasmid expressing the MATERIALS AND METHODS appropriate envelope GP (all plasmids except pMD-RVG-CVS24(B2C), Cells obtained from Manfred Schubert through the Addgene repository (Cambridge, 293T cells (HEK 293T/17, ATCC CRL-11268) and NSC-34 cells were propa- MA, USA),11 and pRRLsincppt-CMV-LacZ-WPRE (abbreviated pLV-LacZ), gated in Dulbecco’s modified Eagle’s medium (Sigma, Gillingham, UK, D6546) acquired from IM Verma through the Addgene repository,47 were kindly supplemented with penicillin/streptomycin (Sigma, P4333), 4 mML-glutamine provided by Professor James Uney, University of Bristol, Bristol, UK). At (Sigma, G7513) and 10% (v/v) fetal bovine serum. For NSC-34 cells, the 16 h post transfection, media was replaced with fresh media supplemented with medium was further supplemented with non-essential amino acids (Sigma, 10 mM sodium butyrate. At 36 h after sodium butyrate induction, vector M7145). To differentiate them, NSC-34 cells were maintained in reduced serum containing media was harvested and filtered through a 45 mmfilter. (1% fetal calf serum) complete medium for 4–10 days. SH-SY5Y (ECACC, For small-scale ‘analytical’ preparations, used for GP incorporation studies, Salisbury, UK, 94030304) cells were maintained in 1:1 Ham’s F12 (Sigma, vectors were purified and concentrated by ultracentrifugation (Beckman, High N4888), Eagle’s minimal essential medium (Sigma, M2279) supplemented with Wycombe,UK,SW-32Ti,82000g, 2 h) through a sucrose cushion (20% 2mM glutamine, 1% non-essential amino acids and 15% fetal bovine serum. sucrose w/v, 10 mM NaCl, 20 mM Hepes (pH 7.4)) of vector containing media Cells were differentiated in complete medium supplemented with 10 mM from a single transfected 150 mm plate, as described by Kutner et al.48 Vector retinoic acid. PC12 (ECACC, 88022401) cells were grown on Collagen IV pellets were resuspended in PBS or PBS supplemented with 1% bovine serum coated tissue culture flasks in RPMI-1640 (Sigma, R8758) supplemented with albumin. Large-scale preparations were concentrated as described by Lami- 10% (v/v) horse serum, 5% (v/v) fetal bovine serum and penicillin/strepto- kanra et al.49 Briefly, vector containing media from 12 transfected 150 mm mycin. PC12 cells were differentiated in complete medium supplemented with plates was pooled and centrifuged overnight (Beckman, F500 rotor, 6000 g). 50 ng ml–1 nerve growth factor. The pellet was resuspended in PBS and repelleted by ultracentrifugation (Beckman, SW-32 Ti rotor, 68 500 g, 1.5 h) and resuspended over several hours –1 Cloning of chimeric GP-expressing plasmids in TSSM formulation buffer (20 mM Tromethamine, 100 mM NaCl, 10 mg ml Chimeric GPs were generated by PCR using a splicing by overlap extension sucrose and 10 mg ml–1 mannitol). Vector preparations were stored at 80 1C. protocol.45 DNA fragments encoding the VSVG and RVG portions of the chimeric constructs described in Table 1 were each amplified by PCR using the proofreading enzyme Pfx (Invitrogen, Paisley, UK). The VSVG_R1 primer Titration of lentiviral vectors (5¢-CGTACGTTACTTTCCAAGTCGGTTCATCTCTATG-3¢) was used to Biological activity of lentiviral vectors carrying the eGFP reporter gene was amplify the C, TC and STC portions of the VSVG gene with the determined by flow cytometry. Briefly, 293T cells were seeded in 12-well plates 5 SOE_VSVG_TM/Cyt_DN (5¢-TGACATGGTGCCGAGTTGGTATCCATCTTTG at 510 cells per well. At 16–24 h after seeding, the cells in a single well were CATTAAATT-3¢), SOE_VSVG_S/TM_DN (5¢-TGGGGAAAGTATAAAAGCTC quantified using a haemocytometer. Cells were transduced for 6 h with a serial –1 TATTGCCTCTTTTTTCTTTATCATAG-3¢) and SOE_VSVG_X/S_DN (5¢-TT dilution of the vector to be quantified in the presence of 8 mgml polybrene. GTTGAAGTTTTTTTTGGTGATACTGGGCTATCCAAA-3¢) primers, respec- At 72 h post transduction, the percentage of eGFP-positive cells was determined tively, using the pMD2-VSVG (provided by Luigi Naldini, Cell Genesys, San by flow cytometry. Biological activity of vectors carrying the LacZ reporter gene Francisco, CA, USA) plasmid as template. The primer RbG_F1 (5¢-C was done using an adapted protocol. At 48–72 h post transduction, cells were ATGGGTCGCGATGCAAACA-3¢) was used to amplify the RVG portion of fixed and stained with X-gal using a b-gal staining kit (Invitrogen). The total the C, TC and STC constructs in conjunction with the SOE_RbG_TM/Cyt_UP number of foci of transduction (consisting of 1–8 LacZ-positive cells each) per (5¢-ATACCAACTCGGCACCATGTCATTAGGGAAAATATCAAC-3¢), SOE_RbG_ well were counted manually. The values from dilutions giving between 30 and S/TM_UP (5¢-GGCAATAGAGCTTTTATACTTTCCCCAGTTCGGGAGAC-3¢) 300 foci of transduction were used to calculate the original vector titre. and SOE_RbG_X/S_UP (5¢-CCCAGTATCACCAAAAAAAACTTCAACAAAATC A similar protocol was used to determine biological activity of LacZ vectors 4 CTCAGCCTCA-3¢) primers, respectively, using the pMD-RVG-CVS24(B2C) on neuronal cells. SH-SY5Y and PC12 cells were seeded at 510 cells per plasmid as template. The corresponding fragments were spliced together by well in a 24-well plate. At 24 h after seeding, the medium was replaced with PCR utilising the overlapping complementary regions encoded within the primers differentiation medium and cells were maintained for 7 to 8 days prior to used to amplify them. The resulting chimera gene was topo cloned using the Zero transduction with vectors. At 72 h post transduction, LacZ-positive foci of Blunt TOPO PCR Cloning kit (Invitrogen). Constructs were verified by restric- transduction were quantified as described above. tion enzyme digest and sequencing, and subcloned into the pMD-RVG-CVS24 The number of physical lentiviral particles present in a preparation was (B2c) plasmid digested with NruIandBsiWI, to replace the C terminal of the estimated using a HIV-1 P24 Antigen ELISA (RETROtek from ZeptoMetrix, RVG open reading frame with that of the chimeric open reading frame. Correct Franklin, MA, USA) or by quantitative PCR using the Lenti-X qRT-PCR insertion was confirmed by restriction digest and sequence analysis. Titration Kit (Clontech, Saint-Germain-en-Laye, France).

Bioinformatics and statistics Analysis of vector GP incorporation by western blot and The amino-acid sequences for the RVG ERA (GenBank: AAA00219.1), CVS- densitometry B2c (GenBank: AAB97691.1) and CVS-N2c (GenBank: AAB97690.1) strains, Equal numbers of vector particles (as determined by P24 ELISA) were lysed by and VSVG Indiana strain (GenBank: ABD73123.1) were aligned using ClustalX boiling in 1 Laemmli buffer (6;259mM Tris pH 6.8, 10% (w/v) SDS, 40% software (ftp://ftp-19bmc.u-strasbrg.fr/pub/ClustalX/) using default settings. (v/v) glycerol, 0.6 M dithiothreitol) and run on a 12% SDS-PAGE gel. Separated Genedoc (version 2.7.000) software (http://www.nrbsc.org/gfx/genedoc/) was proteins were transferred onto nitrocellulose membrane (Hybond-ECL, GE used to display and shade the alignment. Healthcare, Chalfont St Giles, UK), blocked with PBS/Tween-20 (0.1% (v/v)) Statistics were analysed and graphed using GraphPad Prism software (http:// with 5% (w/v) milk powder) and probed with antibodies to RVG (Mouse www.graphpad.com/prism/prism.htm). monoclonal IC5 (Ab82460, Abcam) or mouse monoclonal SNB1 (kind gift

Gene Therapy Enhanced chimeric envelope pseudotyping DCJ Carpentier et al 773 from Dr Edward Wright, UCL, London, UK). Antibody-recognised proteins using 10 objective (NA 0.45) and thalamus images were taken using 4 were detected using a horseradish peroxidase-conjugated anti-mouse IgG objective (NA 0.2). Double fluorescent labelled sections (striatum and sub- secondary (Sigma) and ECL reagent (Pierce, Rockford, IL, USA). To control stantia nigra) were acquired under a confocal laser scanning microscope (TCS that equal levels of P24 protein were loaded membranes were stripped SP5 II, Leica Microsystems). For cell counts, a series of sections through the with Restore Western Stripping Buffer (Pierce) and reprobed with olfactory bulb, the forebrain and midbrain were used for immunostaining. The an anti-HIV-1 gag pr55+p24+p17 (Ab63917, Abcam) detected with a number of immunostained cells in each brain region was counted with a horseradish peroxidase-conjugated anti-rabbit IgG. Bands were quantified computer-assisted imaging program (WS Rasband, ImageJ, US National using ImageJ software on scanned images of exposed films as described on Institutes of Health, USA; http://imagej.nih.gov/ij/). For characterisation of the web link given below; http://lukemiller.org/index.php/2010/11/analyzing- striatal cells around vector injection sites, representative sections were used for gels-and-western-blots-with-image-j/. double immunofluorescence histochemistry. In individual animals, the number of immunostained cells in the fields of interest (40, NA 0.85 objective) was Analysis of transduction pattern in the rat CNS counted with the imaging program. A total of four to six sections obtained All animal procedures were approved by the local ethical committee and from each brain region of individual animals were used for cell counts, and the performed in accordance with the United Kingdom Animals Scientific Proce- average per animal was calculated. dures Act (1986) and associated guidelines. All efforts were made to minimise Statistical analysis of in vivo results the suffering and the number of animals used. The animals were housed under Analysis of variance was used for statistical comparisons. Analysis was carried a 12 h light/dark cycle (light phase, 1900 to 0700 hours) with food and water out using R: A Language and Environment for Statistical Computing (R Devel- available ad libitum. Animals were supplied by Harlan (Blackthorn, UK). opment Core Team, R Foundation for Statistical Computing, Austria; http:// Male Wistar rats (n¼12), weighing 200–250 g at the start of the experiment, www.R-project.org). Data are expressed as mean±s.e.m. values. were used in this study. Before surgery, all animals were deeply anaesthetised by inhalation of a mixture of 3 l oxygen and 3.5% isoflurane (Merial, CONFLICT OF INTEREST Parramata, NSW, Australia) and then received systemic analgesia. They were The authors declare no conflict of interest. placed in a stereotactic frame (Taxic-6000, World Precision Instruments, Hitchin, UK) with the nose bar at 3.3 mm above the ear bars. The anaesthetic ACKNOWLEDGEMENTS mixture was changed to 2 l oxygen and 2.2% isoflurane for the remainder We thank Dr Edward Wright (University College London, London, UK) of the operation. The scalp was cut and retracted to expose the skull. for the kind gift of the SNB1 anti-rabies virus GP antibody, and Professor Craniotomy was performed by drilling directly above the target region to Richard Reynolds and Dr Owain Howell for use of and help with fluorescence expose the cortex. Animals received 4.0 ml of TSSM formulation buffer, microscopy to document histological staining of brain sections. We also containing pseudotyped lentiviral vectors at a concentration of 90 ng ml–1 P24 thank Dr Egle Solito and Enrico Cristante for letting us use several antibodies. protein, via a 26G needle using an infusion pump (UltramicropumpIII and This work was funded by a Seventh Framework Programme European Micro4 Controller, World Precision Instruments) at a flow rate of 0.2 mlmin–1 Research Council Advanced Grant No. 23314 to NDM supporting DCJC, over 20 min. Needle was then allowed to remain on site for an additional 5 min AT and SME. before slow retraction. One single injection was directed into the right striatum using the stereotactic coordinates relative to bregma: anteroposterior, 0.5 mm; mediolateral, 3.0; dorsoventral, 5.0 mm. At the completion of surgery, the skin was sutured. Rats were killed by intraperitoneal injection of an overdose of sodium 1 Canivet M, Hoffman AD, Hardy D, Sernatinger J, Levy JA. Replication of HIV-1 in a pentobarbitone, transcardially perfused with 50 ml saline (0.9% w/v NaCl) plus wide variety of animal cells following phenotypic mixing with murine retroviruses. Virology 1990; 178:543–551. heparin, followed by 250 ml of 4% paraformaldehyde (Sigma Aldrich, St Louis, 2 Lusso P, di Marzo Veronese F, Ensoli B, Franchini G, Jemma C, DeRocco SE et al. MO, USA) in 1 PBS. Brains were removed and post-fixed for 4 h in 4% Expanded HIV-1 cellular tropism by phenotypic mixing with murine endogenous retro- paraformaldehyde, followed by cryoprotection in 10% glycerol and 20% viruses. Science 1990; 247: 848–852. sucrose in 1 PBS for at least 72 h. Brains were subsequently embedded and 3 Chesebro B, Wehrly K, Maury W. Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses. JVirol1990; 64: frozen in OCT (Surgipath FSC22, Leica Microsystems, Wetzlar, Germany). 4553–4557. Coronal sections (40 mm) from the olfactory bulb, the entire striatum, globus 4 Spector DH, Wade E, Wright DA, Koval V, Clark C, Jaquish D et al. Human immuno- pallidus and substantia nigra were cut using a cryostat (Leica Microsystems), deficiency virus pseudotypes with expanded cellular and species tropism. JVirol1990; 64: 2298–2308. mounted on poly-L-lysine coated slides and stored at 20 1C. 5 Zhu ZH, Chen SS, Huang AS. Phenotypic mixing between human immunodeficiency virus and vesicular stomatitis virus or herpes simplex virus. J Acquir Immune Defic Immunohistochemistry Syndr 1990; 3:215–219. Immunohistochemistry was performed on slide mounted brain sections. Tissue 6 Cronin J, Zhang XY, Reiser J. Altering the tropism of lentiviral vectors through pseudotyping. 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Sections were then 10: 2109–2121. blocked for 30 min prior to incubating for 3 h at room temperature with 9 Mitrophanous K, Yoon S, Rohll J, Patil D, Wilkes F, Kim V et al. Stable gene transfer donkey anti-mouse, anti-goat or anti-rabbit secondary antibodies conjugated to the nervous system using a non-primate lentiviral vector. Gene Therapy 1999; 6: 1808–1818. with Alexa fluor 488 or 594 (1:400, Invitrogen). Sections were coverslipped 10 Wong LF, Azzouz M, Walmsley LE, Askham Z, Wilkes FJ, Mitrophanous KA et al. with anti-fade mounting medium Mowiol in the presence of 4,6-diamidino-2- Transduction patterns of pseudotyped lentiviral vectors in the nervous system. Mol Ther phenylindole (DAPI; 0.05 mgml–1) to label nuclear DNA. 2004; 9: 101–111. 11 Mentis GZ, Gravell M, Hamilton R, Shneider NA, O¢Donovan MJ, Schubert M. Transduction of motor neurons and muscle fibers by intramuscular injection of Imaging and analysis HIV-1-based vectors pseudotyped with select rabies virus glycoproteins. J Neurosci Images from single fluorescent labelled sections (olfactory bulb and thalamus) Methods 2006; 157:208–217. 12 Kato S, Inoue K, Kobayashi K, Yasoshima Y, Miyachi S, Inoue S et al. Efficient gene were taken under an epifluorescent microscope (Eclipse 80i, Nikon, Tokyo, transfer via retrograde transport in rodent and primate brains using a human immuno- Japan) equipped with a motorised stage for automated tiling of adjacent fields deficiency virus type 1-based vector pseudotyped with rabies virus glycoprotein. Hum (Prior Scientific, Cambridge, UK). For the olfactory bulb, images were taken Gene Ther 2007; 18: 1141–1151.

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