Journal: Human molecular genetics

Article Title: Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models.

doi: 10.1093/hmg/ddq099

Figure Lengend Snippet: Figure 1. Expression and secretion of functional LV-encoded enzymes in vitro. (A) Schematic of the monocistronic (LV.GFP, LV.GALC, LV.ARSA) and bidir- ectional (bd) LV (bdLV.CTRL, bdLV.GALC or bdLV.ARSA) used to transduce NSC in vitro and for intracerebral injections. FACS (B) and immunofluores- cence analysis (C) using anti-GFP, anti-NGFR and anti-HA antibodies showed robust bdLV-mediated transduction in Twi NSC and simultaneous expression of the two transgenes in bdLV.GALC-transduced NSC (C). (D) Endogenous (437 bp) and vector-encoded GALC mRNA (550 bp) visualized by RT–PCR (see Materials and Methods) in WT and Twi-derived NSC (lane 1 and lane 2: UT and bdLV.GALC-transduced Twi NSC; lane 3: WT NSC; M: marker). (E) Merged confocal picture showing co-localization of HA (red) and LAMP1 (green) in LV-transduced Twi NSC. Nuclei counterstained with DAPI. Scale bars, 50 mm. (F and F′′) The anti-galactocerebroside antibody (GalCer, red) specifically identifies oligodendrocytes but also detects intracellular storage in

Article Snippet: The PDVF membranes were stained with chicken polyclonal anti-GALC antibody (1:2000; expected MW: 50 and 80 kDa); mouse monoclonal anti-GFP antibody (1:1000; A11121, Invitrogen; expected MW: 30 kDa) and goat polyclonal IgG antiactin antibody (1:10 000; I-19, Santa Cruz Biotechnology; expected MW: 46 kDa), revealed by HRP-conjugated goat antimouse (1 20 000; AP124P, Chemicon), donkey anti-chicken (1:5000; SC-2953, Santa Cruz), rabbit anti-goat (1:40 000; AP132P, Chemicon) and incubated with chemiluminescent substrate Amersham ECL PlusTM kit (GE Healthcare).

Techniques: Expressing, Functional Assay, In Vitro, Transduction, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Marker

Journal: Human molecular genetics

Article Title: Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models.

doi: 10.1093/hmg/ddq099

Figure Lengend Snippet: Figure 2. Robust transduction, transgene expression and cross-correction at the injection site. (A) Low-magnification (×0.75) bright-field picture of a cresyl- violet counterstained coronal brain section showing the injection site (EC, arrow). (A′) Distribution of GFP+ cells at the site of injection 3 weeks after bdLV injection. Individual confocal pictures (×10 magnification, 64 pictures for each channel) were composed using the Photomerge tool of Adobe Photoshop CS3. Scale bars: 1.5 mm (A), 0.75 mm (A′). (B) 3D reconstruction of the forebrain (gray), lateral ventricles (light bue), white matter (light yellow) and GFP+- transduced tissue (green) of the brain of a representative bdLV.GALC-treated mouse at 20 days post-injection. Reconstruction was obtained from sequential sections analyzed for stereology using the Neurolucida software, which returned the enclosed volumes for each region: forebrain, 114.33 mm3; lateral ventricles, 0.455 mm3; white matter, 4.65 mm3; GFP+ tissue, 0.99 mm3. (C and D) Higher magnification confocal pictures taken at the injection site of bdLV.CTRL (C) and bdLV.GALC-injected (D) Twi mice showed GFP+ cells with variable morphology. Green, GFP; nuclei counterstained with DAPI (blue). (E–G) Representative confocal pictures after Z-stack analysis showing GFP+ cells (green) expressing markers of astrocytes (E; GFAP), neurons (F; NeuN) and oligodendrocytes (G; GST-p). Arrows and yellow-orange color indicate co-localization of signal. Scale bars: 30 mm (C–G, shown in C). (H) 100% stacked column chart showing percentages of neurons (NeuN), astrocytes (GFAP) and oligodendrocytes (GST-p) within the GFP+ cell population in bdLV-injected Twi and WT mice. (I) Representative confocal picture after Z-stack analysis shows GFP (green) and HA (red) co-expression (arrows and yellow-orange color). Note the presence of surrounding HA+GFP2 cells. (J and J′) Co-localization of HA (red) and Lamp1 (violet) in GFP+ cells (green) indicates lysosomal localization of the bdLV-encoded GALC. Nuclei counterstained with DAPI (blue in J′). Scale bars: 30 mm (shown in J). (K and L) Representative confocal pictures showing GFP2-

Article Snippet: The PDVF membranes were stained with chicken polyclonal anti-GALC antibody (1:2000; expected MW: 50 and 80 kDa); mouse monoclonal anti-GFP antibody (1:1000; A11121, Invitrogen; expected MW: 30 kDa) and goat polyclonal IgG antiactin antibody (1:10 000; I-19, Santa Cruz Biotechnology; expected MW: 46 kDa), revealed by HRP-conjugated goat antimouse (1 20 000; AP124P, Chemicon), donkey anti-chicken (1:5000; SC-2953, Santa Cruz), rabbit anti-goat (1:40 000; AP132P, Chemicon) and incubated with chemiluminescent substrate Amersham ECL PlusTM kit (GE Healthcare).

Techniques: Transduction, Expressing, Injection, Software

Journal: Human molecular genetics

Article Title: Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models.

doi: 10.1093/hmg/ddq099

Figure Lengend Snippet: Figure 4. Widespread distribution of GFP and GALC proteins in CNS tissues of bdLV-injected mice. (A) Schematic of brain and spinal cord dissection in bdLV.CTRL- and bdLV.GALC-injected Twi mice and from age-matched untreated (UT) WT and Twi controls. The arrow indicates the injection site (see also Results). Contro, controlateral non-injected hemisphere. (B) WB analysis using an anti-HA antibody after immunomagnetic enrichment of the HA-tagged GALC from total tissue lysates revealed a band of about 80 kDa in aTel, pTel and SC tissues from bdLV.GALC-treated mice (#1, #2 and #3 represent tissues from different mice) that was absent from bdLV.CTRL-treated WT or Twi controls. (C and D) WB using the anti-GALC antibody revealed bands of 80 and 50 kDa in tissues from all the CNS regions of bdLV.GALC-treated Twi mice (C). These bands were undetectable in tissues derived from bdLV.CTRL-treated Twi mice (D). The GFP protein was detected in tissues from WT and Twi mice injected with bdLV.CTRL (C and D). b-Actin was used as normalizer. (E) Densi- tometry of WB bands (see Materials and Methods) confirmed the complete rescue of GALC expression in all the CNS regions of bdLV.GALC-injected mice.

Article Snippet: The PDVF membranes were stained with chicken polyclonal anti-GALC antibody (1:2000; expected MW: 50 and 80 kDa); mouse monoclonal anti-GFP antibody (1:1000; A11121, Invitrogen; expected MW: 30 kDa) and goat polyclonal IgG antiactin antibody (1:10 000; I-19, Santa Cruz Biotechnology; expected MW: 46 kDa), revealed by HRP-conjugated goat antimouse (1 20 000; AP124P, Chemicon), donkey anti-chicken (1:5000; SC-2953, Santa Cruz), rabbit anti-goat (1:40 000; AP132P, Chemicon) and incubated with chemiluminescent substrate Amersham ECL PlusTM kit (GE Healthcare).

Techniques: Injection, Dissection, Derivative Assay, Expressing

Journal: Human molecular genetics

Article Title: Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models.

doi: 10.1093/hmg/ddq099

Figure Lengend Snippet: Figure 5. Enzymatic reconstitution in CNS tissues of bdLV.GALC-treated mice. (A) Increased GALC activity was measured in bdLV.GALC-treated WT mice (gray columns) with respect to bdLV.CTRL-treated mice (white columns) in almost all CNS regions. In bdLV.GALC-treated Twi mice (red columns), GALC activity was rescued to 50% of the WT levels in brain regions comprising the injection site and rostral to it (aTel, OB). Complete enzymatic correction was achieved in brain regions caudal to the injection site (pTel, Pons, Cb). Similar levels of enzymatic activity were measured in the controlateral (contro) non- injected hemisphere (red striped columns). GALC activity was undetectable in bdLV.CTRL-treated Twi mice (black columns). ∗P , 0.05, ∗∗P , 0.01,∗∗∗P , 0.001. Two-way ANOVA followed by Bonferroni post-test (since no significant differences were found between GALC levels in the injected and controlateral hemispheres for each CNS region, these values were averaged in this analysis). (B) Gel-filtration on Sephadex S300-chromatography analysis on representative CNS tissues (aTel controlateral and SC) from untreated WT, bdLV.CTRL- and bdLV.GALC-treated Twi mice. The peak of activity (600– 700 kDa) corresponding to GALC enzyme, as demonstrated by measuring GALC activity, was absent in bdLV.CTRL Twi tissues but was restored to 60– 75% (measured as the area under the peak) of the WT levels in CNS tissues from bdLV.GALC-treated Twi mice.

Article Snippet: The PDVF membranes were stained with chicken polyclonal anti-GALC antibody (1:2000; expected MW: 50 and 80 kDa); mouse monoclonal anti-GFP antibody (1:1000; A11121, Invitrogen; expected MW: 30 kDa) and goat polyclonal IgG antiactin antibody (1:10 000; I-19, Santa Cruz Biotechnology; expected MW: 46 kDa), revealed by HRP-conjugated goat antimouse (1 20 000; AP124P, Chemicon), donkey anti-chicken (1:5000; SC-2953, Santa Cruz), rabbit anti-goat (1:40 000; AP132P, Chemicon) and incubated with chemiluminescent substrate Amersham ECL PlusTM kit (GE Healthcare).

Techniques: Activity Assay, Injection, Chromatography

Journal: Human molecular genetics

Article Title: Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models.

doi: 10.1093/hmg/ddq099

Figure Lengend Snippet: Figure 7. Mechanisms of GALC distribution in CNS tissues. (A) bdLV.GALC-treated mice showed levels of GALC activity similar to those measured in WT CSF. GALC activity was undetectable in the CSF of Twi mock-treated mice. (B) bdLV genome, assessed by qPCR in bdLV.GALC- and bdLV.ARSA-injected mice, was detected only in tissues comprising the injected region (left aTel). (C) RT–PCR using: (i) primers in the GFP coding region and (ii) two pairs of primers designed on a sequence of the bdLV cassette (expected size 550 bp) and on a sequence of the endogenous GALC (expected size 470 bp) revealed bdLV-encoded GFP and GALC mRNA only in the left aTel. b-Actin was used as normalizer.

Article Snippet: The PDVF membranes were stained with chicken polyclonal anti-GALC antibody (1:2000; expected MW: 50 and 80 kDa); mouse monoclonal anti-GFP antibody (1:1000; A11121, Invitrogen; expected MW: 30 kDa) and goat polyclonal IgG antiactin antibody (1:10 000; I-19, Santa Cruz Biotechnology; expected MW: 46 kDa), revealed by HRP-conjugated goat antimouse (1 20 000; AP124P, Chemicon), donkey anti-chicken (1:5000; SC-2953, Santa Cruz), rabbit anti-goat (1:40 000; AP132P, Chemicon) and incubated with chemiluminescent substrate Amersham ECL PlusTM kit (GE Healthcare).

Techniques: Activity Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Human molecular genetics

Article Title: Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models.

doi: 10.1093/hmg/ddq099

Figure Lengend Snippet: Figure 8. Detection of disease lesions and pathology. Histochemistry using biotinylated lectins (RCA-1; glycolipid storage and globoid cells) and indirect immu- nofluorescence analysis using anti-F4/80, anti-Iba1 (microphages and microglia) and anti-GFAP (astrocytes) antibodies were performed on selected region- matched rostro-caudal tissue slices (A) from bdLV.CTRL- and bdLV.GALC-treated and on untreated WT and Twi mice. Pictures of defined areas in each slice (indicated by blue boxes for RCA-1 and red boxes for the other antigens) were taken, and the total immunopositive area in each picture (expressed in pixels) was calculated using the ImageJ software. Tissue slices in which the primary antibodies were omitted (for Iba1 and GFAP) or tissue slices from untreated WT mice (for F4/80 and RCA-1) were used to set the signal threshold. CC, corpus callosum; Hip, hippocampus; Cb, cerebellum; SC, spinal cord. (B and C) Quantification of lectin- (B) and F4/80 (C) immunopositive area indicated a dramatic increase of tissue storage (globoid cells) and activated macrophages/micro- glia in bdLV.CTRL-treated Twi mice when compared with WT littermates that were significantly reduced in all the CNS regions of bdLV.GALC treated mice. (D and E) Representative pictures showing lectin+ (D; cerebellum) and F4/80+ (E; corpus callosum) cells in WT mice, bdLV.GALC- and bdLV.CTRL-treated Twi mice. (F and G) A moderate reduction of immunoreactivity for GFAP (astroglia; F) and Iba-1 (microglia; G) was found in bdLV-GALC-treated Twi mice with respect to mock-treated littermates, together with a partial normalization of cell morphology towards the WT phenotype (representative pictures of the corpus callosum are shown). Scale bars: 2 mm (A, CC and Cb/Pons), 2.5 mm (A, Hip), 1.5 mm (A, SC); 250 mm (D, E, F and G). ∗P , 0.05, ∗∗P , 0.01, ∗∗∗P , 0.001. Two-way ANOVA followed by Bonferroni post-test.

Article Snippet: The PDVF membranes were stained with chicken polyclonal anti-GALC antibody (1:2000; expected MW: 50 and 80 kDa); mouse monoclonal anti-GFP antibody (1:1000; A11121, Invitrogen; expected MW: 30 kDa) and goat polyclonal IgG antiactin antibody (1:10 000; I-19, Santa Cruz Biotechnology; expected MW: 46 kDa), revealed by HRP-conjugated goat antimouse (1 20 000; AP124P, Chemicon), donkey anti-chicken (1:5000; SC-2953, Santa Cruz), rabbit anti-goat (1:40 000; AP132P, Chemicon) and incubated with chemiluminescent substrate Amersham ECL PlusTM kit (GE Healthcare).

Techniques: Software