LV production and titration.
Vesicular stomatitis virus–pseudotyped (VSV-pseudotyped) LV stocks were produced by transient cotransfection of the transfer constructs pRRLsin.cPPT.hPGK.eGFP.Wpre (26
) or pRRLsin.cPPT.hPGK.ARSA.Wpre, the late-generation packaging construct pCMVΔR8.74, and the pMD2.G envelope construct in 293T cells, followed by ultracentrifugation of conditioned medium, as described (27
). Stocks were titered by Southern blot analysis, endpoint expression titer in HeLa cells, and p24 immunocapture assay, as described (28
C57BL/6 and congenic C57BL/6 Ly45.1 mice were purchased from Charles River Laboratories Inc. (Calco, Italy) and maintained in germ-free conditions. As2–/–
MLD mice were bred in the H.S. Raffaele animal research facility by intercrossing the homozygous offspring of two carrier mice obtained by rederivation (embryo transfer) of As2–/–
) with C57BL/6 females. Thus, the MLD mice used in this study have a mixed C57BL6/129 genetic background. WT C57BL6/129 hybrid mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and used as the most appropriate controls for functional studies. All procedures were performed according to protocols approved by the Animal Care and Use Committee of the Fondazione San Raffaele del Monte Tabor (IACUC 163) and communicated to the Ministry of Health and local authorities according to Italian law.
Transduction of hematopoietic progenitors and BMT.
Six-week-old male immunocompetent C57BL/6 Ly45.1 and As2–/– mice were killed with CO2, and the BM was harvested by flushing the femurs and the tibias. Hematopoietic progenitors were purified using the Enrichment of Murine Hematopoietic Progenitors kit (Stem Cell Technologies Inc., Vancouver, British Columbia, Canada). For transduction, 1 × 106 cells/ml were exposed to increasing doses of the phosphoglycerate kinase–GFP (PGK-GFP) LV (from 1 × 107 to 1 × 108 HeLa transducing units/ml [TU/ml]) and PGK-ARSA LV (3 μg viral p24/ml) in Stem Span SFEM expansion medium (Stem Cell Technologies Inc.), in the absence of serum and cytokines for 12 hours. Vector- or mock-transduced cells (106 cells/mouse) were injected via the tail vein into 6-week-old lethally irradiated (a total of 8 Gy divided into two administrations) C57BL/6 (Ly45.2) or As2–/– female mice. We performed clonogenic assays by plating 1 × 104 and 5 × 104 hematopoietic progenitors in a methylcellulose-based medium (MethoCult M3434; Stem Cell Technologies Inc.). Ten days later, colonies were scored for GFP expression by fluorescence microscopy, plucked, and lysed for PCR analysis for the detection of LV sequences.
Transduced cells were grown for at least 4 days before FACS analysis to reach steady-state GFP expression and to rule out pseudo transduction. Before FACS analysis, cells were washed and resuspended in PBS containing 30 μM propidium iodide (PI) (Becton Dickinson and Co., Franklin Lakes, New Jersey, USA) and 2% FBS. For immunostaining, 1 × 105 cells were blocked in 5% rat serum (Stem Cell Technologies Inc.), 2% FBS in PBS for 15 minutes at 4°C. After blocking, R-phycoerythrin-conjugated (RPE-conjugated) antibodies (IgG isotype control, anti–Sca-1 and anti-CD45.1, all from PharMingen, San Diego, California, USA) were added to a final concentration of 1–5 μg/ml and the cells incubated for 30 minutes at 4°C, then washed, stained with PI, and analyzed by three-color flow cytometry. Only viable, PI-negative cells were used for the analysis.
To analyze engraftment, 12 and 24 weeks after transplant blood and marrow samples were subjected to immunostaining and FACS analysis following red blood cell lysis with ammonium chloride. Cells were stained as described above, with RPE-conjugated antibodies (IgG isotype control, anti–Sca-1, anti-CD45.1, anti-CD4, anti-CD8, anti-CD11b, and anti-B220, all from PharMingen) and analyzed.
At sacrifice we performed clonogenic assays by plating 1 × 104 and 5 × 104 BM cells in a methylcellulose-based medium (MethoCult M3434; Stem Cell Technologies Inc.). Ten days later, colonies were scored for GFP expression by fluorescence microscopy, plucked, and lysed for PCR analysis for the detection of LV sequences. Genomic DNA extracted from plucked hematopoietic colonies was subjected to PCR analysis for the HIV-1 leader region of the LV construct. GAPDH amplification was used to assess DNA integrity. Primers for the LV amplification were as follows: forward, 5′-TGAAAGCGAAAGGGAAACCA-3′; reverse, 5′-CCGTGCGCGCTTCAG-3′. The PCR product length was 64 bp. Primers for GAPDH amplification were as follows: forward, 5′-CGCACTTTCTTGTGCAGTG-3′; reverse, 5′-GTTCAGCTCTCTGGGATGAC-3′. The PCR product length was 450 bp. The PCR reaction mixture consisted of 20 pmol of each primer, 0.2 mM dNTP, 4 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), and 1 U Ampli-Taq Gold DNA polymerase (Applied Biosystems, Foster City, California, USA). In the case of LV amplification, the first denaturation step was at 95°C for 10 minutes, followed by 30 cycles of 60 seconds at 94°C, 60 seconds at 55°C, 60 seconds at 72°C, with the final step at 72°C for 5 minutes. In the case of GAPDH amplification, the first denaturation step was at 95°C for 10 minutes, followed by 30 cycles of 60 seconds at 94°C, 60 seconds at 56°C, 60 seconds at 72°C, with the final step at 72°C for 5 minutes.
Southern blot analysis.
The BM and the spleen of primary and secondary recipients were harvested for genomic DNA analysis. Twenty micrograms of genomic DNA and a curve of plasmid standards were digested with AflII, which cuts twice in the vector sequence, or with BamHI, which cuts once in the vector sequence. Digested DNA was separated on a 1% agarose gel, transferred to a nylon membrane (Hybond-N; Amersham Biosciences, Piscataway, New Jersey, USA) by capillary transfer, and probed with a radiolabeled woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) probe.
Immunofluorescence and fluorescent microscope analysis.
Animals were killed under deep anesthesia and by intracardiac perfusion with 0.9% NaCl followed by 4% paraformaldehyde (PAF) in PBS, pH 7.4. Organs were fixed for 10–12 hours in PAF, equilibrated for 48 hours in PBS containing 15% sucrose, and then embedded in OCT compound for quick freezing in liquid nitrogen. Six- to ten-micron cryostatic sections were laid on gelatin-coated slides, washed in PBS, and frozen at –80°C. For immunofluorescence staining, sections were blocked with 5% goat serum (Vector Laboratories Inc., Burlingame, California, USA) in PBS containing 1% BSA and 0.1% Triton X-100 (PBS-T). Primary antibodies were diluted in PBS-T with 2% goat serum, 1% BSA as follows: IgG2a κ-isotype control clone A110-2, 1:200; F4/80 (MCAP497; Serotec Ltd., Oxford, United Kingdom), clone CI:A3-1, purified, 1:500; NeuN (MAB377; Chemicon International, Temecula, California, USA), 1:100; glial fibrillary acidic protein (GFAP) (MCA1909; Serotec Ltd.), clone DP46.109, 1:100; myelin basic protein (MBP) (MCA408; Serotec Ltd.), 1:100. For GFP staining we used an affinity-purified rabbit polyclonal antibody (A11122; Molecular Probes Inc., Eugene, Oregon, USA), 1:100. After incubation for 1 hour at room temperature, sections were washed in PBS-T and stained for 1 hour with secondary antibodies (goat α-rabbit AlexaFluor488 and goat α-rat AlexaFluor546; Molecular Probes Inc.) diluted 1:500 in PBS-T, 1% BSA. For CD45.1 staining, we used an RPE-conjugated monoclonal anti-mouse CD45.1 antibody (A20; PharMingen), 1:50. Slides were mounted with 20% Mowiol in PBS. Fluorescent and confocal microscopy were performed using a fluorescent microscope Olympus Provis AX70 and a three-laser confocal microscope (Radiance 2100; Bio-Rad Laboratories Inc., Hercules, California, USA). Fluorescent signals from single optical sections were sequentially acquired and analyzed by Adobe Photoshop 5.5 (Adobe Systems Inc., San Jose, California, USA).
Quantification of regional CNS and PNS engraftment.
Counts of GFP+, F4/80+ cells in the CNS and in the PNS were performed on 10-μm cryostatic sections following the immunofluorescence staining already described. Twenty to thirty brain (cerebrum and cerebellum) sections and 10–15 PNS (acoustic ganglion, dorsal root ganglions, and sciatic nerve) sections per mouse were analyzed from each of three mice per time point. As criteria for cell counts, only GFP+, F4/80+ cellular bodies, and not cellular processes, were counted. Counts were performed twice by two different investigators. An unpaired Student’s t test was performed for statistical evaluation of the data.
To determine ARSA activity in transduced cells and in PBMC of ARSA-transplanted mice, cell pellets were lysed in 0.5 M sodium acetate, pH 5, at 4°C for 2 hours. The ARSA activity was detected using the N
-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate (LRh-CS) as substrate (29
), adapting the method for a pyrene containing cerebroside sulfate (30
). The incubation mixtures, in final volumes of 0.25 ml, contained 25 mM sodium acetate buffer (pH 5.0), 5.5 mM sodium taurodeoxycholate, 5 mM MnCl2
, 50 μg of sample proteins dialyzed against water, and 125 pmol of LRh-CS. The samples and a blank were incubated at 37°C for 16 hours, stopped after addition of 0.5 ml of 0.1 M Na2
, and adsorbed on a reverse-phase column (Sep-Pak C18 cartridge; Waters Corp., Milford, Massachusetts, USA); after exhaustive washing with water, the fluorescent lipids were recovered by elution with 2 ml of methanol and 2 ml of chloroform/methanol, 6:4. The combined eluates were evaporated under a nitrogen stream and resolved into bands by TLC on aluminum-coated silica gel plates using chloroform/ethylacetate/n
-propanol/0.25% KCl/methanol, 25:25:25:9:16 (by volume). The spots corresponding to unreacted sulfatide, galactosyl cerebroside, ceramide, and free acid were scraped off the plate, extracted with chloroform/methanol, and their fluorescence measured on a Jasco FP-770 spectrofluorometer using an excitation wavelength of 565 nm and an emission of 575 nm. ARSA activity was also evaluated by p
-nitrocatecholsulfate (PNC) assay performed on 25 μg of sample proteins incubated with 100 μl of 10 mM PNC for 90 minutes at 37°C. The reaction was stopped with 1 ml of 1 M NaOH, and the fluorescence was measured in a spectrophotometer (515 nm). ARSA activity, expressed in nanomoles per milligram per hour, was normalized for protein content.
Mice were anesthetized with trichoroethanol, 0.02 ml/g of body weight, and placed under a heating lamp to avoid hypothermia. Using tape, we secured the mice on a smooth table to prevent movement artifacts due to the electrical stimulation, the lower limbs gently stretched to facilitate the measurement of distances between proximal and distal points of stimulation. The sciatic nerve motor conduction velocity (MCV) was obtained by stimulating the nerve with steel monopolar needle electrodes. A pair of stimulating electrodes was inserted subcutaneously near the nerve at the ankle; a second pair of electrodes was placed at the greater ischiatic notch to obtain two distinct sites of stimulation, distal and proximal, respectively, along the nerve. The muscular response to the electrical nerve stimulation, called the compound motor action potential (cMAP), was recorded with a pair of recording needle electrodes; the active electrode was inserted in muscles in the middle of the paw, while the reference was placed in the skin between the first and second digit. Motor evoked potentials (MEPs) were recorded with the same montage described for MCV from the paw muscle with transcranial electrical stimulation of the motor cortex. The excitatory volleys descending along the corticospinal pathways evoke a motor potential in the paw muscles through a trans-synaptic depolarization of alpha-motor neurons (cortical MEP, cMEP). The peripheral conduction time (PCT) was obtained with a method based on the F wave latency determination. In fact, the antidromic volley following nerve stimulation excites the alfa-motor neuron, giving rise to spikes traveling orthodromically up to the muscle, where they can be recorded as a late potential called the F wave. The PCT was thus calculated with the formula (F wavelat + cMAPlat – 1)/2. The central conduction time (CCT) was measured as the difference between cMEP and PCT latencies. An unpaired Student’s t test was performed for statistical evaluation of the data.
A motor learning task was performed with an accelerating rotarod apparatus (Ugo Basile, Comerio, Italy). This rotarod equipment is based on a rotating cylinder, 3.2 cm in diameter, covered with textured rubber. Each section is 6.0 cm wide, allowing five mice to be tested simultaneously, one per section. Mice walk forward on the rotating cylinder, at speeds increasing from 4 to 40 rpm over a 5-minute test session. Latencies in falling off the cylinder were measured over 3 days, three trials per session. Statistical analyses were made by one-way ANOVA using Scheffe’s test after significant main effect of the treatment was determined.
Mice were killed by CO2 inhalation. The tissues were removed, fixed in 2% glutaraldehyde, 1% osmium tetroxide, and embedded in Epon/araldite. Semithin (1-μm-thick) sections were stained with Toluidine Blue. Six to seven brain (cerebrum and cerebellum) and sciatic nerve sections per mouse were analyzed from each of two to four mice per condition. Metachromatic deposits larger than 20 μm were counted at ×20 magnification, demyelinated fibers were counted at ×100 magnification. Counts were performed twice by two different investigators. Microscope images were taken with a digital camera and processed by Adobe Photoshop 5.5 software (Adobe Systems Inc.).