Efficient transduction of the peripheral nervous system (PNS) is required for gene therapy of acquired and inherited neuropathies, neuromuscular diseases and for pain treatment. We have characterized the tropism and transduction efficiency of different adeno-associated vectors (AAV) pseudotypes after sciatic nerve injection in the mouse. Among the pseudotypes tested, AAV2/1 transduced both Schwann cells and neurons, AAV2/2 infected only sensory neurons and AAV2/8 preferentially transduced Schwann cells. AAV2/8 expression in the sciatic nerve was detected up to 10 weeks after administration, the latest time point analyzed. The injected mice developed neutralizing antibodies against all AAVs tested; the titers were higher against AAV2/1 than AAV2/2 and were the lowest for AAV2/8, correlating with a higher transgene expression overtime. AAV2/8 coding for ciliary neurotrophic factor (CNTF) led to an upregulation of P0 and PMP22 myelin proteins, four weeks after transduction of injured sciatic nerves. Importantly, CNTF-transduced mice showed a significant increase in both GAP43 expression in sensory neurons, a marker of axonal regeneration, and the compound muscle action potential. These results prove the utility of AAV8 as a gene therapy vector for Schwann cells to treat myelin disorders or to improve nerve regeneration.
Efficient gene transfer to the peripheral nervous system (PNS) is critical for gene therapy of inherited and acquired peripheral neuropathies, accelerating peripheral nerve regeneration or pain treatment. The PNS contains different cell types, mostly postmitotic, and their continuous communication is essential for the accurate function of the whole system. For instance, myelination of the peripheral axons involves reciprocal interactions between Schwann cells and neurons. Thus, while axonal signals regulate myelin production, myelin, synthetized by Schwann cells, regulates axonal diameter, formation of the nodes of Ranvier and nerve conduction velocity.1 In this context, overexpression of a therapeutic protein by the appropriate cell type may be crucial to maintain or enhance both, the crosstalk between different cell types and the PNS function.
Specific cell targeting can be achieved by using different viral vectors that can enter a particular cell type through its specific receptor. It can also be accomplished by using a cell-specific promoter directing the transgene expression or by engineering vectors containing cell-specific ligands. Herpes simplex virus-based vectors were shown to efficiently transduce sensory neurons when injected subcutaneously in animal models, which has lead to the initiation of a phase I clinical trial for pain treatment (for a review, see Srinivasan et al.2). Different serotypes of adeno-associated vectors (AAV) also transduce sensory neurons in the dorsal root ganglia (DRG) through direct administration into the cerebral spinal fluid or via retrograde transport.3, 4, 5, 6 Specific transduction of non-neuronal cell types in the PNS, particularly of Schwann cells, may be of great interest for the treatment of hypo- or de-myelinating diseases, diabetic neuropathy or to overexpress trophic factors for nerve regeneration. Transduction with first-generation adenovirus showed a broad but transient tropism in many tissues, including different cell types in the PNS.7 On the other hand, initial studies reported that VSV-pseudotyped lentiviral vectors transduced mainly neurons in the central nervous system (CNS).8 However, more recent works described the ability of different pseudotypes of lentiviruses to also infect glial cells.9, 10, 11 Among them, transduction of mouse and human Schwann cells has been achieved in vitro12 and in vivo in animal models of peripheral nerve trauma.13, 14
With the aim to study whether AAV vectors are capable of specifically transducing Schwann cells, we tested the biodistribution of AAV1, AAV2 and AAV8 vectors following intraneural administration. Here, we demostrate that AAV8 mostly infects Schwann cells, whereas AAV2 has a unique tropism for sensory neurons, and AAV1 transduces both sensory neurons and Schwann cells when injected into the sciatic nerve of mice. Moreover, we show that AAV8-driven expression of ciliary neurotrophic factor (CNTF) by mouse Schwann cells increases the expression of myelin protein and improves regeneration of injured sciatic nerve shortly after in vivo transduction.
Differential tropism of AAV1, AAV2 and AAV8 after intrasciatic administration
Equal amounts of AAV2/1, AAV2/2 and AAV2/8 pseudotypes (1.8 × 109 viral genomes (vg)) containing the green fluorescent protein (GFP) cDNA driven by the cytomegalovirus (CMV) promoter were injected into the sciatic nerve of mice (a minimum of 10 animals for each viral serotype). Retrograde transport of AAV vectors into sensory neurons was evaluated in lumbar L4–L6 DRG of animals euthanized 3 weeks post administration. GFP expression in sensory neurons was detected in animals injected with AAV2/1 or AAV2/2, but very rare labeled neurons were observed in AAV2/8-injected animals (Figure 1, arrows). The percentage of GFP–positive neurons in DRG sections stained with the pan-neuronal marker PGP9.5, averaged 7.45±1.5% for AAV2/1, 3.81±1.35% for AAV2 and only 0.51±0.40% for AAV8. In addition, AAV1- and AAV8-treated DRG contained GFP-expressing cells that were not labeled for PGP 9.5, and had Schwann cell-like morphology (Figure 1, arrowheads).
Longitudinal sections of the sciatic nerve from the same animals were stained for S100, a Schwann cell marker, or for PGP 9.5 (Figure 2). We found that AAV2/2 GFP expression was detected exclusively in axons, positive for PGP 9.5, along the sciatic nerve. AAV2/1-driven GFP expression was seen in axons and Schwann cells, and AAV2/8 targeted mostly Schwann cells in a range between 3 and 5 mm around the injection site. No differences in virus diffusion were detected between AAV2/1 and AAV2/8. At the injection site, up to 90% of positive Schwann cells were counted with AAV2/8, whereas only 15% for AAV2/1. The percentage of GFP-positive Schwann cells decreased with the distance from the injection site. Half-log lower dose of AAV2/8 vector was injected in the sciatic nerve of another group of animals and colocalization was again confirmed by S100 immunohistochemistry, although with lower efficiency (data not shown), suggesting that this particular tropism is not due to saturation of viral receptors.
Recently, Klein et al. described that AAV8 tropism in the CNS is modified depending on the method of purification used. Although AAV8 vectors purified by iodixanol gradient infected exclusively neurons, CsCl purification changed AAV8 tropism to glial cells.15 We performed intrasciatic injections using 8 × 106 green fluorescent units of CsCl- or iodixanol-purified AAV8–GFP vectors to compare their tropism in the PNS. Three weeks later, GFP mRNA was mostly detected in the sciatic nerve, where nuclei of Schwann cells but not neurons are located, regardless of the purification method employed. However, GFP mRNA levels in the sciatic nerve were higher with CsCl-purified than with iodixanol-purified AAV8 vector; thus, CsCl-purified vectors were used for the following experiments. On the other hand, we could hardly detect GFP mRNA in DRG from animals injected with both AAV8 vectors, indicating that at least through nerve administration, AAV8 transduces mainly Schwann cells, independent of the method of purification (Supplementary Figure S1, Supplementary Information).
Long-term expression of AAV vectors in Schwann cells
AAV2-driven expression in the sciatic nerve peaks at 3 weeks post administration and it is maintained for at least 6 months.16 To determine the stability of expression mediated by AAV1 or AAV8 vectors in Schwann cells, we administered 2.2 × 109 vg of the two serotypes coding for the LacZ gene under the expression of a CMV promoter into the sciatic nerve. Expression was quantified by galactolight assay on sciatic nerve protein extracts at 1, 3, 6 and 10 weeks after administration. As shown in Figure 3a, although data are not statistically significant due to the variability, β-galactosidase expression peaks at week 3 for AAV1 and then is markedly decreased after 6 weeks, whereas AAV8-driven expression is maintained for or at least 10 weeks.
Both viruses drove expression through the same promoter; thus, it seems unlikely that the decrease in AAV1-directed expression in the sciatic nerve would be due to CMV silencing. To test this hypothesis, vector copy number in the peripheral nerve was analyzed in another cohort of animals injected with 1.5 × 109 vg of AAV1CMVGFP or AAV8CMVGFP, and euthanized at 1, 6 and 10 weeks. Vector DNA quantification by real time PCR in sciatic nerves showed an approximately 10-fold difference in viral copy number per cell between AAV1 and AAV8 at week 1 after transduction (Figure 3b). The significant difference was maintained at 10 weeks for both serotypes, despite more than 80% decrease in the number of AAV DNA copies overtime. Efficiency of AAV8 infection in the sciatic nerve is outlined by the fact that the number of vg per cell of AAV8 at week 10 is still higher than those of AAV1 at week 1. These data suggest that the decrease in AAV1 expression may be due to a reduction in the AAV1-transduced cells, but not to promoter silencing.
Development of neutralizing antibodies following intrasciatic AAV administration
AAV vectors were shown to be significantly less immunogenic than adenoviral vectors.17 However, recent studies demonstrated a direct correlation between the virus immunogenicity and its ability to infect dendritic cells, which was superior for AAV2 than for AAV8 serotype.18 Immune response directed against adenoviral or AAV vectors is developed after intracranial injection in the CNS in a dose-dependent manner. Basically, if the viral vectors escape from the CNS to peripheral organs, a systemic adaptive anti-viral immune response mediates an almost complete elimination of the transgene expression from the brain (for a review, see Lowenstein et al.19). Blood–brain barrier confers a particular immunological privilege to the CNS; however, this may not be the case for the PNS since the blood–nerve barrier is more permeable.
Immune response activation in AAV-transduced sciatic nerves was analyzed on histological slides of sciatic nerves 1 week after transduction without evidence of T-cell infiltration (Supplementary Figure S2). At the same time point, inguinal lymph node morphology showed primary lymph follicles in AAV-injected animals, but no secondary follicles, indicative of B-lymphocyte proliferation (Supplementary Figure S3). Altogether, these data suggest that CTL response was not reducing AAV-mediated expression in Schwann cells, which is consistent with the peak expression at 3 weeks post infection for both AAV1 and AAV8 pseudotypes.
To explore the possibility of re-infection of sciatic nerve with AAV vectors, we quantified the levels of circulating neutralizing antibodies present in the blood of AAV-transduced animals. We plotted the percentage of viral infectivity inhibition after incubation with serum of transduced animals, which correlated with the level of neutralizing antibodies (Figure 4). Twenty-seven percent inhibition was observed with a 1:50 dilution of serum from AAV8-infected animals, and with a 1:200 dilution of serum from AAV2-injected mice. More importantly, using serum from AAV1-transduced animals, we did not obtain 50% infection even at the highest serum dilution. Thus, titers of neutralizing antibodies generated after intrasciatic administration were significantly higher in AAV1-transduced animals than in AAV2-injected mice, with the lowest titer corresponding to animals injected with AAV8 (Figure 4). We also observed a dose–response inhibition in animals injected with half-log lower dose of AAV8, where more than 50% expression is obtained already with the undiluted serum.
AAV8–CNTF-transduced Schwann cells promote axonal regeneration and myelin proteins overexpression in injured sciatic nerve
To test whether AAV8 could have a therapeutic potential in a mouse model of PNS regeneration, we injected 3.6 × 109 vg of AAV1 or AAV8CMVLacZ into the sciatic nerve of mice that had undergone nerve crush. β-galactosidase activity was detected in sciatic nerve protein extracts at 1, 3, 6 and 10 weeks after administration (Figure 5). Similar to what was shown for intact nerves, the decrease in AAV8-driven expression overtime is significantly lower than for AAV1, although some of the decreased overtime here may account for transduced Schwann cell proliferation during nerve regeneration.
With the aim of stimulating myelin protein expression by Schwann cells, we cloned the mouse CNTF cDNA into an AAV backbone under the regulation of a CAG promoter. Bioactivity of our CNTF construct is demonstrated in the RT4-DP6 Schwann cell line by quantitative reverse transcriptase-PCR of myelin proteins (Supplementary Figure S4). Significantly higher levels of CNTF correlated with a 2.5-fold increase in P0 and a 1.7-fold increase in PMP22 in the transfected cells.
Next, 4.5 × 109 vg of AAV8–CNTF or AAV8–GFP were administered into the crushed sciatic nerve of mice. Animals were evaluated at 17, 24 and 30 days post injury. A tendency for improved regeneration was seen at 17 and 24 days in CNTF-treated animals, with significant differences achieved at 30 days, as indicated by the higher amplitude of the compound muscle action potentials, compared with GFP-treated mice (Table 1). Increased CNTF immunoreactivity was found in Schwann cells of CNTF-transduced animals compared with GFP-injected nerves (Figure 6a). Moreover, the augmented CNTF mRNA in Schwann cells resulted in twofold higher levels of P0 and PMP22 mRNA in the sciatic nerve (Figure 6b), which agrees with the levels of myelin proteins obtained in vitro (Supplementary Figure S4). On the other hand, CNTF not only stimulated myelin protein mRNA transcription but also overexpression of GAP43 by peripheral neurons, as GAP43 mRNA levels in L4–L6 DRG were twice higher than in GFP-treated mice (Figure 6b). GAP43 is a protein overexpressed by neurons in the process of regeneration and it is located in the growth cones of regenerating axons.
Gene transfer to the CNS has been extensively studied, but transduction of the PNS by viral vectors still remains a field to be intensely explored. Among the cell types that constitute the PNS, Schwann cells, similar to their homologs in the CNS, the oligodendrocytes, are the supporting cells producing myelin. Currently, many investigations are being focused on these cell types and their role in dysfunctions of the nervous system. For instance, they participate in the development of some neurodegenerative diseases affecting myelination.20 In addition, studies in diabetic patients without evidence of neuropathy showed demyelination without fiber loss or axonal atrophy, suggesting that Schwann cells could be primarily involved in the development of diabetic neuropathy.21, 22 Moreover, in the PNS, activated Schwann cells are naturally secreting neurotrophic factors essential for nerve regeneration. In this regard, Schwann cells are the only cells producing CNTF in the PNS. Evidence of CNTF autocrine effect on Schwann cells or oligodendrocytes has been reported, although data have been controversial. Disrupted CNTF signaling delays myelination of mouse cranial motor neurons,23 however, CNTF fails to promote myelination in vivo.24 Here, we show that CNTF gene transfer to Schwann cells increases expression of myelin proteins and activates their differentiation in vitro and in vivo.
Naturally produced CNTF is non-secreted, but it is released to the extracellular space when Schwann cells are lysed after PNS injury. The CNTF receptor, α-ret, is located mainly in neurons, where CNTF has a paracrine effect promoting cell survival and axonal growth.12 Wild-type CNTF protein lacks the secretion signal sequence.25 To stimulate secretion, many groups have engineered CNTF constructs by adding signal sequences from various secreted proteins.12, 26 Non-secreted CNTF produced by Schwann cells may have different effects than secreted CNTF. Indeed, some detrimental effects have been reported following systemic CNTF injection, such as induction of cachexia in mice27 or dose-dependent deleterious effects of secreted CNTF in a mouse model of retinal degeneration.28 Thus, expression of CNTF by the appropriate cell type may be crucial to obtain the beneficial effects of this cytokine; among them, the stimulation of nerve regeneration. In contrast to previous works, we did not add any secretion signal to our construct to promote natural expression and release of CNTF by Schwann cells. Our results suggest that overexpression of CNTF by Schwann cells through an AAV8 vector may have both an autocrine effect in Schwann cells by inducing overexpression of myelin proteins, and a paracrine action in neurons, stimulating regeneration of their axons.
The sciatic nerve crush is a well-characterized model of peripheral nerve regeneration. After a lesion, nerve fibers in the distal stump degenerate. Myelin and axon debris are removed by a process called Wallerian degeneration. If the endoneurial tubes remain intact, anatomical and functional recovery is enabled. In the final steps of fiber regeneration, remyelination of the regenerated fibers needs Schwann cell differentiation and myelin protein synthesis. Impairment of peripheral nerve regeneration contributes to peripheral neuropathies in several diseases, like diabetes, alcoholism and Charcot–Marie–Tooth, among others. Moreover, speeding peripheral nerve regeneration by gene therapy may also help recovery in traumatic injuries, reducing the painful healing period.
During axonal regeneration, numerous changes in gene expression occur also in the neuronal cell bodies (reviewed in Navarro29). Among them, GAP43 expression in the nerve growth cone is needed for axonal regeneration. We found an increase in GAP43 mRNA in DRG, presumably transcribed by sensory neurons (although we cannot rule out the possibility of satellite cell transduction), but not in the spinal cord, where the somas of motoneurons are located. This may result from a technical limitation due to the dilution of GAP43 mRNA produced by motoneurons in the whole spinal cord tissue that, compared with sensory neurons in DRG, represent a much smaller proportion of cells and thus of mRNA content. Indeed, our electrophysiological data demonstrate improved motor recovery in CNTF-injected mice, which is in agreement with previous studies describing its role in stimulation of motoneuron regeneration in vitro and in vivo.30, 31
Intrasciatic administration of AAV8 provides a useful tool for specific Schwann cell transduction, compared with other viral vectors described so far. Through this route of administration, we did not observe the neuronal tropism described for this AAV serotype after intracranial injection in the CNS, through intrathecal or intramuscular administration.5, 32 Differences in the distribution of AAV8 receptor between axons and soma of sensory and motor neurons may explain this discrepancy. Another possibility would be associated to the particular characteristics of peripheral neurons; thus, any virus entering the axon in the peripheral nerve, needs to retrogradely travel up to the nuclei of the neuron, located in the DRG or the spinal cord, before its genome can be expressed. Differences in the retrograde transport capacity of the different AAV serotypes could also account for their variation in sensory or motor neuron expression.
Despite some experimental variability, expression driven by AAV8 vector is considerably stable as no significant decrease was observed between 3 and 10 weeks post administration, the latest time point analyzed. In contrast, AAV1 expression was dramatically diminished by week 10. Vector copy number per cell was lowered between weeks 1 and 10 for both viruses in the sciatic nerve, but we did not detect inflammation indicative of a T-cell response against the transduced cells (Supplementary Figure S3), although specific immunohistochemistry is needed to confirm this hypothesis. The vector copy number per cell did not exactly correlate with β-galactosidase activity in the sciatic nerve at the same time points (Figures 3a and b), which could be explained by two reasons. It is possible that a percentage of the vg copies present at week 1 were not expressing the reporter protein yet, as the peak of expression is at week 3. On the other hand, AAV1 also infects sensory neurons; thus, although a nuclear localization signal was used to drive β-galactosidase protein into the nuclei of the producing cells, when expression is strong enough, some β-galactosidase leakage could be found in the cytoplasm and axons of the producing cells. In this case, the β-gal activity detected in the sciatic nerves transduced with AAV1 may be due to both neuronal and Schwann cell expression, whereas β-gal activity obtained from AAV8 transduction may be exclusive of cells whose nuclei are located in the sciatic nerve, mainly Schwann cells.
Development of circulating antibodies against AAV vectors has been described after intravascular or intramuscular administration. We also detected neutralizing antibodies against the three serotypes tested in the serum of injected mice at 3 weeks, but the titers differed among the serotypes. The most immunogenic was AAV1, followed by AAV2, whereas AAV8-injected mice developed the lowest antibody titers against this vector. As expected, a dose response was observed for AAV8, depending on the titer of virus injected. On the other hand, we cannot rule out the possibility of immune response development against marker genes, as previously described.33
In summary, we provide evidence that intranerve administration of AAV8 is a useful tool for local and specific Schwann cell transduction, and it proves to be efficient for stimulating expression of genes involved in peripheral nerve myelination and regeneration in the injured mouse nerve.
Materials and methods
AAV vector construction, production and titration
CNTF cDNA was amplified from mouse sciatic nerve mRNA using the following primers that allow the addition of an XhoI and a BamHI restriction site at each end of the amplified fragment (CNTF-Fwd: 5′-IndexTermCTCGAGGGATCCATGGCTTTCGCAGAGCAATCAC-3′, CNTF-Rev: 5′-IndexTermCTCGAGGGATCCCTACATTTGCTTGGCCCCATAA-3′). CNTF cDNA was then cloned into XhoI and BamHI sites between the internal terminal repeats of AAV2, under the regulation of the chicken β-actin promoter and the enhancer of CMV (CAG). The woodchuck hepatitis virus responsive element was added at the 3′ end to stabilize mRNA expression.34
AAV2/1 and AAV2/8 CMV-LacZ as well as AAV2/1, AAV2/2 and AAV2/8 CMV-GFP and AAV2/8 CAG-CNTF were generated as previously described35 by triple transfection of HEK 293-AAV cells (Stratagene, Carlsbad, CA, USA) with branched polyethylenimine (Sigma, St Louis, MO, USA), with the plasmid containing the internal terminal repeats of AAV2, the AAV-helper plasmid containing Rep2 and Cap for each serotype (kindly provided by JM Wilson, University of Pennsylvania, Philadelphia, PA, USA), and the pXX6 plasmid containing helper adenoviral genes.36 Vectors were purified by CsCl or iodixianol gradients.35 Encapsidated DNA was quantified by a PicoGreen assay (Invitrogen, Carlsbad, CA, USA) following denaturation of the AAV particles (M Monfar et al., manuscript in preparation), and the titers were calculated as vg per ml. Titers for rAAVGFP vector in IU per ml were measured in QBI-HEK 293A cells (Q-Biogene, Carlsbad, CA, USA) by counting transduction events 72 h after vector exposure.
Animals and surgery
Female Institute of Cancer Research; Caesarean Derived-1 mice (8–12-weeks old) were anesthetized by intraperitoneal injection of ketamine (10 mg per kg of body weight; Imalgene 500, Rhône-Merieux, Lyon, France) and xylazine (1 mg per kg of body weight; Rompun, Bayer, Leverkusen, Germany). After sciatic nerve exposure, local anesthesia with Bupivacaine 0.5% (B. Braun, Melsungen, Germany) was applied. A volume of 3 μl of viral vectors was directly injected into the sciatic nerve through a 33-gauge needle and a Hamilton syringe connected to a Micropump (Micro4, World Precision Instruments, Sarasota, FL, USA) at a rate of 400 nl min−1. The injection site was approximately 45 mm from the tip of the third toe. The needle remained in place at the injection site for 1 additional min, before it was slowly removed. For nerve injury, the sciatic nerve was exposed at the mid-thigh and crushed three times for 30 s using fine forceps, at a distance of 42 mm from the tip of the third toe. A volume of 3 μl of viruses was loaded into the endoneurium just distal to the crush site. All mice were fed ad libitum with a standard diet (Teklad Global, Harlan Teklad, Madison, WI, USA). Animal care and experimental procedures were approved by the Biosafety and the Ethical Committees of the Universitat Autònoma de Barcelona.
Reinnervation of target organs was tested at 17, 24 and 30 days after nerve crush, by nerve conduction tests.37, 38 Under anesthesia, the sciatic nerve was stimulated at the sciatic notch, and the compound muscle action potentials were recorded from anterior tibialis and plantar muscles. The latency, indicative of conduction velocity, and the amplitude, indicative of amount of regeneration, of the compound muscle action potential were measured. Recovery of pain sensitivity was tested by light pricking with a needle in five areas, from the proximal pawpad to the tip of the second digit. A score to pinprick was assigned from no response (0), reduced or inconsistent response (1) to normal reaction (2) in each area tested, and summed to assess the extension of sensory reinnervation.37, 38
Neutralizing antibody titers against AAVs in mouse serums
Serum from 3-weeks-injected mice with each AAV pseudotype was serially diluted with serum from non-injected animals and incubated for 30 min at 37 °C with a multiplicity of infection of 1 (AAV1 2.9 × 106 vg; AAV2 2.24 × 106 vg; AAV8 1.54 × 108 vg or 3.07 × 107 vg for AAV8 1/5 low dose). Vector transgene expression was quantified after 72 h incubation with QBI-HEK 293A cells.
Anesthetized animals were perfused with phosphate-buffered saline, followed by 4% paraformaldehyde in phosphate-buffered saline. Cryo-protected sciatic nerves and DRG were embedded in Tissue-Tek Oct Compound (Miles, Elkhart, IN, USA). Sections of sciatic nerves with 10 mm thickness were incubated with primary antibody dilutions at 1:1000 for S100 (DakoCytomation, Glostrup, Denmark), 1:500 for PGP 9.5 (UltraClone Ltd, Isle of Wight, UK) and 1:100 for CNTF (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Goat anti-rabbit Alexa Fluor 568 was used as secondary antibody (1:500; Molecular Probes, Invitrogen, Carlsbad, CA, USA). Nuclear staining was obtained with TO-PRO-3 (1:100; Molecular Probes). Fluorescence was detected with a laser-scanning confocal microscope (TCs SP2; Leica Microsystems GmbH, Heidelberg, Germany).
CNTF blot analysis
Sciatic nerves were sonicated and homogenized in RIA lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodiumdeoxycholate and Complete Mini EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined by BCA Protein Assay (Pierce, Rockford, IL, USA), and 50 μg of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis gel (Bio-Rad, Hercules, CA, USA). Polyvinyldene fluoride membranes were incubated with anti-CNTF and anti-goat conjugated to horseradish peroxidase (1:2000, DakoCytomation) combined with western blotting detection reagent (ECL Plus; Amersham, Freiburg, Germany). Pixel intensity of the obtained bands was quantified by GeneSnap software for Gene Genius Bio Imaging System (Syngene, Cambridge, UK).
Galacto-Light Plus System kit (Applied Biosystems, Carlsbad, CA, USA) was used to quantitatively evaluate β-galactosidase activity. A wet weight of 70 μg of homogenized sciatic nerve was incubated for 60 min at 50 °C to inactivate endogenous β-galactosidase activity. Light units were obtained in a Victor-3 fluorescence Luminoskan RS (Labsystems, Ramat-Gan, Israel) and normalized to microgram of protein.
Reverse transcriptase-PCR assay
The RT4-D6P2 T cells or sciatic nerves were homogenized with Trizol (Invitrogen) or Qiazol (Qiagen, Hilden, Germany), respectively, to obtain total RNA. Messenger RNA was retrotranscribed to cDNA (Omniscript RT Kit, Qiagen) and analysis of expression was performed by real time PCR (Smart Cycler II; Cepheid Sunnyvale, CA, USA) with FastStart Sybrgreen Master (Roche Diagnostics). Primer sequences used: r36B4Fwd718: 5′-IndexTermATGGATACAAAAGGGTCCTGGC-3′; r36B4Rv830: 5′-IndexTermAGCCGCAAATGCAGATGGATC-3′; m36B4Fwd718: 5′-IndexTermATGGGTACAAGCGCGTCCTG-3′; m36B4rv830: 5′-IndexTermAGCCCGCAAATGCAGATGGATC-3′; mCNTF-Fwd: 5′-IndexTermGACCTGACTGCTCTTATGGAATC-3′; mCNTF-Rv: 5′-IndexTermGCCTCAGTCATCTCACTCCAG-3′; mGAP-43Fwd: 5′-IndexTermAGCCTAAACAAGCCGATGTGCC-3′; mGAP-43Rv: 5′-IndexTermTTCGTCTACAGCGTCTTTCTCCTCC-3′; GFP-Fwd: 5′-IndexTermTGCTTCAGCCGCTACCCCGAC-3′; GFP-Rv: 5′-IndexTermTGTCGCCCTCGAACTTCACCTC-3′.
PCR amplifications were performed as follows: heat inactivation (5 min, 95 °C); followed by 45 cycles of 95 °C, 15 s; 58 °C, 30 s; 72 °C, 30 s. Fluorescence detection of product was performed at the end of the PCR extension and melting curves were analyzed by monitoring the continuous decrease in fluorescence of the SYBR Green signal. PCR products were verified for a single amplification product using melting curve analysis, and the molecular weight of each product was confirmed by agarose electrophoresis. Quantification relative to 36B4 controls was calculated using the Pfaffl method.39
DNA was extracted from sciatic nerve and DRG with 0.1 mg per ml of proteinase K (Roche Diagnostics), followed by phenol/chloroform extraction. RT primers for cyclophilin B, as housekeeping gene, or GFP were as follows: mCyclophilinB-Fwd6009: 5′-IndexTermTCAACCTCTCCTCTCCTGCC-3′; mCyclophilinB-Rv6141: 5′-IndexTermGGTTTCTCCACTTCGATCTTGC-3′; CMVAAV2-Fwd: 5′-IndexTermAGCAGAGCTGGTTTAGTGAACC-3′; GFPAAV2-Rv: 5′-IndexTermTGCTCACCATGGTGGCGACC-3′. Viral genome copies per cell were calculated using a standard curve generated from known amounts of a plasmid DNA containing a CMV-GFP sequence or a 500 bp cyclophilin PCR product (CyclophilinB-Fwd5617: 5′-IndexTermCATGCCTATGGTCCTAGCTT-3′ and CyclophilinB-Rv6141) purified by Geneclean (Q-Biogene) in 10 ng per μl of salmon's sperm DNA (Sigma) and assuming that 1 μg of mouse genomic DNA contains 3 × 105 haploid genomes.
Student's t-test or two-way analysis of variance with Bonferroni post hoc tests was performed for each set of data.
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We are in debt with M Monfar and MM Segura for critically reading this manuscript. We thank the vector core of the University Hospital of Nantes and the Vector Production Unit at CBATEG (Universitat Autònoma de Barcelona) that were supported by the Association Française contre les Myopathies (AFM) for producing AAV vectors. JH, LA and GP were recipients of predoctoral fellowships (JH from the AFM: AFM2008/13622AE; LA and GP from the Generalitat de Catalunya: 2006FI00762 and 2009FI_B 00219, respectively). AB was a beneficiary of the Ramon y Cajal Program. This work was supported by the Instituto de Salud Carlos III (PI051705 and PS09730 to AB, PI081162 to MC, PI080598 to EU, and RETICS TERCEL to XN), the Generalitat de Catalunya (SGR 2009-1300) and the UAB (EME04-07).
The authors declare no conflict of interest.
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Homs, J., Ariza, L., Pagès, G. et al. Schwann cell targeting via intrasciatic injection of AAV8 as gene therapy strategy for peripheral nerve regeneration. Gene Ther 18, 622–630 (2011). https://doi.org/10.1038/gt.2011.7
- Schwann cells
- AAV8 serotype
- PNS regeneration
- AAV tropism
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