AAV vector construction. Vector DNA plasmid pAAV.CMV.NT-3 (gift from B.K.K.) was used to generate single-stranded rAAV1.CMV.NT-3. It contains the human NT-3 CDS (GeneBank designation
NTF3) under the control of the CMV promoter cloned between AAV2 inverted terminal repeats. To generate self-complementary (sc) AAV vectors, AAV DNA plasmid vectors pscAAV.CMV.NT-3 were generated as follows: the NT-3 coding sequence was polymerase chain reaction (PCR) amplified from plasmid, the pAAV.CMV.NT-3 vector using forward (5′-accttgcggccgccaccatgtccatcttgttttatg-3′) and reverse (5′-catatgcggccgcctcatgttcttc cgatttttctcgacaaggcacaca-3′) primers. The NT-3 PCR fragment was then digested with Not I and ligated into the self-complementary pAAV.CMV.X5 (b54) vector from which the X5 cDNA was removed by Not I digestion. For generating self-complementary DNA vector plasmid pscAAV.tMCK.NT3, the NT-3 cDNA was amplified from plasmid pAAV.CMV.NT-3 by PCR using forward (5′-atgtcggtacctgcagggatatcca ccatgtccatcttgttttatgtga-3′) and reverse (5′-tcagtggcgcgccgaaaaaacctcccacacctccc-3′) primers. The resulting NT-3 cDNA PCR fragment was then digested with Kpn I and Asc I enzymes and cloned into a self-complementary pscAAV.tMCK.aSG vector plasmid from which the αSG transgene was removed by Kpn I and Asc I digestion. The final constructs were confirmed by restriction digestion and sequencing. All vectors include a consensus Kozak sequence, an SV40 intron, and synthetic polyadenylation site (53 bp). The tMCK promoter (713 bp) was a kind gift from Dr. Xiao Xiao (University of North Carolina, Chapel Hill, NC).
19 (link) It is a modification of the previously described CK6 promoter
46 (link) and includes a modification in the enhancer upstream of the promoter region containing transcription factor binding sites. The enhancer is composed of 2 E-boxes (right and left). The tMCK promoter modification includes a mutation converting the left E-box to a right E-box (2R modification) and a 6 bp insertion (S5 modification).
rAAV Vector production. AAV1 vector production was accomplished using a standard 3 plasmid DNA/CaPO
4 precipitation method using HEK293 cells. Two hundred and ninety-three cells were maintained in DMEM supplemented with 10% fetal bovine serum and penicillin and streptomycin. The production plasmids were: (i) pAAV.CMV.NT-3, pscAAV.CMV.NT-3, or pscAAV.tMCK.NT-3 (ii) rep2-cap1 modified AAV helper plasmid encoding the cap 1 serotype, and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I/II RNA genes. A quantitative PCR-based titration method was used to determine an encapsidated vg titer utilizing a Prism 7500 Taqman detector system (PE Applied Biosystems, Grand Island, NY).
47 (link) The primer and fluorescent probe targeted the tMCK and CMV promoters and were as follows: tMCK forward primer, 5′-ACCCGAGATGCCTGGTTATAATT-3′; tMCK reverse primer, 5′-TCCATGGTGTACAGAGCCTAAGAC-3′; and tMCK probe, 5′-FAM-CTGCTGCCTGAGCCTGAGCGGTTAC-TAMRA-3′; CMV forward primer, 5′-TGGAAATCCCCGTGAGTCAA-3′; CMV reverse primer, 5′-CATGGTGATGCGGTTTTGG-3′; and CMV probe, 5′-FAM-CCGCTATCCACGCCCATTGATG-TAMRA-3′.
Animals, procedures and treatment groups. Tr
J mice (B6.D2-Pmp22
Tr-J/J) and C57BL/6 wild type were obtained from Jackson Laboratory (Bar Harbor, ME). All animal experiments were performed according to the guidelines approved by The Research Institute at Nationwide Children's Hospital Animal Care and Use Committee. The design of the experimental groups comparing single-stranded and self-complementary AAV1.NT-3 vectors, treatment duration, doses, and promoters is outlined below: (i) for the nerve regeneration study, 9–12-week-old Tr
J mice were injected in the left gastrocnemius muscle with either PBS or 1 × 10
11 vg of ssAAV1.CMV.NT-3 (
n = 12). At 3 weeks postinjection, under isoflurane anesthesia, left sciatic nerves were exposed and crushed with a fine forceps at a level 5 mm distal to the sciatic notch to generate a regeneration paradigm as previously described.
18 (link) Functional recovery, measured weekly by grip strength obtained from the limb harboring the crushed nerve and the morphological assessment of nerve regeneration were the primary endpoints of this study. At 20 weeks, postcrush mice were euthanized for tissue and serum collection for NT-3 ELISA enumeration. (ii) In this set of experiments, the effect of NT-3 gene therapy on the sciatic nerve motor conduction parameters and on the motor functions (ipsilateral and simultaneous bilateral grip strength) were investigated with endpoint correlative histopathology. Six- to 8-week-old Tr
J mice received 1 × 10
11 vg of ssAAV1.CMV.NT-3 or PBS in the right quadriceps muscle (
n = 14 in each group). The left sciatic nerve conduction studies were performed at baseline age and were repeated at 20 and 40 weeks post-gene transfer. At 20 weeks, four vector-injected and five PBS-injected mice were euthanized for tissue collection for the assessment of NF cytoskeleton and NF phosphorylation studies using ultrastructural morphometry and western blot. Functional status of the remainder mice were monitored using rotarod between 23 and 40 weeks, and following endpoint electrophysiology, mice were euthanized for harvesting left sciatic nerve and distal leg muscles. (iii) The efficacy of scAAV1.NT-3 under control of the CMV promoter versus the muscle-specific tMCK promoter both given at three doses, within a half-log range (3 × 10
9 vg, 1 × 10
10 vg, and 3 × 10
10 vg) was assessed using endpoint electrophysiological and morphological studies. A total of 177 Tr
J mice in 7 cohorts (
n = 23–29 in each cohort) were generated, receiving i.m. injections of the self-complimentary vectors into the right gastric muscle at low dose, intermediate dose, or high dose with either promoters as indicated above or PBS. Technically acceptable quality nerve conduction studies were obtained from the left sciatic nerves in 171 mice. At the end of each study, mice were euthanized for tissue and serum collection for NT-3 ELISA. MF density determinations were done in high-dose cohorts (
n = 13 with CMV,
n = 26 with tMCK, and
n = 12 with PBS).
Serum NT-3 ELISA. Serum collected from PBS and AAV1.NT-3 injected mice was assayed for NT-3 levels using a capture ELISA assay. Briefly, Immunlon4 plates were coated with 100 µl of a monoclonal anti-human NT-3 capture antibody (Cat# MAB267, 4 µg/ml, R&D Systems, Minneapolis, MN) in BupH carbonate buffer for 6 hours at 25 °C. Plates were subsequently blocked with PBS + 1% BSA + 5% sucrose overnight at 2–8 °C. The next day, plates were washed four times with PBS + 0.05% Tween20 (PBS-T) and a NT-3 standard (recombinant human NT-3, Cat# 267-N3, R&D Systems) was prepared using serial twofold dilutions in the range of 10–1,280 pg/ml in 20 mmol/l Tris, 150 mmol/l NaCl, 0.1% BSA, 0.05% Tween-20 and applied to the plate (100 µl volume). Animal sera were diluted 1:20 and 1:50 using the same dilution buffer used for the NT-3 standard and 100 µl added to the plate. Standards and serum samples were incubated at room temperature (25 °C) with gentle shaking for 2 hours ± 10 minutes. Following four PBS-T washes, 100 µl of a diluted goat anti-NT3-biotin detection antibody was added to each well and incubated 90 minutes ± 10 minutes. at RT (0.2 µg/ml of polyclonal goat anti-NT3-biotin detection antibody; Cat# BAF267; R&D Systems). Following PBS-T washes, 100 µl of a 1:1,000 dilution (PBS diluent) of extra-avidin-HRP developer solution was added to the wells and incubated for 60 minutes ± 10 minutes at RT (extra-avidin-HRP; Cat# E2896; Sigma, St Louis, MO). After washing, plates were developed by adding 100 µL of RT TMB substrate solution in the dark for 15 minutes ± 1 minutes (1-step ultra TMB-ELISA; Cat# 34028; Thermo, Waltham, MA). The reaction was stopped by adding 50 µl of 2N H
2SO
4, and the optical density at 450 nm determined for each well on a Bio-tek Synergy 2 ELISA plate reader running the Gen5 2.0 Data Analysis Software package (Bio-tek US, Winooski, VT). NT-3 serum concentrations were extrapolated from the NT-3 standard curve using a best fit algorithm.
Motor function testing. Tr
J mice were tested for baseline motor function within 1 week prior to receiving i.m. injection of ssAAV1.CMV.NT-3 or PBS. Motor function tests included bilateral simultaneous hindlimb grip power and that of the left hind paw using a grip strength meter (Chatillon Digital Meter; Model DFIS-2; Columbus Instruments, Columbus, OH) as we have used in our previous studies.
21 (link) Bilateral or unilateral grip strength was assessed by allowing the animals to grasp a platform followed by pulling the animal until it releases the platform; the force measurements were recorded in four separate trials. Measurements were performed on the same day and time of each week. Endpoint bilateral and ipsilateral grip strength measurements were done in two sessions (morning and afternoon), three trials in each per day for 3 consecutive days prior to obtaining the nerve conduction studies. The mean of these measurements were used to correlate with conduction studies.
Rotarod testing. Mouse motor function and balance was tested weekly by using the accelerating rotarod (Columbus Instruments). Mice were trained on the rotarod apparatus for 2 weeks to acclimate to testing protocol prior to data collection. A fixed rotation protocol at 5 rpm constant rotation was used, and the average of the three trials per session was recorded.
Nerve conduction studies. The nerve conduction studies were performed under isofluorane anesthesia. Temperatures were recorded with an infrared thermometer (Fisher Scientific, Pittsburgh, PA), and body temperature was maintained between 32 and 36 °C using a heating pad. Following body temperature equilibration, left sciatic nerve conduction studies were obtained using a XLTEK NeuroMaX 1002 electromyograph (Ontario, Canada) and Rhythmlink disposable subdermal needle recording electrodes (for both stimulation and recording) as we described previously.
21 (link) The stimulating electrodes were placed at the proximal and distal stimulation sites (i.e., the left sciatic notch and just above the ankle, respectively), and a third pair of recording electrodes was positioned in the foot pad between the second and third digits of the left foot. The latency, duration, negative area under curve, and conduction velocity values of the recorded sciatic motor responses were determined. A caliper was utilized to measure the interelectrode distances, and these distances were used in calculations of intersegmental velocity. In addition, onset latency, duration, and amplitude were also calculated.
Processing of sciatic nerve for histopathological analysis. For the nerve regeneration study, mice were killed quickly by an overdosage of xylazine/ketamine anesthesia at 20 weeks postcrush. The sciatic nerves from crushed and intact sites were removed under a dissecting microscope, fixed in glutaraldehyde; tissue blocks were marked for proximodistal orientation and processed for plastic embedding for light and electron microscopy using standard methods established in our laboratory.
48 (link) In all other experiments, left sciatic nerves were removed and processed in the same manner.
MF density determinations. Quantitative analysis at the light microscopic level was performed on 1 µm thick cross sections from regenerating and intact uncrushed sciatic nerves using a microscope-mounted video camera at ×1,600 magnification and an image analysis software (Bioquant TCW98 image analysis software; R&M Biometrics, Nashville, TN) as previously described.
29 (link) Data assessing regeneration response were obtained from the second segment, at a level ~4 mm distal to the crush. The mid sciatic nerve segments were analyzed from uncrushed intact nerves in all cases. Four randomly selected areas were analyzed in each mice. MF densities (mean number ± SE/mm
2) and composites of MF axon size distribution histograms were generated in rAAV1.NT-3 and PBS-injected groups.
g ratio of the MF. The
g ratio refers to the ratio of axonal diameter/fiber diameter, and lower
g ratios represent axons with thicker myelin.
49 (link) For
g ratio determinations, three representative areas of cross sectional images of mid sciatic nerves from three ssAAV1.CMV. NT-3- and PBS-injected Tr
J mice and wild type were captured at ×100 magnification, and the shortest axial lengths as axon diameters and fiber diameters were recorded with a calibrated micrometer, using the AxioVision, 4.2 software (Zeiss) as we described previously.
21 (link) The
g ratios and axon diameters are displayed in a scattergram.
SC density. One micrometer thick, plastic embedded cross-sections were used for MF and SC nuclei counts. Three randomly selected areas in five AAV1.CMV.NT-3- and PBS-injected Tr
J mice were photographed at ×100, and the number of MF and SC nuclei not in contact with the MFs was determined. Morphologic criteria used for identification of SC nuclei included homogenous, rounded, ovoid, or bean-shaped appearance with irregular contour. Nuclei with irregular contour and dense peripheral zones belonging to fibroblasts were excluded. The SC densities were estimated as number per mm
2 of the endoneurial area, by adding the number of SC nuclei belonging to unmyelinated fibers or at a promyelination stage with 1:1 axon-SC relationship to the number of MFs as we reported previously.
14 (link) SC nuclei belonging to the MFs were excluded.
NF packing density determinations. Ultrastructural morphometric studies were performed using cross sectional images of sciatic nerves at ×52,000 final magnification. NF density histograms were generated by determining the number of NFs per unit hexagonal area in randomly selected myelinated axons from treated and untreated Tr
J mice and wild-type mice as previously described.
18 (link) Ten randomly selected MFs with axon diameters between 3.6 and 5.0 µm at 20 weeks posttreatment were analyzed in each group.
Histological analysis of muscle. Gastrocnemius and tibialis anterior muscles from ssAAV1.CMV.NT-3 and PBS-injected Tr
J mice (
n = 3 in each group) were removed and 12 µm thick cross cryostat sections were stained for succinic dehydrogenase for generation of muscle fiber size distribution histograms as previously described.
21 (link) Over 2,000 fibers were analyzed in each group.
NF cytoskeleton and phosphorylation. Sciatic and spinal nerves and roots from ssAAV1.CMV.NT-3 and PBS-injected Tr
J mice were used for quantitative western blot analysis of NF proteins with NF-H-specific antibodies. Briefly, the tissues were harvested and immediately frozen over dry ice. Tissues were homogenized in radio immunoprecipitation assay buffer (50 mmol/l Tris-HCl pH 8.0, 1% NP-40, 150 mmol/l NaCl, 0.5%sodium deoxycholate, 1% sodium dodecyl sulfate, 1 mmol/l ethylene glycol tetraacetic acid, 1 mmol/l Na3VO4, 1 mmol/l NaF, phenylmethylsulfonyl fluoride (1:250), Complete protease inhibitor (1:25), and 25.5 mmol/l sodium pyrophosphate) using blue tip and Kontes pestle. Protein concentrations were determined using RC/DC method (BioRad Laboratories, Hercules, CA). For sodium dodecyl sulfate polyacrylamide gel electrophoresis, 5 µg of protein was run on 3–8% Tris-acetate NuPage gels (Invitrogen, Grand Island, NY) and transferred to PVDF membrane (Amersham Biosciences, Pittsburgh, PA). After blocking for 1 hour in 5% nonfat dry milk in TBST (100 mmol/l Tris-HCl, pH 8.0, 167 mmol/l NaCl, 0.1% Tween), the western blots were incubated with diluted primary antibodies against total NF-H (AB1989, COOH-terminal antibody from Chemicon; diluted 1:500), hyperphosphorylated NF-H (SMI-31 from Sternberger; diluted 1:20,000) and hypophosphorylated NF-H (SMI-35 from Sternberger; diluted 1:10,000). Blots were washed and incubated in appropriate horseradish peroxidase–conjugated secondary antibodies at a dilution of 1:2,000. GAPDH was used as loading control (Millipore, Billerica, MA; diluted 1:500). Immunoreactive bands were visualized with the use of ECL Plus Western blotting detection system (GE Healthcare, Pittsburgh, PA) and Hyperfilm ECL (Amersham Biosciences). Signal intensities were measured with ImageQuant software (GE Healthcare).
Statistical analysis. For comparisons between ssAAV1.CMV.NT-3 gene transfer and PBS-treated Tr
J groups, statistical analysis were performed in Graph pad Prism 4 software, using one-way analysis of variance followed by Bonferroni multiple
post hoc comparisons. Unpaired or paired Student's
t-test was performed when applicable. Differences between the means were considered significant at two-tailed test. Significance level was set at
P < 0.05. Summary statistics were reported as mean ± SEM.
For the studies comparing the efficacy of scAAV1.NT-3 under control of the CMV promoter versus the muscle-specific tMCK promoter both given at three doses, the following analyses were used: (i) Spearman correlation to study the relationship between outcomes, (ii) Kruskal–Wallis test to compare outcomes among all groups (PBS, CMV low dose/intermediate dose/high dose and tMCK low dose/intermediate dose/high dose), and (iii) Mann–Whitney
U-test to compare outcomes between each group and PBS (control) group, and Bonferroni correction to adjust for multiple comparisons. Two-way analysis of variance is used to study the effects of gene vectors and doses on outcomes. All tests are conducted by SAS 9.2 (by SAS Institute, Cary, NC).
SUPPLEMENTARY MATERIALFigure S1. Serum levels of NT-3 in Tr
J mice at 23 weeks postinjection (shown as individual mice) compared to PBS-treated Tr
J controls (numbers 567, 570, 573, and 591) are shown in individual mice.
Figure S2. One micrometer thick, toluidine blue-stained representative cross sections of intact/uncrushed (
a,
b) and regenerating (
c,
d) sciatic nerves from Tr
J mice injected with PBS (
a,
c) and AAV1.NT-3 (
b,
d) at 20 weeks. Thinly myelinated and naked axons are indicated with arrows in PBS-treated intact and regenerating nerves (
a,
c). AAV1.NT-3 gene therapy results in an increase of axons with thicker myelin (arrows) in intact nerves (
b) and an apparent increase in the small myelinated fibers (arrows) in regenerating nerves (
d).
Figure S3. Composite histograms showing myelinated fiber distribution in the regenerating (
a) and contralateral intact (
b) sciatic nerves from Tr
J mice at 20 weeks post AAV1.NT-3 gene therapy showing an increase in the subpopulation of axons <4 µm in diameter in AAV1NT3 group compared to PBS-control.
Figure S4. Neurogenic changes in the gastrocnemius muscle from a PBS-treated Tr
J (
a) showing atrophic angular fibers of either histochemical fiber types (arrows) or fiber type atrophy (asterisk). Reinnervation induced changes (asterisks mark fiber type groupings) at 40 weeks post AAV1.NT-3 gene therapy (
b).
Figure S5. Muscle fiber size histograms from tibialis anterior (
a) and gastrocnemius (
b) muscles at 40 weeks post AAV1.NT-3 gene therapy. Both muscles showed an increase in fiber diameter (
c) as histologic evidence of nerve regeneration into the muscle compared to PBS-injected control group.
Figure S6. Representative tracings of sciatic motor nerve conduction from a wild-type and Tr
J mouse at baseline and endpoint at 40 weeks postvector injection.
Table S1. Sciatic nerve electrophysiology in Tr
J mice following AAV1. NT-3 gene transfer at 24 weeks.
Sahenk Z., Galloway G., Clark K.R., Malik V., Rodino-Klapac L.R., Kaspar B.K., Chen L., Braganza C., Montgomery C, & Mendell J.R. (2013). AAV1.NT-3 Gene Therapy for Charcot–Marie–Tooth Neuropathy. Molecular Therapy, 22(3), 511-521.