Gene replacement therapy in a model of Charcot-Marie-Tooth 4C neuropathy

Charcot-Marie-Tooth type 4C is a demyelinating neuropathy caused by mutations of SH3TC2. Schiza et al. report that intrathecal injection of a lentiviral vector for targeted SH3TC2 delivery into Schwann cells leads to functional and morphological improvements in a disease model. This study provides proof of principle for treating demyelinating neuropathies.


Cloning of lentiviral vectors
SH3TC2 cDNA cloned in pcDNA3 under CMV promoter was used to add the myc-tag downstream of SH3TC2 cDNA by PCR amplification using forward and reverse primers as follows: pcBspE-F (5'ctgagctccggattttcaataagctgacagagctgc-3'); pcmyc-R2 (5'tggccctcgagcagaagctgatcagcgaggaggacctgtaagatatccat-3'). A PCR product of 536bp was gelextracted, purified (Gel extraction kit, Qiagen), digested with BspEI and EcoRV restriction enzymes, and the SH3TC2.myc tag was ligated into the pcDNA vector. Correct localisation of SH3TC2.myc tag was confirmed by cell transfection into Hela cells (Supplementary Fig. 1). The lentiviral vector backbone originating from pCCLsin.PPT.hPGK.GFP.pre, in which hPGK promoter was replaced by the myelin-specific rat myelin protein zero (Mpz) promoter was used to clone the SH3TC2.myc construct. The Gibson Assembly master mix (NEB) was used to ligate the two fragments together. The SH3TC2.myc sequence was PCR-amplified with 283G-F (5'gacgctctgccaagcttgataccgatatccaagcttggtaccatcgcatggaatgggtggctgcttctgc-3') and 283G-R (5'gagcagaagctgatcagcgaggaggacctgtaagatatcaagcttatcgcgatacc-3'). Correct orientation and the in-frame positioning of the Mpz promoter and SH3TC2.myc in the full vector (LV-Mpz-SH3TC2.myc) was further confirmed by direct sequencing analysis (Fig. 1A).

Vector Production
A total of 5x10 6 293T cells were seeded in 10 cm plates 24 h prior to transfection in Iscove modified Dulbecco culture medium with 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 mg/ml) in a 5% CO2 incubator. One hour prior to transfection the culture 2 medium was changed. A total of 64 µg of plasmid DNA was used for the transfection per dish: 16 µg of the envelope plasmid pMD2-VSVG, 16 µg of the packaging plasmid CMV∆R8.74 and 32 µg of the transfer vector plasmid. The precipitate was formed by adding the plasmids to a final volume of 540 µl and 60 µl of 2.5 M CaCl 2 and then adding drop wise 600 µl of 2x HEPESbuffered saline. The precipitate was added immediately to the cultures. The medium was then replaced after 6 h with fresh medium containing 1 mM Na butyrate. The conditioned medium was collected 60 h after transfection, cleared by low-speed centrifugation, and filtered through 0.22 µm-pore-size filters. The media was concentrated down from 6 ml to 250 µl volume by using the Lenti-X columns. Real time PCR and ELIZA were used to determine titer concentrations of the lentiviral vector which ranged from 5.3x10 9 to 1.5x10 11 before proceeding with gene delivery experiments.

Intraneural and intrathecal vector delivery
Intraneural vector delivery was performed by direct intraneural injection into surgically exposed mid-sciatic nerves distal to the sciatic notch, with pulled atraumatic glass pipettes filled with 10μl of the lentiviral vector using a microinjector, in order to minimize trauma to the nerves. For intrathecal injections, a small skin incision along the lower lumbar spine level was done to visualize the spine; the lentiviral vector was delivered into the L5-L6 intervertebral space of anesthetized mice. A flick of the tail was considered indicative of successful intrathecal administration. A 100-μL Hamilton syringe connected to a 30-gauge needle was used to inject 30 μL of lentiviral stock. Animals were euthanized 4 or 8 weeks post-injection, and dissected tissues were analysed by immunohistochemistry, immunoblotting, and RNA expression analysis. 3

Vector copy number determination
Genomic DNA was extracted from lumbar roots and sciatic nerves of mice 4 weeks after intrathecal vector delivery using the Qiagen DNA Mini (Thermo Fisher Scientific). The extracted DNA was analysed for yield and purity using a Nanodrop 1000 spectrophotometer.
Approximately 20 ng of DNA were used as template for two quantitative PCR assays on an

Immunoblot analysis
Fresh lumbar roots and sciatic nerves were collected at 4 weeks post-injection and lysed in icecold RIPA buffer (10 mM sodium phosphate, pH 7.0, 150 mM NaCl, 2 mM EDTA, 50 mM sodium fluoride, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS) containing a mixture of protease inhibitors (Roche). 150ng of lysed proteins were fractionated by 12% SDS/PAGE and then transferred to a Hybond-C Extra membrane (GE Healthcare Life Sciences) using a semidry transfer unit. Nonspecific sites on the membrane were blocked with 5% non-fat milk in PBS with Tween 20 (PBST) for 1 h at RT. Immunoblots were incubated with anti-myc (1:1,000; Santa Cruz) and anti-GAPDH (1: 4,000; Santa Cruz Biotechnology) antibodies at 4 °C overnight. After washing, the immunoblots were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antiserum (Jackson ImmunoResearch, diluted 1:3000) in 5% milk-PBST for 1 h. The bound antibody was visualized by an enhanced chemiluminescence system (GE Healthcare Life Sciences).

Behavioural analysis
Rotarod Test. Motor balance and coordination was determined using an accelerating rotarod apparatus (Ugo Basile). LV-Mpz.SH3TC2.myc and LV-Mpz.Egfp-treated mice were tested 8 weeks post-injection. Training of animals consisted of three trials per day with 15-min rest period between trials, for 3 consecutive days. Mice were placed on the rod, and the speed was gradually increased from 4 to 40 rotations per minute (rpm). The trial lasted until the mouse fell from the rod or after the mouse remained on the rod for 600 s and was then removed. Testing was performed on the fourth day using two different speeds, 20 and 32 rpm. The latency to fall was calculated for each speed.
Foot Grip Test. This test was performed 8 weeks post-injection. To measure grip strength, a mouse was held by the tail and lowered towards the apparatus (Ugo Basile, Italy) until it grabbed the grid first with all four paws and then with only the front paws. Mice were gently pulled back until they released the grid. Each session consisted of three consecutive trials. Measurements of the force in g were indicated on the equipment. Hindlimb force was calculated by subtracting the forelimb force from the force generated by all four limbs.
Results of all behavioral tests of LV-Mpz.SH3TC2.myc injected mice were compared to LV-Mpz.Egfp injected littermates using the Mann-Whitney U test. Significance was defined as p< 0.05 in all comparisons.

Assessment of transcriptional changes in treated Sh3tc2 -/mice
Bilateral sciatic nerves and lumbar spinal roots were dissected as above, fresh frozen and stored previously reported to be dysregulated in Sh3tc2 -/mice was analysed first in untreated Sh3tc2 -/compared to 11-week old wild type mice (Supplementary Fig. 10). Subsequently, the expression of the most significantly dysregulated genes was compared between full and mock treatment groups using quantitative PCR. All probes used for quantitative PCR were obtained from TaqMan Gene Expression Assay. Control probe for GAPDH was included. Each sample was loaded in triplicate and contained 11.25ng of cDNA, 1 μL of TaqMan Gene Expression Assay, and 10 μL of TaqMan® Gene Expression Master Mix (end volume 20 μL). Samples were analysed using the 7900HT Fast Real-Time PCR System (hold at 55˚C for 2 min and at 95˚C for 10 min, followed by 40 cycles at 95˚C for 15 s and at 60˚C for 1 min). Data was normalized to GAPDH using the 2 -ΔΔCT method.