i-Fect in vivo!
USPTO Patent Application 20080031911-Smad Signaling and Neuro-regeneration
United States Patent 20070105806
Pub from The Journal of Neuroscience
June 13, 2007, 27(24):6363-6373
Inventors: Fan Wang, Zhigang He
USPTO Application #: 20080031911
Inhibition of Smad2/3 Signaling Promotes Axonal Regeneration after Spinal Injury in Rats-Gene Expression Knockdown in vivo!
One hour after the spinal cord is lesioned, the rats in the SB-505124 group receive a bolus injection of SB-505124 (30 mg/kg) in 0.9% saline administered via a tail vein. The treatment is repeated every 24 hours on days 1 through 7 post-lesion. Vehicle only control rats undergo the same treatment but are injected with an equal volume of 0.09% saline in a tail vein. At the same treatment time points as the SB-505124 group, the Smad2/3 siRNA group and corresponding controls receive 10 .mu.l rat Smad2/3 siRNA (Dharmacon, Lafayette, Colo.), mismatch siRNA, or transfection reagent only delivered to the spinal cord via the catheters. The siRNA (or mismatch siRNA control) complexes are prepared immediately prior to administration by mixing the RNA solution (200 .mu.M in annealing buffer) with a transfection reagent, i-Fect ™ . (Neuromics, Edina, Minn.), in a ratio of 1:4 (w:v). At this ratio, the final concentration of RNA as an RNA/lipid complex is 2 .mu.g in 10 .mu.l.
"The effect of siRNAs against Nav1.8, formulated with iFECT, on complete Freund's adjuvant-induced tactile hypersensitivity was evaluated in rats (FIG. 5). Adult male Sprague-Dawley rats received an injection of CFA (150 uL) into the hindpaw on day 0. siRNAs against Nav1.8 were then administered by intrathecal bolus to the lumbar region of the spinal cord on days 1, 2 and 3; specifically, for each bolus injection, 2 ug of siRNA was complexed with iFECT transfection reagent (Neuromics, Minneapolis Minn., USA) at a ratio of 1:4 (w:v) in a total volume of 10 uL. Five groups of rats (with 5 rats per group) were treated with either siRNA (AL-DP-6049, AL-DP-6209, AL-DP-6217 or AL-DP-6218; Table 1), or PBS, in the presence of iFECT. Tactile hypersensitivity was expressed as tactile withdrawal thresholds which were measured by probing the hindpaw with 8 calibrated von Frey filaments (Stoelting, Wood Dale Ill., USA) (0.41 g to 15 g). Each filament was applied to the plantar surface of the paw. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength and calculated with a Dixon non-parametric test (see Dixon, W. J. (1980) "Efficient analysis of experimental observations" Annu Rev Pharmacol Toxicol 20:441-462; Chaplan, S. R., F. W. Bach, et al.(1994) "Quantitative assessment of tactile allodynia in the rat paw" J Neurosci Methods 53:55-63). Tactile thresholds were measured before CFA injection to assess baseline thresholds, and then on day 4 after CFA and treatment with test articles. In rats treated with PBS, tactile hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal threshold, as expected. In rats treated with AL-DP-6209, tactile thresholds were nearly normalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-6209, is efficacious in vivo against inflammation-induced hyperalgesia. Treatment with the Nav1.8 siRNA, AL-DP-6217, resulted in the average tactile threshold trending towards baseline, with one of five rats demonstrating a normal tactile response. AL-DP-6049 and AL-DP-6218 did not significantly alter tactile thresholds compared to PBS treatment, in this experimental paradigm."
tsA-201 cells plated at high density in 10 cm dishes were transfected with CaV2.2e[37a] or CaV2.2e[37b] (+ ß1b + 2–1 + eGFP, in which eGFP is enhanced green fluorescent protein) as described in our previous work (Beedle et al., 2004). On the next day, dishes were transfected with 6 µg of siRNA using i-Fect (Neuromics, Minneapolis, MN). Cells were split 24 h after siRNA transfection and subjected to whole-cell patch-clamp analysis on the next day. Electrophysiological recordings were conducted with 20 mM barium as the charge carrier as described in detail in our previous work (Beedle et al.,2004; Altier et al., 2006).
In vivo injection of siRNA
Rats were anesthetized with halothane (5%) and received three percutaneous intrathecal injections (L5–L6) of 6-FAM–siRNA (60 µg/rat in 10 µl final volume of DEPC water) over 2 d. To localize siRNA in DRG tissues, rats were anesthetized with sodium pentobarbital (200 mg/kg, i.p.) and transcardially perfused with 4% paraformaldehyde in 100 mM PBS, pH 7.4. DRG (T10–L6) were removed and fixed overnight at 4°C, placed in 25% sucrose in PBS for 24 h at 4°C, embedded in OCT compound (Miles, Elkhart, IN), and sectioned at 25 µm. Confocal images were obtained from fixed DRG slices mounted for visualization. Images were acquired using a Zeiss LSM-510 META confocal microscope using a 20x objective in the inverted configuration. For all confocal images, a regular phase transmission image was obtained. 6-FAM–siRNA was observed using argon laser excitation (488 nm) and emission (505–530 nm) filter sets.
The Journal of Neuroscience, June 13, 2007, 27(24):6363-6373; doi:10.1523/JNEUROSCI.0307-07.2007