Conceived and designed the experiments: LJM MR AW. Performed the experiments: BN BW JS. Analyzed the data: MR BW JS LJM AW. Contributed reagents/materials/analysis tools: LJM MR. Wrote the paper: LJM AW MR.
Current address: Vinca Institute for Nuclear Sciences, University of Belgrade, Belgrade, Serbia
The authors have declared that no competing interests exist.
Multiple sclerosis (MS) is a debilitating inflammatory disease of the central nervous system (CNS) characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that bind oligodendrocytes and promote both cell signaling and remyelination of CNS lesions in an MS model involving chronic infection of susceptible mice by Theiler’s encephalomyelitis virus and in the lysolecithin model of focal demyelination. This intriguing result raises the possibility that molecules with binding specificity for oligodendrocytes or myelin components may promote therapeutic remyelination in MS. Because of the size and complexity of IgM antibodies, it is of interest to identify smaller myelin-specific molecules with the ability to promote remyelination
MS is a debilitating neurological disease with a prevalence of about 0.1% in the Western world
While the origin of MS remains unresolved, therapy and cure present even more urgent challenges. Therapies for relapsing MS include plasma exchange to remove pathogenic immunoglobulins and/or treatment with anti-inflammatory drugs such as glatiramer acetate, β interferon, mitoxantrone, and natalizumab
A fortuitous observation led to the discovery of antibodies that promote remyelination
Aptamers are folded, single-stranded nucleic acids with activities that, like folded proteins, depend on their three-dimensional shapes and surface features
We used
A. A pool of >1012 100-nucleotide fluorescent (*) single-stranded DNA aptamers containing 60 nucleotides of random sequence is generated by a PCR and mixed with a suspension of crude mouse myelin. Cycles of affinity purification and amplification (
We analyzed the specificity of anti-myelin DNA aptamer 3064 and controls 3060 and 3202 by assessing the binding of fluorescent aptamers to crude myelin protein suspension using centrifugal sedimentation to recover bound aptamers (
A. Binding of fluorescent aptamers 3064 (filled circles), 3060 (filled squares) and 3202 (filled triangles) to crude mouse myelin proteins detected by sedimentation of the insoluble myelin fraction. Mean and standard deviation are shown for three repeats. B. Coomassie staining of crude myelin proteins (myelin; CBB) separated by SDS-polyacrylamide gel electrophoresis and results of western and southwestern blotting with the indicated antibodies or fluorescent aptamers. Mobilities of molecular weight standards are indicated at left and apparent molecular weights of aptamer-reactive proteins are indicated at right.
It is interesting to note that DNA aptamer 3064 is observed to bind preferentially to an intact myelin suspension (
The binding properties of anti-myelin DNA aptamer 3064 are similar to certain natural human IgM autoantibodies that promote remyelination in the murine TMEV model of MS
The indicated 5′-fluoresceinated, 3′-biotinylated DNA aptamers (lanes 4, 8, 12) were folded and then incubated with different amounts of streptavidin to produce multimeric aptamer complexes. Aptamer:streptavidin concentration ratios were 1∶1 (lanes 1, 5, 9), 4∶1 (lanes 2, 6, 10), and 20∶1 (lanes 3, 7, 11). Mobilities of aptamer monomer and complexes containing one to four aptamers (1–4; see Fig. 1C) are indicated. Note that complexes with two bound aptamers display two distinct mobilities due to cis vs. trans binding arrangements on the streptavidin tetramer.
To explore the biodistribution of DNA aptamer 3′-biotin conjugates, healthy FVB mice (
Control animals (“buffer injection” group in each panel) were injected with the equivalent buffer solution lacking aptamer conjugate. Conjugates were extracted from duplicate mice (middle and right group in each panel) and detected by the indicated number of PCR cycles as described in
To study aptamer conjugate effects on CNS remyelination, TMEV-infected SJL/J mice were treated by i.p. injection of various aptamer conjugates (500 µl of 1 µM conjugate solution; 0.5 nmol) twice per week for a total of 10 doses starting 27 weeks after Theiler’s virus infection. Central nervous system pathology was then assessed 5 weeks after completion of aptamer injections. Results are shown in
Scale bar in lower right panel is 100 micrometers. After blinded micrograph review of specimens from control and aptamer-treated animals the percent of spinal cord quadrants showing demyelination or remyelination was determined as reported in
aptamer | biotin | streptavidin | N | Demyelination | remyelination |
(specificity) | |||||
3064 | + | + | 10 | 41.1±6.4 | 34.9±6.1 |
(myelin) | |||||
3060 | + | + | 7 | 38.2±8.7 | 8.8±4.5 |
(Nickel) | |||||
3202 | + | + | 8 | 51.8±26.4 | 4.2±2.3 |
(dT40control) | |||||
3064 | – | + | 9 | 42.0±5.8 | 8.5±3.4 |
(myelin) | |||||
3060 | – | + | 8 | 50.0±4.6 | 10.3±4.4 |
(Nickel) |
Table indicates lesion status, by animal, after blinded review of neuropathology. N: number of mice. Data express percentage of spinal cord quadrants showing at least 75% of the lesion was remyelinated (mean +/− SEM). Statistical comparisons: P<0.002 remyelination - (ANOVA on Ranks). Dunn’s comparison to 3202 (control) showed statistical difference (p<0.05) against 3064 with biotin/streptavidin. There was no statistically significant difference in remyelination between 3202 control and 3064 without biotin.
This aptamer-induced remyelination can be compared with prior effects obtained using much larger and more labile human IgM autoantibodies. In the latter case, a single dose (0.6 nmol) IgM antibody promoted ∼60% remyelination vs. 15% observed for negative control
A number of important mechanistic issues remain to be better understood for both antibodies and DNA aptamers that appear to promote remyelination. These include the question of whether the agents reach the demyelinated regions of the CNS and function directly or indirectly, their actual target epitope(s), and their detailed dose-response profiles. Evidence of spleen uptake in healthy mice (
The molecular mass of the anti-myelin DNA aptamer tested here is ∼13,000. If fully tetramerized with streptavidin (mass ∼52,800) the resulting complex with mass ∼104,800 is still about 10-fold smaller than the IgM antibodies we have previously shown to promote remyelination. IgM antibodies are more difficult to manufacture, are more likely to be immunogenic, but have been shown to cross the blood-brain barrier. DNA aptamers can be prepared by chemical synthesis, but future studies will be required to assess the immunogenicity and tissue distribution of the aptamer conjugates shown here to promote remyelination. These considerations motivate further exploration of DNA aptamer reagents for MS treatment.
CNS tissue from strain SJL mice (5 g) is homogenized in 0.32 M sucrose containing 2 mM EGTA (pH 7.5) using a tissue grinder followed by a Dounce homogenizer to yield a final volume of 100 ml. 17 ml of homogenate is layered onto 3 ml of 0.85 M sucrose containing 2 mM EGTA and subjected to centrifugation at 28,000 rpm for 1 h at 4°C. Material is collected from the interface and homogenized in a total volume of 240 ml solution containing 2 mM EGTA. After centrifugation, the pellet is homogenized in 5 ml of a solution containing 10 mM EGTA, the volume brought to 400 ml in 10 mM EGTA, and the solution stirred for 15 min at 4°C. After centrifugation for 15 min at 10,000 rpm, homogenization and centrifugation is repeated. The resulting pellet is homogenized in 100 ml of solution containing 0.85 M sucrose and 2 mM EGTA. The homogenate is overlayed with 3 ml solution containing 0.32 M sucrose and 2 mM EGTA, subjected to centrifugation at 28,000 rpm for 90 min. After repeated homogenization and washing the myelin is isolated from the 0.32 M/0.75 M interface of a discontinuous sucrose gradient, washed with distilled water, and resuspended in 50 mM Tris-HCl containing 2 mM EGTA.
The initial round of selection employed 2.5 nmol (∼1×1015 molecules) of random oligonucleotide library LJM-2772. Oligonucleotides were heated to 90°C for 1 min in PBS containing 1 mM MgCl2, placed on ice for 15 min, and then incubated for 8 min at room temperature to allow folding. 200 µL mouse myelin suspension (10 µg) was pelleted by centrifugation for 5 min at 6500 rpm (microcentrifuge). The pellet was washed twice by resuspension in 500 µL binding buffer (20 mM Tris-HCl, pH 7.6, 10 mM NaCl, 0.5 mM KCl). The DNA library (5 µM in round 1, 300 nM in subsequent rounds) was then incubated for 30 min with gentle agitation in a 500 µL binding reaction with 10 µg myelin suspension in 500 µL binding buffer. The suspension and bound aptamers was washed twice with 1 mL binding buffer by 6500 rpm centrifugation. To the pellet was added 400 µL 2× PK buffer (300 mM NaCl, 2.5 mM EDTA, 2% SDS), followed by agitation, and extraction with 400 µL phenol:chloroform (1∶1, v:v). DNA was precipitated from the aqueous phase by addition of ethanol. A portion of the recovered DNA was amplified by PCR to establish the optimal number of amplification cycles. After the first round, PCR was performed with a fluorescein-labeled primer, allowing quantitation of library recovery by fluorescence spectroscopy. The upper primer sequence was 5′-F-ATAC2AGCT2AT2CA2T2 (F indicates fluorescein). The lower primer sequence was 5′-A20-X2-AGAT2GCACT2ACTATCT (X indicates GLEN Research spacer phosphoramidite 10-1909). PCR reactions (100 µL) employed Taq DNA polymerase, primers at 10 µM final concentration, and incubation for 5 min at 94°C, followed by cycles of 30 s at 94°C, 30 s at 47°C, and 30 s at 72°C. A second aliquot of recovered DNA was then amplified for the optimum number of cycles to prepare aptamer for the next selection round. Single-stranded fluorescent aptamer was obtained by precipitation of PCR reactions from ethanol, followed by denaturing polyacrylamide gel electrophoresis. The fluorescent DNA band was cut from the gel, diced, and eluted in TE buffer at 37°C for 2–12 h, followed by precipitation from ethanol and quantitation by UV spectrometry. After 11 selection cycles, PCR was performed and the resulting duplex DNA was ligated into the pGEM-Teasy cloning vector (Promega, Madison, WI), cloned, and sequenced.
To promote intramolecular folding, aptamer stock solutions (5 µM) in PBS were heated to 90°C and MgCl2 was then added to a final concentration of 1 mM and solutions were allowed to cool room temperature. Myelin stock was diluted in PBS and sonicated on ice. Fluorescent folded aptamers were added to different amounts of myelin suspension and incubated at 37° C for 3 h. Insoluble material with bound aptamers was recovered by 30 s centrifugation in the microcentrifuge. The pellet was washed with 100 µL PBS and again recovered by centrifugation. After phenol extraction and ethanol precipitation fluorescent aptamers were quantitated in black plastic 96-well plates using a Typhoon Fluorescent Imaging system (GE). For Southwestern blotting, 15 µg crude myelin protein was separated in each lane of a 10% Bis-Tris SDS polyacrylamide gel in MES buffer. After electrophoresis, duplicate lanes were either stained with Coomassie blue dye or transferred to PVDF membrane by electroblotting. Western blotting was performed by standard methods using antibodies with the indicated specificities. For southwestern blots, membranes were blocked for 30 min at 37° C in TBST buffer containing with 1% BSA, 10% non-fat dry milk, 2 mg/mL sonicated and heat-denatured salmon testis DNA, and Tween 20 detergent. Folded fluorescent aptamers (5 µM final concentration) were then added in PBS containing 1 mM MgCl2 and incubated with PVDF membranes for 14 h followed by washing in TBST buffer containing Tween 20 detergent and then washing with 0.5× TBE buffer. Fluorescein fluorescence was then detected on membranes using the Typhoon Fluorescent Imaging system (GE). Tryptic peptide mass fingerprinting was performed in the Mayo Clinic Proteomics Center.
All studies conformed to Mayo Clinic and National Institutes of Health animal use guidelines and were reviewed and approved by the Mayo Clinic Institutional Animal Care and Use Committee as protocol A29509. Eight-week-old female SJL/J mice (Jackson Laboratories, Bar Harbor, ME) received a single intracerebral injection of 2 × 105 plaque-forming units of the Daniel’s strain of Theiler’s Myeloencephilitus Virus (TMEV) in Dulbecco’s phosphate buffered saline (DPBS; 10 µL). The resulting encephalitic-like infection resulting in greater than 98% incidence of demyelination with increasing neurologic deficits progressing over several months
DNA oligonucleotides LJM-3064b (40 nt), LJM-3060b (43 nt) and LJM-3202b (40 nt) were synthesized DMT-off at 1 µmol scale using 3′ biotinTEG control pore glass support (Glen Research 20-2955). Oligonucleotides were cleaved from the support and deprotected in hot ammonia, then dried and purified by reverse phase HPLC and sterilized by precipitation from ethanol. Groups of mice received 500-µL intraperitoneal (i.p.) injections of the 3′ biotin conjugated aptamer (1 µM) combined with streptavidin (0.25 µM) in Calcium-free D-PBS (Invitrogen) supplemented with magnesium chloride (1 mM). Injections were twice per week for 5 weeks. Briefly, sterile aptamer solution (1 µM) in Calcium-free D-PBS supplemented with MgCl2 (1 mM) was heated to 90° C for 1 min, placed on ice for 15 min, and then incubated for 8 min at room temperature to allow aptamer folding. Streptavidin stock solution (Jackson Immune Research; 1 mg/mL; 18 µM in Calcium-free D-PBS) was added to a final concentration of 0.25 µM and incubated with gentle agitation for 30 min at 37° C immediately prior to intraperitoneal injection into mice. The final aptamer injection solution (500 µL) contained streptavidin: 13.8 µg/mL (0.25 µM), aptamer-3′-Biotin: 13.4 (12.7–14.2) µg/mL (1.2–1.5 µM) in Calcium-free D-PBS supplemented with MgCl2 (1 mM). Each treatment therefore consisted of 6.9 µg (125 pmol) streptavidin, 6.7 µg (500 pmol) aptamer-3′-biotin, and 47.6 µg (500 µmol) MgCl2.
Healthy FVB mice (38 d) or TMEV-infected SJL/J mice (6 months post infection) were used for aptamer biodistribution studies. DNA aptamer-streptavidin conjugates were extracted 4 h or 12 h after a single i.p. injection of 500 µL 1 µM biotinylated DNA aptamer 3064 prepared as low-order conjugates conjugate with streptavidin in Calcium-free D-PBS containing 1 mM MgCl2. Control animals were injected with the equivalent solution lacking aptamer conjugate. Animals were killed by sedation with isoflurane vapors followed by IP injection of 0.1 mL sodium barbital (constituting a lethal dose). Tissues (80–150 mg/organ)were harvested and homogenized in Qiagen plasmid preparation buffer P1 (300 µL), followed by addition of Qiagen plasmid preparation buffer P2 (300 µL), and then precipitation of genomic DNA, proteins and cellular debris by addition of Qiagen plasmid preparation buffer P3 (300 µL). After centrifugation, the clarified supernatant was treated with proteinase K (0.1 mg/ml final concentration) overnight at 55° and nucleic acids precipitated by addition of an equal volume of isopropanol and centrifugation. DNA aptamer 3064 was detected by PCR using primers:
LJM-4556 (5′-CTAGACTAGA2GCTGAGCTGCTAGACTAGA2GCTGAGCTG4TCG2CG3TG3) and LJM-4557 (5′- ACGT2ACGT2ATGACATGACACGT2ACGT2ATGACATGACAC3AGAGACA2GAC2AC) and Failsafe Kit (Epicentre, Madison, WI) condition J or L with the first five cycles annealed at 40° C and subsequent cycles annealed at 60° C. An aptamer-specific PCR product of 120 bp is generated from 40-nucleotide aptamer 3064.
Mice were euthanized with sodium pentobarbital and perfused intracardially with Trump’s fixative (phosphate-buffered 4% formaldehyde/1% glutaraldehyde, pH 7.4). Spinal cords were removed, cut into 1 mm blocks and every third block fixed and stained with osmium tetroxide and embedded in araldite plastic (Polysciences, Warrington, PA). One-micrometer-thick cross-sections were cut from each block, mounted onto glass slides, and stained with 4% paraphenylene-diamine to visualize myelin
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We thank L. Pease, A. Bieber N. Becker, J. Lindor, B. Bergen, and M. Doerge for excellent advise, discussion, and technical expertise.