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. Jan-Dec 2017;13:1744806917727917.
doi: 10.1177/1744806917727917.

Intrathecal Administration of AYX2 DNA-decoy Produces a Long-Term Pain Treatment in Rat Models of Chronic Pain by Inhibiting the KLF6, KLF9 and KLF15 Transcription Factors

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Free PMC article

Intrathecal Administration of AYX2 DNA-decoy Produces a Long-Term Pain Treatment in Rat Models of Chronic Pain by Inhibiting the KLF6, KLF9 and KLF15 Transcription Factors

Julien Mamet et al. Mol Pain. .
Free PMC article

Abstract

Background: Nociception is maintained by genome-wide regulation of transcription in the dorsal root ganglia—spinal cord network. Hence, transcription factors constitute a promising class of targets for breakthrough pharmacological interventions to treat chronic pain. DNA decoys are oligonucleotides and specific inhibitors of transcription factor activities. A methodological series of in vivo–in vitro screening cycles was performed with decoy/transcription factor couples to identify targets capable of producing a robust and long-lasting inhibition of established chronic pain. Decoys were injected intrathecally and their efficacy was tested in the spared nerve injury and chronic constriction injury models of chronic pain in rats using repetitive von Frey testing.

Results: Results demonstrated that a one-time administration of decoys binding to the Kruppel-like transcription factors (KLFs) 6, 9, and 15 produces a significant and weeks–month long reduction in mechanical hypersensitivity compared to controls. In the spared nerve injury model, decoy efficacy was correlated to its capacity to bind KLF15 and KLF9 at a specific ratio, while in the chronic constriction injury model, efficacy was correlated to the combined binding capacity to KLF6 and KLF9. AYX2, an 18-bp DNA decoy binding KLF6, KLF9, and KLF15, was optimized for clinical development, and it demonstrated significant efficacy in these models.

Conclusions: These data highlight KLF6, KLF9, and KLF15 as transcription factors required for the maintenance of chronic pain and illustrate the potential therapeutic benefits of AYX2 for the treatment of chronic pain.

Figures

Figure 1.
Figure 1.
Illustration of DNA decoy action in chronic pain treatment. In a chronic pain state (a), TFs involved in the maintenance of neuronal sensitization bind to specific DNA sequences (TF binding site) on the genome of DRG and spinal cord cells and upregulate or downregulate (pathologic nociception regulation) hundreds of genes (sensitization-related genes). As a result, the genomic status of those cells is shifted toward a pathologic set of gene regulations that are responsible for the maintenance of neuronal sensitization and chronic pain (transcriptomes associated with long-term neuronal sensitization). DNA decoys (green) are synthetic, double-stranded oligonucleotides that mimic the binding sites of their TF target on the genome. The local intrathecal administration of specific DNA decoys (b) can prevent the binding of TF to their binding sites on the genome and interrupt the maintenance of the transcriptomes associated with long-term neuronal sensitization, resetting gene regulation toward a normal transcriptome and a normal neuronal sensitivity. TF: transcription factor.
Figure 2.
Figure 2.
Effects of the triplex/duplex in the phase 1 of the decoy screening in the SNI model. Decoys’ performance relating to the dual efficacy criteria are shown: von Frey values normed on POD 14 values before the first injection (white bars), and von Frey values normed on vehicle values at each tested day (black curves). Blue dashed lines mark the normed threshold value of 1 to assess decoys performance (black curves) against vehicle. Red dashed lines mark the normed threshold value of 0.75 (i.e., 25 % reduction) to assess decoys performance (white bars) against POD14 preinjection von Frey values. von Frey values were normalized individually for each rat, and data are presented as means for each group; corresponding raw von Frey values are presented in supplemental Table 3. The triplexes meeting the predefined criteria of potential efficacy are shown in (a) while the ones that did not are shown in (b). Each triplex contained 100 nmoles of each decoy and the duplex TLX12 contained 150 nmoles of each decoy. Dosing occurred once at day 14 (day 15 for TLX10) and day 17 (day 18 for TLX10); n = 3 for each triplex/duplex and n = 4 for vehicle. This study was performed at the Stanford University. POD: post-surgery day.
Figure 3.
Figure 3.
(a) Effects of TLX1, TLX4, TLX5, and TLX8 in the phase 2 of the decoys’ screening in the SNI model. Triplexes contained 100 nmoles of each decoy and were dosed once at post-surgery day (POD) 14 and 23. (b) Effect of TLX1 and of its individual decoys tested in parallel in the phase 3 of the screening. t test versus vehicle, *p < 0.05, n = 5–6 per group. (c) Effect of TLX5 and of its individual decoys tested in parallel in the phase 3 of the screening, t test versus vehicle, *p < 0.05, n = 3 per group. In all graphs, decoys’ performance relating to the dual efficacy criteria are shown: von Frey values normed on POD14 preinjection values are displayed as white bars, and von Frey values normed on vehicle values at each tested day are displayed as black curves. Blue dashed lines mark the normed threshold value of 1 to assess decoys performance (black curves) against vehicle, red dashed lines mark the normed threshold value of 0.75 (i.e., 25 % reduction) to assess decoys performance (white bars) against POD14 preinjection von Frey values. von Frey values were normalized individually for each rat, and data are presented as means for each group; corresponding raw von Frey values are presented in supplemental Table 3. In (b) and (c) sections, TLX1 and TLX5 contained 100 nmoles of each decoy, individual decoys were dosed at 200 nmoles and injections occurred once at day 14 and 17. Inclusion/exclusion based on predetermined criteria: two rats in (a) and three rats in (b) did not develop hypersensitivity following surgery, with POD14 von Frey values ≤5 and were excluded. The experiment in (c) was performed in two sequential cohorts of three animals per group with equal representation of testing conditions. All vehicle-treated animals of the first cohort showed a reduction of von Frey values in the 50% range or below, with an average of 59% reduction between POD14 and POD20 leading to the exclusion of that cohort (vehicle- plus decoy-treated animals). This study was performed at the Stanford University. KLF: Kruppel-like transcription factor; POD: post-surgery day.
Figure 4.
Figure 4.
(a) KLF ELISA linearity and sensitivity. Increasing amounts of Hela nuclear protein extracts were incubated with a fixed amount of KLF decoy 1 biotinylated probe (12.5 µmoles) and either 1:1000 (square) or 1:200 (black and white circles, two separate experiments) dilution of a specific KLF4 antibody. The signal produced by the 1:200 dilution plateaus after 15 µg of protein. Regressions of the linear signal portion for each antibody dilution are shown as dotted line (R2 = 0.87 and 0.91 and 1:1000 and 1:200, respectively). (b) KLF ELISA specificity control 1. The specificity of KLF binding is illustrated by the positive binding of reference decoys 1 and 2 to KLF4 and the lack of binding to KLF4 of two different mutant decoys lacking KLF binding sites. (c) KLF ELISA specificity control 2. Specificity of the assay was further demonstrated by the inhibition of KLF4 binding to the biotinylated KLF decoy 1 probe in the presence of increasing amount of free KLF decoy 1 competitor. This study was performed at Adynxx. (d) Decoys’ KLF binding patterns. Pie graph representation of the proportionate binding of each specific KLF relative to the total binding to all bound KLFs for the corresponding decoy. Binding data significantly different from the binding level of mutant decoy 1 (p ≤ 0.05), or with a trend of statistical significance (p ≤ 0.1), were used to generate the graphs (see Table 2), n ≥ 5. This study was performed at Adynxx. (e) Effect of decoys with differential KLF binding patterns in the spared nerve injury (SNI) model. Mean + SEM values of normalized total responses to repetitive von Frey hair application for animals groups treated with vehicle (white triangle), KLF reference decoy 1 (black triangle), KLF reference decoy 2 (black circle), CACCC-box decoy (black square), KLF decoy 1 (X mark) and 2 (white square) are displayed. Normalization of von Frey hair responses was performed for each rat individually against its preinjection value measured on post-surgery day 14, and the corresponding raw von Frey values are presented in supplemental Table 4. Articles were injected intrathecally once on day 14 and 17 (arrows). Testing of KLF decoys 1 and 2 and reference decoy 1, which presented the strongest activity level, was pursued until von Frey values started to resolve from a plateau level toward baseline in the vehicle group (day 48). Preinjection data before day 14 are combined across groups, 200 nmoles of each decoy or vehicle were injected once at days 14 and 17 (arrows), t test versus vehicle: *p ≤ 0.05, data distribution over the testing periods: ANOVA, all groups: p < 0.0001, Dunnett’s multiple comparison test against vehicle: p < 0.0001 for all groups except for KLF ref 2 (p = 0.53), n = 5 per group. This study was performed at AfaSci, Inc. (f). Range of effect of the KLF decoys in the SNI model. Mean + SEM values of response for each individual von Frey hair out of five stimulations from the day of the first injection until the last day of testing for each decoy plotted from the experiment displayed in (e) above, ref = reference, t test versus vehicle, *p < 0.05, ANOVA, for all groups at each hair level from 6 to 26 g: p < 0.05, Dunnett’s multiple comparison test against vehicle: ‡p < 0.05. KLF: Kruppel-like transcription factor; VF: von Frey hair.
Figure 5.
Figure 5.
(a) Effect of variants of KLF decoy 1 in the SNI model. Mean + SEM values of normalized total responses to repetitive von Frey hairs application for animal groups treated with vehicle (triangle), KLF decoy 1 (X mark), 1.6.5 (white circle), 1.6.2 (square), and 1.9 (black circle) decoys are displayed. Normalization of von Frey hair responses was performed for each rat individually against its preinjection value measured on post-surgery day 14, and the corresponding raw von Frey values are presented in supplemental Table 5. Articles were injected intrathecally once on post-surgery day 14 (arrow); Preinjection data before day 14 are combined across groups, t test versus vehicle: *p ≤ 0.05, data distribution over the testing period: ANOVA, all groups: p < 0.0001, Dunnett’s multiple comparisons test against vehicle: p < 0.05 for all comparisons except for decoy 1.9, n = 4 per group. This study was performed at AfaSci, Inc. (b) Range of effect of the variants of KLF decoy 1 in the SNI model. Mean + SEM values of response for each individual von Frey hair out of five stimulations from the day of injection until the last day of testing for each decoy plotted from the experiment displayed in (a) above, ref = reference, t test versus vehicle, *p < 0.05, ANOVA for all groups at each hair level from 6 to 26 g: p < 0.05, Dunnett’s multiple comparison test against vehicle: ‡p < 0.05. (c) Efficacy pattern of KLF decoys in the SNI and CCI pain models. Mean efficacy values for each decoy tested in the SNI and CCI models are plotted. Efficacy is calculated as the percentage of reduction of normalized von Frey hair response values compared to vehicle during the entire testing period (area under the curve) in each study and model, n = 4–6 per decoy and model. (d) Linear regression of KLF decoy's efficacy in the SNI model in relation to KLF15/KLF9 binding ratio. Efficacy is measured as described in (c) above. When a decoy efficacy was tested in independent studies, the value used for the plot is an average of the efficacy measured across those studies. Similarly, when a decoy binding was repeated throughout several phases of the sequence development process, the value used for the plot is an average of the binding measured across those phases. Each dot represents values of a given decoy, linear regression (red line), coefficient of linear regression R2 ∼ 0.7. (e) Linear regression of KLF decoy efficacy in the CCI model in relation to KLF6 + KLF9 binding. This plot was constructed using the principles described in (c). Each dot represents values of a given decoy, coefficient of linear regression R2 ∼ 0.6. Studies included in (c), (d), and (e) were performed at the Stanford University and AfaSci, Inc. CCI: chronic constriction injury; KLF: Kruppel-like transcription factor; SNI: spared nerve injury; VF: von Frey hair.
Figure 6.
Figure 6.
(a) Comparison of AYX2 (white bars) and KLF decoy 1 (black bars) binding features for KLF6, KLF9 and KLF15. Mean + SEM binding efficiency for each KLF (binding level in OD450 unit divided by the decoy size in bp) is shown in the graph; n ≥ 5. This study was performed at Adynxx, Inc. (b) Effect of ascending AYX2 doses in the SNI model. Mean + SEM values of total responses to repetitive von Frey hair application for groups treated with vehicle (white triangle) or AYX2 50 (black triangle), 100 (X mark), 200 (black circle), or 300 nmoles (white circle) are displayed. In this experiment, the 50-nmole AYX2 dose is considered without effect based on the fact that von Frey responses remain stable following injection and do not increase nor decrease. Articles were injected intrathecally once on day 14 (arrow), t test versus vehicle: *p ≤ 0.05, data distribution over the testing period: ANOVA, all groups: p < 0.0001, Dunnett’s multiple comparisons test against vehicle: p < 0.05 for all comparisons, n = 4 per group. (c) Range of effect of increasing dose levels of AYX2 in the SNI model. Mean + SEM values of response for each individual von Frey hair out of five stimulations over the testing period from the day of injection until the last day of testing plotted from the experiment displayed in (b) above, t test versus vehicle, *p < 0.05, ANOVA for all groups at each hair level from 4 to 26 g: p < 0.0005, Dunnett’s multiple comparison test: ‡p < 0.05. (d) Effect of ascending AYX2 dose levels in the CCI model. Mean + SEM values of total responses to repetitive von Frey testing for animals groups treated with vehicle (white triangle) or AYX2 200 (black circle) or 300 nmoles (white circle) are displayed. Articles were injected intrathecally once on day 14 (arrow), t test versus vehicle: *p ≤ 0.05, data distribution over the testing period: ANOVA, all groups: p < 0.0001, Dunnett’s multiple comparisons test against vehicle: p < 0.05 for all comparisons, n = 4 per group. (e) Range of effect of increasing dose levels of AYX2 in the CCI model. Mean + SEM values of response for each individual von Frey hair out of 5 stimulations over the testing period from the day of injection until the last day of testing plotted from the experiment displayed in (d) above, t test versus vehicle, *p < 0.05, ANOVA, for all groups at each hair level from 6 to 26 g: p < 0.05, Dunnett’s multiple comparison test against vehicle: ‡p < 0.05. Studies presented parts (b)–(e) were performed at AfaSci, Inc. KLF: Kruppel-like transcription factor; VF: von Frey hair.

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