Gangliosides for standardizing and increasing the sensitivity of cells to botulinum neurotoxins in in vitro test systems

Abstract

The present invention pertains to a method for standardizing the sensitivity of induced pluripotent stem cell (iPS)-derived neurons to a neurotoxin polypeptide, comprising the steps of: a) cultivating different batches of induced pluripotent stem cell-derived neurons in a cell culture medium comprising GT1b for at least 3 hours; b) contacting the different batches of induced pluripotent stem cell-derived neurons of step a) with a neurotoxin polypeptide; c) cultivating the different batches of induced pluripotent stem cell-derived neurons of step b) for at least 24 hours in the presence of GT1b under conditions which allow for the neurotoxin polypeptide to exert its biological activity, thereby standardizing the sensitivity of the induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide. The invention further relates to a method for the generation of induced pluripotent stem cell-derived neurons having a standardized sensitivity to a neurotoxin polypeptide, comprising the steps of: a) providing different batches of induced pluripotent stem cell-derived neurons; b) cultivating the different batches of induced pluripotent stem cell-derived neurons of step a) in a cell culture medium comprising GT1b for at least 3 hours, thereby standardizing the sensitivity of the induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide. In addition, encompassed by the present invention is a method for determining the biological activity of a neurotoxin polypeptide, comprising the steps of: a) cultivating induced pluripotent stem cell-derived neurons in a cell culture medium comprising GT1b for at least 3 hours; b) contacting the induced pluripotent stem cell-derived neurons of step a) with a neurotoxin polypeptide; c) cultivating the induced pluripotent stem cell-derived neurons of step b) for at least 24 hours in the presence of GT1b under conditions which allow for the neurotoxin polypeptide to exert its biological activity; and d) determining the biological activity of the neurotoxin polypeptide in said cells. Finally, the invention relates to the use of GT1b for a) standardizing the sensitivity of different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide; or b) reducing the variability of the sensitivity of different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide.

Claims

1. A method for standardizing the sensitivity of induced pluripotent stem cell (iPS)-derived neurons to a neurotoxin polypeptide, comprising the steps of: a) measuring the sensitivity of neurons from different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide to establish variability in sensitivity across the different batches; b) cultivating neurons from said different batches of induced pluripotent stem cell-derived neurons in a cell culture medium comprising GT1b for at least 3 hours; c) contacting the neurons of step b) with a neurotoxin polypeptide; d) cultivating neurons of step c) for at least 24 hours in the presence of GT1b under conditions which allow for the neurotoxin polypeptide to exert its biological activity; e) measuring the sensitivity of the different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide in step d) to establish a reduction in variability in sensitivity across the different batches relative to the variability in sensitivity across the different batches in step a).

2. The method of claim 1, wherein the reduction in the variability of the sensitivity of the different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide is an at least 1.5-fold or at least 2-fold reduction.

3. The method of claim 1, wherein the induced pluripotent stem cell-derived neurons are human induced pluripotent stem cell-derived neurons.

4. The method of claim 1, wherein the different batches of induced pluripotent stem cell-derived neurons differ in the number of passages, the number of freeze/thaw cycles, the cultivation conditions, the storage time, the growth time, the differentiation conditions, or combinations thereof.

5. The method of claim 1, wherein the cell culture medium comprises Neurobasal medium, B27 Supplement (2%), and Glutamin or Glutamax (1%).

6. The method of any of claim 1, wherein GT1b is added in a concentration of 1 to 300 M.

7. The method of claim 1, wherein the neurotoxin polypeptide is BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H or TeNT, or a subtype thereof.

8. The method of claim 1, wherein the sensitivity of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide is measured by quantification of the neurotoxin-cleaved substrate.

9. The method of claim 8, wherein neurotoxin-cleaved substrate is quantified by Immuno-Western blot analysis, SDS-PAGE Immunoblot analysis or ELISA.

10. A method for determining the biological activity of a neurotoxin polypeptide, comprising the steps of: a) measuring the sensitivity of neurons from different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide; b) cultivating neurons from said different batches of induced pluripotent stem cell-derived neurons in a cell culture medium comprising 1 to 300 M GT1b for at least 3 hours; c) contacting the neurons of step b) with a neurotoxin polypeptide selected from BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H or TeNT, or a subtype thereof; d) cultivating neurons of step c) for at least 24 hours in the presence of 1 to 300 M GT1b under conditions which allow for the neurotoxin polypeptide to exert its biological activity; e) measuring the sensitivity of the different batches of induced pluripotent stem cell-derived neurons to a neurotoxin polypeptide in step d) wherein the sensitivity of the neurons of step d) is increased at least 2-fold, in comparison to the sensitivity neurons from step a).

Description

(1) The Figures show:

(2) FIG. 1: SiMa cells were cultivated and intoxicated as described in Example 2 and the ratio of cleaved to uncleaved SNAP-25 was determined by Western Blot analysis. On the X-axis the concentration of the Botulinum Neurotoxin type is given, whereas on the Y-axis the relative amount of cleaved SNAP-25, i.e. the ratio of cleaved to uncleaved SNAP-25 is plotted. The circles symbolize SiMa cells cultivated without GT1b, the squares symbolize SiMa cells cultivated with 30 M GT1b. The cultivation with GT1b led to an increase in sensitivity of about 10-fold.

(3) FIG. 2: SH-SY5Y cells were cultivated and intoxicated as described in Example 2 and the ratio of cleaved to uncleaved SNAP-25 was determined by Western Blot analysis. On the X-axis the concentration of the Botulinum Neurotoxin type is given, whereas on the Y-axis the relative amount of cleaved SNAP-25, i.e. the ratio of cleaved to uncleaved SNAP-25 is plotted. The circles symbolize SH-SY5Y cells cultivated without GT1b, the squares symbolize SH-SY5 cells cultivated with 30 M GT1b. The cultivation with GT1b led to an increase in sensitivity of about 2-fold.

(4) FIG. 3: PC12 cells were cultivated and intoxicated as described in Example 2 and the ratio of cleaved to uncleaved SNAP-25 was determined by Western Blot analysis. On the X-axis the concentration of the Botulinum Neurotoxin type is given, whereas on the Y-axis the relative amount of cleaved SNAP-25, i.e. the ratio of cleaved to uncleaved SNAP-25 is plotted. The circles symbolize PC12 cells cultivated without GT1b, the squares symbolize PC12 cells cultivated with 30 M GT1b. The cultivation with GT1b led to an increase in sensitivity of about 1.4-fold.

(5) FIG. 4: Neuro2A-cells were cultivated and intoxicated as described in Example 2 and the ratio of cleaved to uncleaved SNAP-25 was determined by Western Blot analysis. On the X-axis the concentration of the Botulinum Neurotoxin type is given, whereas on the Y-axis the relative amount of cleaved SNAP-25, i.e. the ratio of cleaved to uncleaved SNAP-25 is plotted. The circles symbolize Neuro2A cells cultivated without GT1b, the squares symbolize Neuro2A cells cultivated with 30 M GT1b. At the given neurotoxin concentrations, no complete dose response curve could be observed as well as no increase in sensitivity with GT1b.

(6) FIG. 5: NG108-15-cells were cultivated and intoxicated as described in Example 2 and the ratio of cleaved to uncleaved SNAP-25 was determined by Western Blot analysis. On the X-axis the concentration of the Botulinum Neurotoxin type is given, whereas on the Y-axis the relative amount of cleaved SNAP-25, i.e. the ratio of cleaved to uncleaved SNAP-25 is plotted. The circles symbolize NG108-15-cells cultivated without GT1b, the squares symbolize NG108-15-cells cultivated with 30 M GT1b. The cultivation with GT1b led to an increase in sensitivity of about 1.6-fold.

(7) The invention will now be illustrated by the following examples which shall, however, not be construed as limiting the scope of the present invention.

EXAMPLES

Example 1

(8) iCell neurons were thawed and plated according to the Cellular Dynamics International (CDI) user manual on 96 well plates from 4 different cell batches. 24 hours (h) after plating the medium was replaced by either fresh maintenance medium as described in the user manual or by the same medium supplemented with 30 M GT1b.

(9) After further 72 h incubation time, the medium was removed and replaced by fresh medium containing BoNT/A in varying concentrations. If cells were grown on GT1b containing medium the fresh medium also contained 30 M GT1b.

(10) 72 h after start of the intoxication, the medium was aspirated and the cells were lysed by addition of 25 l SDS sample buffer.

(11) The percentage of cleaved SNAP-25 was determined by SDS-PAGE immunoblot analysis, as described in Pellett et al., 2010 (loc. cit.).

(12) The EC50 (concentration of BoNT/A yielding half maximum cleavage of SNAP-25) was calculated by plotting the percent cleaved SNAP-25 versus the BoNT/A concentration.

(13) The resulting EC50 values of the different cell batches with and without addition of GT1b are shown in Table 1.

(14) TABLE-US-00001 TABLE 1 EC50 without GT1b EC50 with GT1b Batch 02 2.84 U/ml 0.65 U/ml Batch 03 5.37 U/ml 0.89 U/ml Batch 04 6.40 U/ml 0.77 U/ml Batch 05 5.47 U/ml 0.68 U/ml Mean 5.02 U/ml 0.75 U/ml RSD 30.3% 14.6%

(15) Despite the higher sensitivity resulting from the addition of GT1b lowering the EC50 from 5.0 to 0.75 U/mL, the relative standard deviation of the EC50 values of the batches is reduced from 30% to 15%.

Example 2

(16) Cultivation and differentiation of SiMa cells (see FIG. 1): A vial containing SiMa-cells was thawed and re-suspended in culture medium (90% RPMI 1640+10% h.i. FBS+2 mM L-glutamine+/30 M GT1b) to a final density of 30,000 cells/mL. The cells were seeded on poly-D-lysine coated 96-well microtiter plates at 3,000 cells/well and incubated for 72 hours at 37 C., 95% O.sub.2/5% CO.sub.2 under a saturated water vapor atmosphere. After 72 hours, the medium was exchanged to serum-free medium (MEM+2% B27+1% N2+2% Non-essential amino acids+2 mM L-glutamine+/30 M GT1b) containing Botulinum neurotoxin type A in concentrations ranging from 1.0*10.sup.9 to 5.65*10.sup.15 M. After 72 hours of incubation as indicated above, the medium was removed, the cells were re-suspended in lysis buffer (20 mM Tris/HCl, 20 mM NaCl, 2 mM MgCl.sub.2, 0.5% Triton X-100, 5 U/mL benzonase at pH 8.0), mixed with RotiLoad 1 SDS sample buffer and subjected to Western Blot analysis to determine the ratio of cleaved SNAP-25/uncleaved SNAP-25 as described in Whitemarsh et al. (2012), Toxicol. Sci. 126, 426-35, using an antibody generated in mice (Synaptic Systems SySy111111).

(17) Cultivation and differentiation of SH-SY5Y cells (see FIG. 2): A vial containing SH-SY5Y-cells was thawed and re-suspended in culture medium (85% MEM:F12+15% h.i. FBS+/30 M GT1b) to a final density of 60,000 cells/mL. The cells were seeded on uncoated 96-well microtiter plates at 6,000 cells/well and incubated for 24 hours at 37 C., 95% O.sub.2/5% CO.sub.2 under a saturated water vapor atmosphere. The medium was then supplemented with Nerve Growth factor (100 ng/ml) and Aphidicoline (0.3 mM)+/30 M GT1b. This medium was exchanged every 2-3 days. After 17 days of incubation, the medium was exchanged to fresh medium containing Botulinum neurotoxin type A in concentrations ranging from 1.0*10.sup.9 to 5.65*10.sup.15 M. After 72 hours of incubation as indicated above, the medium was removed, the cells were re-suspended in lysis buffer (20 mM Tris/HCl, 20 mM NaCl, 2 mM MgCl.sub.2, 0.5% Triton X-100, 5 U/mL benzonase at pH 8.0), mixed with RotiLoad 1 SDS sample buffer and subjected to Western blot analysis to determine the ratio of cleaved SNAP-25/uncleaved SNAP-25 as described in Whitemarsh et al. (2012), Toxicol. Sci. 126, 426-35, using an antibody produced in mice (Synaptic Systems SySy111111).

(18) Cultivation and differentiation of PC12 cells (see FIG. 3): A vial containing PC12 cells was thawed and re-suspended in culture medium (85% RPMI 1640+10% horse serum+5% h.i. FBS+/30 M GT1b) to a final density of 25,000 cells/mL. The cells were seeded on collagen coated 96-well microtiter plates at 2,500 cells/well and incubated for 72 hours at 37 C., 95% O.sub.2/5% CO.sub.2 under a saturated water vapor atmosphere. The medium was then supplemented with Nerve Growth factor (100 ng/ml)+/30 M GT1b. This medium was exchanged every 2-3 days. After 11 days of incubation, the medium was exchanged to fresh medium containing Botulinum neurotoxin type A in concentrations ranging from 1.0*10.sup.9 to 5.65*10.sup.15 M. After 72 hours of incubation as indicated above, the medium was removed, the cells were re-suspended in lysis buffer (20 mM Tris/HCl, 20 mM NaCl, 2 mM MgCl.sub.2, 0.55% Triton X-100, 5 U/mL benzonase at pH 8.0), mixed with RotiLoad 1 SDS sample buffer and subjected to Western blot analysis to determine the ratio of cleaved SNAP-25/uncleaved SNAP-25 as described in Whitemarsh et al. (2012), Toxicol. Sci. 126, 426-35, using an antibody generated in mice (SYNAPTIC Systems SySy111111).

(19) Cultivation and differentiation of Neuro2A cells (see FIG. 4): A vial containing Neuro2A cells was thawed and re-suspended in culture medium (90% DMEM+10% h.i. FBS+/30 M GT1b) to a final density of 20,000 cells/mL. The cells were seeded on 96-well microtiter plates at 2,000 cells/well and incubated for 24 hours at 37 C., 95% O.sub.2/5% CO.sub.2 under a saturated water vapor atmosphere. The medium was exchanged by serum-free DMEM+/30 M GT1b followed by 3 days of incubation at 37 C. Then the medium was exchanged to fresh serum-free medium containing 0.2% BSA+/30 M GT1b and Botulinum neurotoxin type A in concentrations ranging from 1.0*10.sup.9 to 5.65*10.sup.15 M. After 72 hours of incubation as indicated above, the medium was removed, the cells were re-suspended in lysis buffer (20 mM Tris/HCl, 20 mM NaCl, 2 mM MgCl.sub.2, 0.5% Triton X-100, 5 U/mL benzonase at pH 8.0), mixed with RotiLoad 1 SDS sample buffer and subjected to Western blot analysis to determine the ratio of cleaved SNAP-25/uncleaved SNAP-25 as described in Whitemarsh et al. (2012), Toxicol. Sci. 126, 426-35, using an antibody produced in mice (SYNAPTIC Systems SySy111111).

(20) Cultivation and differentiation of NG108-15 cells (see FIG. 5): A vial containing SH-SY5Y cells was thawed and re-suspended in culture medium (90% DMEM+10% h.i. FBS+/30 M GT1b) to a final density of 60,000 cells/mL. The cells were seeded on 96-well microtiter plates at 6,000 cells/well and incubated for 72 hours at 37 C., 95% O.sub.2/5% CO.sub.2 under a saturated water vapor atmosphere. The medium was then supplemented with dibutyryl-cAMP (1 mM)+/30 M GT1b. This Medium was exchanged every 2-3 days. After 5 days of incubation, the medium was exchanged to fresh medium containing Botulinum neurotoxin type A in concentrations ranging from 1.0*10.sup.9 to 5.65*10.sup.15 M. After 72 hours of incubation as indicated above, the medium was removed, the cells were re-suspended in lysis buffer (20 mM Tris/HCl, 20 mM NaCl, 2 mM MgCl.sub.2, 0.5% Triton X-100, 5 U/mL benzonase at pH 8.0), mixed with RotiLoad 1 SDS sample buffer and subjected to Western blot analysis to determine the ratio of cleaved SNAP-25/uncleaved SNAP-25 as described in Whitemarsh et al. (2012), Toxicol. Sci. 126, 426-35 using an antibody generated in mice (Synaptic Systems SySy111111).