NERVE GROWTH FACTOR MUTANT

Abstract

Provided is a nerve growth factor mutant, wherein the nerve growth factor mutant is an amino acid sequence as shown by any one of SEQ ID No: 3 to SEQ ID No: 21 in the sequence listing. The advantage of the nerve growth factor mutant lies in that the mutation of a nerve growth factor can alleviate side effects such as pain, falling within the field of biological pharmacy.

Claims

1. (canceled)

2. A nucleic acid comprising a nucleotide sequence encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor.

3. A nucleic acid encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor.

4. The nucleic acid according to claim 2, wherein the wild-type human nerve growth factor comprises an amino acid sequence of SEQ ID No: 2.

5. The nucleic acid according to claim 3, wherein the wild-type human nerve growth factor comprises an amino acid sequence of SEQ ID No: 2.

6. The nucleic acid according to claim 2, wherein the nucleic acid has a nucleotide sequence of SEQ ID No: 22.

7. The nucleic acid according to claim 2, wherein the nucleic acid has a nucleotide sequence which is from nucleotide position 364 to 726 of SEQ ID NO: 22.

8. An expression vector, comprising the nucleic acid according to claim 2.

9. The expression vector according to claim 8, wherein the expression vector is selected from the group consisting of a DNA vector and a virus vector.

10. The expression vector according to claim 9, wherein the DNA vector is selected from the group consisting of a DNA plasmid vector, a liposome bound thereto, a molecular conjugate bound thereto, and a polymer bound thereto.

11. The expression vector according to claim 10, wherein the DNA plasmid vector is a eukaryotic expression vector.

12. The expression vector according to claim 9, wherein the virus vector is selected from the group consisting of an adeno-associated virus vector, a lentivirus vector and an adenovirus vector.

13. A method for expressing the expression vector according to claim 8, comprising transfecting the expression vector into a host cell, and culturing the resulting recombinant cell to express the expression vector.

14. A host cell, comprising the expression vector according to claim 8.

15. The host cell according to claim 14, wherein the host cell is a mammalian cell.

16. The host cell according to claim 15, wherein the mammalian cell is a Chinese hamster ovary cell, a human embryonic kidney 293 cell, a COS cell or a Hela cell.

17. A pharmaceutical composition, comprising a nucleic acid comprising a nucleotide sequence encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor; a nucleic acid encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor; the expression vector of claim 8 or a host cell comprising the expression vector; and a pharmaceutically acceptable excipient.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is formulated for injection.

19. A method of treating or inhibiting a nervous system disease comprising: administering a nucleic acid comprising a nucleotide sequence encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor; a nucleic acid encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor; the expression vector of claim 7 or a host cell comprising the expression vector to a subject in need thereof.

20. A method of reducing the weight of a subject comprising: administering a nucleic acid comprising a nucleotide sequence encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor; a nucleic acid encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor; the expression vector of claim 8 or a host cell comprising the expression vector to a subject in need thereof.

21. A method of providing a long-acting nerve growth factor to a subject comprising: administering a nucleic acid comprising a nucleotide sequence encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor; a nucleic acid encoding a nerve growth factor mutant, wherein the nerve growth factor mutant comprises Phe12Glu with reference to the amino acid positions set forth in a wild-type human nerve growth factor; the expression vector of claim 8 or a host cell comprising the expression vector to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1 is a result of the SDS PAGE electrophoresis of the wild-type hNGF purified by Superdex 75 column in Example 4.

[0059] FIG. 2(A) and FIG. 2(B) are results of the activity measurement of the mutants in Example 5.

[0060] FIG. 3 (A) and FIG. 3 (B) are results of the pain threshold measurement of the short-term administrated to mice in Example 7.

[0061] FIG. 4 (A), FIG. 4 (B) and FIG. 4 (C) are results of the pain threshold measurement of the long-term administrated to mice in Example 7.

[0062] FIG. 5 is a result of the behavioral experiments of the mice injected with a mutant and a wild-type hNGF respectively in Example 8 by observing the leg lifting maintenance time.

DETAILED DESCRIPTION

Example 1: Plasmid Construction of Wild-Type hNGF and its Mutants

[0063] 1. Construction of expression plasmid containing DNA sequence of wild-type hNGF

[0064] The DNA sequence of wild-type hNGF was synthesized (SEQ ID NO: 1 in the sequence listing), and the target sequence was amplified by PCR using primers (F: GGAATTCATGTCCATGTTG (SEQ ID NO: 41), R: CAAGCTTTCAGGCTCTTCT (SEQ ID NO: 42)). The PCR product was digested with EcorI (NEB #R0101S), and then the resulting digested product was subjected to a secondary digestion with Hind III (NEB #R0104S). The pcDNA3.1(−) expression vector was digested in the same manner. The digested vector and the fragments amplified by PCR were subjected to agarose gel electrophoresis. The target fragments were cleaved, and the digested vector and the target DNA fragments were respectively recovered by using a DNA gel recovery kit (TIANGEN, #DP209-03) and were ligated by a DNA ligase kit (Takara/6022) at 16° C. for 1 h, to complete the plasmid construction of the wild-type hNGF.

[0065] 2. Construction of expression plasmid containing DNA sequence of hNGF mutants

[0066] In the same manner as the above, the plasmids of all mutants were synthesized and constructed. The DNA sequences of the mutants were nucleotide sequences from SEQ ID No: 22 to SEQ ID No: 40 in the sequence listing.

Example 2: Transformation and Extraction of Plasmids Containing hNGF and its Mutants

[0067] 1. Transformation

[0068] The plasmids containing hNGF and its mutants constructed in the above Example 1 were subjected to a heat shock transformation. The top 10 competent cells (Tiangen/CB104-02) were taken out from the −70° C. refrigerator and immediately thawed on ice, and 50 μl of competent cells were taken for transformation. 2 μl of the plasmid was added to the 50 μl of competent cells, mixed by flicking, subjected to an ice bath for 30 min, and then subjected to a dry bath at 42° C. for 90 s, during which the centrifuge tube was not shaken, and the centrifuge tube was immediately placed on ice for 2 min after taking out of the dry bath. 500 μl of antibody-free LB (Luria-Bertani)/SOC (Super Optimal broth with Catabolite repression) medium was added, and cultured at 37° C. for 45 min on a shaker at 150 rpm/min. All the liquid in the centrifuge tube was poured onto the LB plate and spread evenly. The plate, after drying, was inverted in an incubator for 16 h.

[0069] 2. Large scale extraction of plasmid

[0070] The single colonies obtained in the above 2.1 experiment were picked up, inoculated into 500 μl of LB liquid medium and cultured at 37° C. for 7 h, and the bacterial solution was sent for sequencing. The correct bacterial solution confirmed by sequencing was subjected to a lot of shaking, and 500 μl of the bacterial solution was inoculated into 500 ml of LB medium and cultured at 37° C. for 16 h. The overnight cultured solution was collected by centrifugation at 4° C., centrifuged at 6000×g for 10 min and the supernatant was completely discarded. The plasmid was large-scale extracted by using a Plasmid Maxi Kit (purchased from QIAGEN, Cat. No. 12163), and the concentration was measured for use.

Example 3: Expression of Wild-type hNGF and Its Mutants

[0071] The wild-type hNGF and its mutant plasmids large-scale extracted in the above Example 2 were transfected into 293F cells, and the expression supernatant was collected and quantified on day 4 after transfection.

[0072] Experimental Procedures:

[0073] 1. One day before transfection, 900 ml of 293F cells in total were inoculated at 0.5×10.sup.6/ml in 300 ml/bottle.

[0074] 2. Cells were counted on the day of the transfection, and the cell density was about 1.0×10.sup.6/ml with a viability of 99% or more.

[0075] 3. Transfection: 36 ml of a cell culture medium was taken into an 125 ml culture flask; 360 ug of plasmid was added and mixed evenly; and then 1080 ug of PEI was added and mix evenly, leave it to stand at room temperature for 15 min, mixed with cells at about 12.3 ml/bottle, and incubated at 37° C., in 8% CO.sub.2, under 120 RPM.

[0076] 4. On the fourth day after the transfection, the cell supernatant was collected and centrifuged at 10000 g for 20 min.

[0077] 5. The supernatant was collected and filtered at 0.45 um, to obtain a protein supernatant of wild-type hNGF and its mutants.

[0078] 6. SDS-PAGE detection, quantification by silver nitrate staining.

Example 4: Purification of Wild-Type hNGF and its Mutants

[0079] The protein supernatant of the NGF and its mutants obtained in the above Example 3 was purified.

[0080] 1. Cation exchange chromatography: the protein supernatant of wild-type hNGF and its mutants was first adjusted to pH 4.0 with acetic acid and water. A CM Sepharose FF column was fully equilibrated with 0.05 mol/L acetate buffer (pH 4.0) and then loaded. After the loading was completed, it was rinsed with an equilibration solution to the baseline, and then impure peaks were eluted to the baseline with an equilibration solution of 0.05 mol/L Tris-HCl (pH 9.0), and finally subjected to a gradient elution with 0.05 mol/L Tris-HCl and 0.05 mol/L Tris-HCl-0.4 mol/L NaCl (pH 9.0). The target peak was collected according to the ultraviolet absorption, in which the collection was started when the number shown on the UV detector began to rise, and stopped when the number was lowered to the baseline.

[0081] 2. Hydrophobic chromatography: a Butyl Sepharose 4 FF column was well equilibrated with 0.02 mol/L phosphate (pH 6.8)-1.5 mol/L sodium chloride buffer. Into the target peak solution collected in step 1, a sodium chloride solid was added, such that the final concentration of sodium chloride in the solution was 1.5 mol/L. After the sodium chloride was fully dissolved, the sample was loaded at a speed of 120 cm/h. After the loading was completed, it was rinsed with an equilibration solution to the baseline, and then the target peak was collected by elution with 0.02 mol/L phosphate (pH 6.8).

[0082] 3. Gel exclusion chromatography: Superdex 75 prep grade chromatography column was fully equilibrated with 0.05 mol/L phosphate-0.15 mol/L sodium chloride buffer at pH 6.8. Then, the target peak collected in step 2 was loaded, in which the collection was started when the number shown on the UV detector began to rise from the baseline, and stopped when the number was lowered to the baseline.

[0083] The SDS PAGE of the wild-type hNGF purified by Superdex 75 column is shown in FIG. 1, indicating that the prepared NGF has high purity. The samples with target protein peaks of the collected wild-type hNGF and its mutants were concentrated to 0.4 mg/ml by using an ultrafiltration tube, and stored at 4° C. for subsequent experiments.

Example 5: Measurement for the Activity of Wild-Type hNGF and its Mutants by Chicken Embryo Method

[0084] 1. Measurement for the activity of wild-type hNGF and its mutants activity by chicken embryo dorsal root ganglion method

[0085] The wild-type hNGF (amino acid sequence is shown in the sequence listing) and its mutants (amino acid sequences were shown in the sequence listing) samples obtained in the above Example 4 were diluted. A solution: 6 ng of the extracted wild-type hNGF and its mutants samples were dissolved by adding 1 ml of a serum-free DMEM medium. B solution: 50 μl of A solution was added with 4.95 ml of serum-free DMEM medium. C solution: 60 μl of B solution was added with 2.94 ml of serum-free DMEM (3 ml in total) to achieve a final concentration (3 AU/ml). A and B solutions were diluted in a centrifuge tube, and C solution was placed in a cell bottle. C solution was used as a No. 1 bottle, and was further diluted by a factor of 3 to No. 2, No. 3, No. 4, No. 5 and No. 6 solutions to be tested. Each solution to be tested was added into one culture bottle at 2 ml/bottle. At the same time, a serum-free DMEM culture medium was used as a blank control, and the standard product purchased from the National Institute of Food and Drug Control was used as a positive control (reference product). After an 8-day-old chicken embryo dorsal root ganglion was added, the culture bottle was placed in a saturated humidity incubator in a 5% CO.sub.2 and at 37° C., and the results were observed after 24 h.

[0086] The content of NGF per ml of the sample to be tested when growing best is used as 1 activity unit (AU). The titer was calculated from end-point judgment, which was deemed as the best dilution for growing taken from the 3rd and 4th dilutions back counted from the dilution having the negative control result. The reference product is a standard product purchased from the National Institute of Food and Drug Control, in which the capacity of each is 1000 AU.

[0087] The formula for calculating the specific activity of NGF is shown as follow:

[0088] specific activity of the sample to be tested (AU/mg)=activity of the reference product (AU/ml)×[pre-dilution factor of the sampler activity at the dilution point of the corresponding reference product (AU/ml)/actual activity of the reference product (AU/ml)]

[0089] The results of the measurements are shown in FIGS. 2(A) and 2(B). The results showed that the hNGF mutants Phe12Glu, Lys32Leu, Arg59Leu, Asp65Ala, Lys74Leu, Lys88Leu, Lys88Gly, Gln96Glu, Phe101Ala, Arg114Leu all retained wild-type activity and even a higher activity.

Example 6: Measurement of the Activity of NGF and its Mutants by TF-1 Cell Method

[0090] The detailed operation method was performed in accordance with the method in Example 1 of a patent entitled “Method for Quantitatively Measuring Nerve Growth Factor Activity” with a publication number of CN103376248A, and the test results of the specific activity were shown in the following table.

TABLE-US-00001 TABLE 1 Sample Name Specific Activity (U/mg) by Cell Method Wild-type hNGF 430,000 Lys74Leu 767,000 Phe12Glu 620,000 Lys88Gly 590,000 Gln96Glu 430,000

Example 7: Detection for Whether NGF and its Mutants Cause Pain (Pain Threshold)

[0091] Experimental principle: qualified mouse having a normal response to pain was screened, and injected a certain dose of NGF sample (wild-type or its mutants). The pain threshold of curved claw response in mouse by mechanical stimulation was determined, and subjected to a statistical analysis, and finally whether the sample caused mouse hyperalgesia was determined.

[0092] 7-1. Observation of Short-Term Pain-Causing Condition

[0093] I. Experimental material

[0094] Dynamic Plantar Aesthesiometer (Ugo Basile, Italy), model 37450.

[0095] II. Experiments

[0096] 1. Screening of qualified mice

[0097] SPF grade CD-1 mice were ordered, in which the mice were male weighed 30-35 g.

[0098] By the Dynamic Plantar Aesthesiometer [Ugo Basile, Italy, model: 37450], the experimental animals were screened for qualified mice, in which the mean threshold of the left and right feet is between 7.5 and 10 and the P value for the threshold of the left and right feet in a same mouse is more than 0.05.

[0099] Mice were randomly divided into experimental groups and blank control groups, in which the experimental groups were divided into subgroups according to various samples and administration doses, and each group had 10 mice.

[0100] 2. Design of administration for NGF samples

[0101] 2.1. Screening of the pain-causing dosage of wild-type samples

[0102] Drug formulation: a positive control NGF wild-type samples and each mutant sample were diluted by using sample stock solutions (50 mM PB, 150 mM NaCl, pH 6.8).

[0103] Blank control: stock solution of NGF samples.

[0104] Mode of administration and dosage: 20 μl were administered plantar subcutaneously to the left and right feet of mice respectively.

[0105] The minimum dose for a short-term administration was 1.25 μg per mouse, while the corresponding higher dose was administered to determine the ability to cause pain, see the dose labeled in FIG. 3 (B).

[0106] 3. Measurement of pain threshold

[0107] Mechanical threshold measurements were performed at 1 h and 2 h after administration respectively, and values were recorded for observing the short-term administration (within 2 h).

[0108] 4. Statistical analysis for the results

[0109] GraphPad Prism software was used for graph drawing and statistical analysis for the results. The difference in the mechanical thresholds between each dose group and the control group were compared, and the ability to cause pain of wild-type samples and mutants samples were analyzed.

[0110] As can be seen from FIG. 3(A), when the minimum dose of administration was 1.25 μg per mouse, the control group had no pain, while the wild-type positive control group had a pain threshold of obviously less than 5 at 1 h, and the pain was obvious; the pain threshold for each mutant experimental group was about 7, and there was no significant difference as compared with the negative control group, indicating that the short-term injection of mutants were basically no painful;

[0111] As can be seen from FIG. 3(B), when the administration dose of the control group and the wild-type was 1.25 μg per mouse and the administration dose of the experimental group was increased, the control group had no pain, while the wild-type positive control group had a pain threshold of obviously less than 5 at 1 h, and the pain was obvious; the pain threshold for each mutant experimental group was about 7, and there was no significant difference as compared with the negative control group, indicating that the short-term injection of mutants were also basically no painful in the case of increasing the administration dose.

[0112] 7-2. Observation of Long-Term Pain-Causing Condition

[0113] Three of the above mutants No. 1 (Phe12Glu), No. 2 (Lys88Gly) and No. 3 (Arg114Leu) were randomly selected for long-term pain-causing test. Except that the three doses for this experiment were: 0.2 μg per mouse, 0.5 μg per mouse and 1.25 μg per mouse, once a day for 3 weeks, during which the pain threshold was measured continuously, the rest all were performed according to the method in 7-1 “observation of short-term pain-causing condition”.

[0114] The results are shown in FIGS. 4(A), 4(B) and 4(C), indicating that as for the mutant Phe12Glu, the pain threshold was not significantly reduced within 14 days, and no pain was observed, while only a short-term pain was observed for a medium dose (0.5 μg per mouse) after 14 days; as for the mutant Lys88Gly and the mutant Arg114Leu, no obvious pain threshold change was showed during the test; and as for the wild-type NGF, the pain thresholds of three doses were reduced within 17 days gradually, and pain gradually appeared. In view of this, the mutants of the present disclosure have a significant pain reducing effect over the wild-type.

Example 8. Behavioral Test of Whether Wild-Type hNGF and its Mutants Cause Pain

[0115] The wild-type hNGF and its mutants samples were administered in the joints of the mice, and whether the samples cause pain were examined by leg lifting maintenance time and number of the mice according to behavior.

[0116] Experiments

[0117] 1. Ordering of mouse

[0118] SPF grade CD-1 mice were ordered, in which the mice were male weighed 30-35 g. They were randomly divided into experimental groups, blank control groups (abbreviated as control groups) and positive control groups, in which each group was divided into seven subgroups according to the dose, and each group was randomly selected for 6 mice.

[0119] 2. Administration dose and time

[0120] 2.1. Administration dose

[0121] Positive control: the wild-type hNGF was diluted with a sample stock solution (50 mM PB, 150 mM NaCl, pH 6.8) to 1.25 μg/10 μl group;

[0122] Experimental group: the preparation method of the mutant drug was the same as the positive control, and the mutants Phe12Glu, Lys88Gly and Arg114Leu were diluted to 1.25 μg/10 μl group and 0.5 μg/10 μl group;

[0123] Blank control: normal saline.

[0124] 2.2. Mode of administration

[0125] Drugs were injected into the joint in hind legs of the mice, in which 10 μl was administered into each joint cavity.

[0126] 2.3. Time of administration

[0127] Each dose group was administered in a single continuous 3-4 days. That is, the administration was performed at 10 am on the first day, and at the same time points on the 2nd, 3rd, and 4th days thereafter.

[0128] 3. Behavioral observation

[0129] Observation was performed at 2nd and 4th hours after the administration of each experimental group, and at the same time points on the 2nd, 3rd, and 4th days of administration.

[0130] Observation indicators: the numbers of the mouse spontaneous leg lifting within 2 min and the maintenance time (s) of each leg lifting were used to calculate the accumulated time of leg lifting.

[0131] 4. Statistical analysis for the results

[0132] A two-way ANOVA of GraphPad Prism software was used to compare the leg lifting maintenance time, and analyze whether different samples cause pain.

[0133] The experimental results are shown in FIG. 5. The wild-type hNGF group may cause obvious pain at each time point after the injection of the drug. There is no leg-lifting behavior or pain abnormality in the continuous administration of each dose group of the mutant, thereby determining that the mutants did not cause pain. Chi-square analysis showed that there was a significant difference between the positive control group and the experimental group at each detection time point.