GENE SEQUENCE CONSTRUCT USED FOR TREATMENT OF CENTRAL NERVOUS SYSTEM DISEASES

Abstract

A gene sequence construct used for the treatment of central nervous system diseases: by means of the construction of an auto-processing expression vector, tyrosine hydroxylase (TH), GTP-cyclohydrolase I (GCH1), aromatic amino acid dopa decarboxylase (AADC), and so on may be simultaneously expressed; proteins are connected by means of an auto-processing unit (APU); the use of a viral vector to introduce the construct into a target cell may ultimately result in the high-efficiency expression of tyrosine hydroxylase (TH), GTP-cyclohydrolase I (GCH1), aromatic amino acid dopa decarboxylase (AADC), and so on having independent functions, being used in the prevention or treatment of Parkinson's disease, Alzheimer's disease and other neurodegenerative diseases.

Claims

1. A gene sequence construct, characterized in that the construct comprises two or more nucleotide sequences that are related to the treatment of a central nervous system disease, and wherein the two or more nucleotide sequences are linked by an auto-processing unit (APU).

2. The gene sequence construct of claim 1, characterized in that said auto-processing unit (APU) comprises an N-terminal auto-processing domain and/or a C-terminal auto-processing domain.

3. The gene sequence construct of claim 2, characterized in that said N-terminal auto-processing domain comprises Intein, B-type bacterial intein-like domain (BIL), Furin sequence, or a derivative thereof.

4. (canceled)

5. The gene sequence construct of claim 1, characterized in that said two or more nucleotide sequences related to the central nervous system disease comprise two or more of the nucleotide sequences of tyrosine hydroxylase (TH), GTP-cyclohydrolase I (GCH1), aromatic amino acid dopa decarboxylase (AADC), or a nervous system growth factor; wherein the two or more nucleotide sequences are linked by an auto-processing unit (APU).

6. The gene sequence construct of claim 5, characterized in that the nervous system growth factor comprises nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), or a GDNF family molecule.

7. The gene sequence construct of claim 5, characterized in that the construct comprises the nucleotide sequences of tyrosine hydroxylase (TH), GTP-cyclohydrolase I (GCH1), and aromatic amino acid dopa decarboxylase (AADC); wherein at least two of the nucleotide sequences are linked by an auto-processing unit (APU).

8. A viral vector genome, characterized in that said viral vector genome comprises the gene sequence construct of claim 1, said viral vector comprises a lentiviral vector or an adeno-associated viral vector.

9. A lentiviral vector system, characterized in that said lentiviral vector system comprises a genome comprising the gene sequence construct of claim 1.

10. A biological product comprising the gene sequence construct of claim 1.

11. The gene sequence construct of claim 2, wherein said C-terminal auto-processing domain comprises a 2A peptide or a 2A-like peptide.

12. The gene sequence construct of claim 11, characterized in that, the 2A peptide or 2A-like peptide comprises a 2A peptide derived from foot-and-mouth disease virus (F2A), a 2A peptide derived from porcine teschovirus virus (P2A), a 2A peptide derived from insect virus (T2A), or a 2A peptide derived from equine rhinitis virus (E2A).

13. The gene sequence construct of claim 5, wherein the gene sequence construct comprises the following modes of construction: TH.sub.-APU-CH1.sub.-APU-AADC; TH.sub.-APU-CH1.sub.-other sequence-AADC; TH.sub.-other sequence-CH1.sub.APU-AADC; TH.sub.-APU-AADC.sub.-APU-CH1; TH.sub.-other sequence-AADC.sub.-APU-CH1; TH.sub.-APU-AADC.sub.-other sequence-CH1; CH1.sub.-APU-TH.sub.-APU-AADC; CH1.sub.-APU-TH.sub.-other sequence-AADC; CH1.sub.-other sequence-TH.sub.-APU-AADC; CH1.sub.-APU-AADC.sub.-APU-TH; CH1.sub.-APU-AADC.sub.-other sequence-TH; CH1.sub.-other sequence-AADC.sub.-APU-TH; AADC.sub.-APU-TH.sub.-APU-CH1; AADC.sub.-APU-TH.sub.-other sequence-CH1; AADC.sub.-other sequence-TH.sub.-APU-CH1; AADC.sub.-APU-CH1.sub.-APU-TH; AADC.sub.-APU-CH1.sub.-other sequence-TH; or AADC.sub.-other sequence-CH1.sub.-APU-TH, wherein the other sequence comprises a linker peptide coding sequence, an internal ribosome entry site (IRES), a promoter, or an intein coding sequence.

14. The gene sequence construct of claim 6, wherein said GDNF family molecule comprises a naturally occurring analog of GDNF, neurturin, persephin, or artemin.

15. The gene sequence construct of claim 1, further comprising a promoter.

16. The gene sequence construct of claim 15, wherein the promoter is a constitutive promoter selected from the group of a CMV promoter, a phosphoglycerate kinase promoter, and a thymidine kinase promoter.

17. The gene sequence construct of claim 1, wherein the promoter is a tissue-specific promoter selected from the group consisting of a synapsin promoter, a CD68 promoter, or a GFAP promoter.

18. A method of treating or prevent of a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of the biological product of claim 10, thereby treating or preventing the neurodegenerative disease.

19. The method of claim 18, wherein the neurodegenerative disease comprises Parkinson's disease.

20. The method of claim 18, wherein the neurodegenerative disease comprises Alzheimer's disease.

21. A method of producing dopamine, comprising contacting a cell with the biological product of claim 10, thereby producing dopamine.

Description

DESCRIPTION OF DRAWINGS

[0037] In order to more clearly explain the technical solutions of the examples of the present invention or in the existing technology, provided below is a brief introduction of the drawings used to describe the examples or the existing technology. Apparently, the drawings described below are only certain examples of the present invention. For those of ordinary skill in the art, they may also obtain other drawings based on these drawings without creative work.

[0038] FIG. 1 is a schematic diagram of a gene sequence construct for gene therapy of Parkinson's disease of the present invention.

[0039] FIG. 2 is a schematic diagram of the structures of the constructs of the present invention.

[0040] FIG. 3. Western blot detection results for aromatic amino acid dopa decarboxylase (AADC), GTP-cyclohydrolase I (GCH1), and tyrosine hydroxylase (TH) proteins after transduction of 293T cells.

[0041] FIG. 4. Western blot detection of aromatic amino acid dopa decarboxylase (AADC), GTP-cyclohydrolase I (GCH1), and tyrosine hydroxylase (TH) proteins after transduction of SH-SY5Y cells.

[0042] FIG. 5. HPLC detection results of dopamine DA production after viral transduction of SH-SY5Y cells.

[0043] Among them in FIG. 1, the coding sequences for the expression of aromatic amino acid dopa decarboxylase (AADC), GTP-cyclohydrolase I (GCH1), and tyrosine hydroxylase (TH) are included. The proteins are linked by auto-processing peptide (P2A). The genes are transcribed, translated, and can produce independent aromatic amino acid dopa decarboxylase (AADC-P2A), GTP-cyclohydrolase I (GCH1-P2A), and tyrosine hydroxylase (TH), after being processed by auto-processing peptide. P2A: porcine teschovirus virus 2A auto-processed peptide.

[0044] In FIG. 2: AADC: aromatic amino acid dopa decarboxylase; GCH1: GTP-cyclohydrolase I; TH: tyrosine hydroxylase; P2A: porcine teschovirus virus 2A auto-processed peptide; Synapsin, CMV, SV40 and PGK are all promoters.

[0045] In FIG. 3: Blank: cells without viral transduction; GFP: cells transduced with CMV promoter-EGFP virus; PD-1: cells transduced with Synapsin promoter-AADC-P2A-GCH1-P2A-TH virus; PD-2: cells transduced with CMV promoter -AADC-P2A-GCH1-P2A-TH virus; P: cells transduced with CMV promoter-AADC-SV40 promoter-TH-PGK promoter-GCH1 virus; Endo-TH: endogenous TH of the cells; Exo-TH: exogenous overexpressed catalytic domain of TH.

[0046] In FIG. 4: Blank: cells without viral transduction; GFP: cells transduced with CMV promoter-EGFP virus; PD-1: cells transduced. with Synapsin promoter-AADC-P2A-GCH1-P2A-TH virus; PD-2: cells transduced with CMV promoter -AADC-P2A-GCH1-P2A-TH virus; P: cells transduced with CMV promoter-AADC-SV40 promoter-TH-PGK promoter-GCH1 virus; Endo-TH: endogenous TH of the cells; Exo-TH: exogenous overexpressed catalytic domain of TH.

[0047] In FIG. 5: GFP: cells transduced with GFP virus; PD-2: cells transduced with CMV promoter-AADC-P2A-GCH1-P2A-TH virus.

DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with examples. The following examples explain the present invention and the present invention is not limited to the following examples.

Examples

[0049] I. As shown in FIG. 2, the construction of various constructs:

[0050] KL0039 vector, synthetic CMV enhancer-synapsin promoter-AADC-P2A-GCH1-P2A-TH and AADC-SV40 promoter-TH-PGK promoter-GCH1 sequences (where TH is a truncated form of TH); wherein, CMV enhancer-synapsin promoter-AADC-P2A-GCH1-P2A-TH is ligated into pUC57 vector (pUC57-synapsin-AGT). Here, the KL0039 vector is a lentiviral transfer vector, derived from existing lentiviral vectors or after partial modifications as needed.

[0051] 1. Construction of PD1 vector

[0052] A PCR product was amplified from the sequence from WPRE to cPPT using KL0039 as template and primers Age-F and Sal-R and purified after electrophoresis. The primer sequences are: Age-F, CTGAGTGCCATTGGATGAcaatcaacctctggattaca; Sal-R, gattactattaataactactcacgcatgctcttctcca. Plasmid pUC57-synapsin-AGT was digested with AgeI and SalI, and the 4.1-kb fragment was recovered. The ligation products of the purified PCR product and synapsin-AGT fragment by T4 DNA ligase were used to transform DH5 competent cells. Transformant colonies were screened by PCR and the positive clones were further confirmed by sequencing.

[0053] 2. Construction of PD2 vector

[0054] Using KL0039 vector as template and primers SnaBI-F:

[0055] TCAGtacgtattagtcatcgctat and SpeI-R:

[0056] CGATactagtgagctctgcttatataga, a PCR product (245 bp) of CMV promoter was amplified and purified after electrophoresis. Double digestion by SnaBI and SpeI was performed on the purified PCR product of CMV promoter and plasmid pUC57-synapsin-AGT, respectively, and the fragments of CMV promoter and pUC57-AGT were purified from the digestion products. The ligation product of the two fragments by T4 DNA ligase was used to transform DH5 competent cells. Transformant colonies were screened by PCR and the positive clones were further confirmed by sequencing. The positive clone was named pUC57-CMV-AGT. Then, a PCR product was amplified from the sequence from WPRE to cPPT using KL0039 as template and AgeF+SalR and purified after electrophoresis. Double digestion by AgeI and SalI was performed on the purified PCR product and plasmid pUC57-CMV-AGT, respectively, and the fragments of the PCR product and CMV-AGT were purified from the digestion products. The ligation product of the two fragments by T4 DNA ligase was used to transform DH5 competent cells. Transformant colonies were screened by PCR and the positive clones were further confirmed by sequencing.

[0057] 3. Construction of P vector: the AADC-SV40 promoter-TH-PGK promoter-GCH1 sequence was used to replace the AGT sequence in PD2 vector.

[0058] 4. GFP vector: the EGFP sequence was cloned and used to replace the AGT sequence in PD2 vector.

[0059] II. Evaluation of the differential expression of target proteins in 293T and SH-SY5Y cells after transduction with various constructs

[0060] Lentiviral four-plasmid system was used to transiently transfect 293T cell line, packaging GFP (CMV promoter-EGFP), PD1 (synapsin promoter-AGT), PD2 (CMV promoter-AGT) lentivirus and positive control virus P (CMV promoter-AADC-SV40 promoter-TH-PGK promoter-GCH1), respectively. The initial viruses were concentrated after purification and transduced into 293T cells after dilution. The titers were determined using RT-PCR (WPRE/ALB). The vector titers of all constructs were similar, ranging from 3.4E+09TU/m1 to 8.74E+09TU/ml.

[0061] In order to assess the expression levels of target proteins, 293T cells and SH-SY5Y cells were transduced with the lentiviruses at MOI=10 and MOI=20, respectively. The cells were harvested 72 hours after transduction and cell lysates were used for Western blot analysis of AADC, GCH1, and TH. The results show that a relatively low level of endogenous TH, but no endogenous AADC and GCH1, was detected in 293T cells. The molecular weights of all target proteins were consistent with the expected values. Compared with no virus transduction Blank and GFP virus transduction, high levels of expression of all three target proteins were detected from cells transduced with PD2 viral vector. Although cells transduced with P viral vector expressed the highest level of AADC, the other two target proteins GCH1 and TH were barely detected. As expected, no expression of the three target proteins was detected in 293T cells transduced with PD1 viral vector, in which the synapsin promoter used is neuron-specific (FIG. 3). Further, we assessed the expression of targeted proteins in SH-SY5Y cells after transduction with the three different constructs. Endogenous TH and AADC with expected molecular weights were detected, but not endogenous GCH1. Similar to the results from transduced 293T cells, high levels of expression of all three target proteins were detected from cells transduced with PD2 viral vector, when compared with no virus transduction Blank and GFP virus transduction. Although cells transduced with P viral vector expressed the highest level of AADC, the other two proteins GCH1 and TH were still barely detected. For cells transduced with PD1 viral vector, only low levels of exogenous AADC and TH were expressed, and GCH1 was barely detected (FIG. 4).

[0062] III. Evaluation of the differential catecholamine production in SH-SY5Y cells after transduction with various constructs

[0063] In neurons, DA is converted primarily by monoamine oxidase (MAO) to dihydroxyphenylacetic acid (DOPAC). The levels of catecholamine were measured by mass spectrometry in the supernatants of two cultured cells, SH SY5Y cells and 293T cells, after transduction with lentiviral vectors. The SH SY5Y cells were transduced by viruses and the media were replaced after overnight. The supernatants were collected after being cultured until the third day and centrifuged at 4500 rpm for 5 minutes. The clear supernatants were transferred to 1.5 mL centrifugation tubes and stored in freezer at 80 C. before testing. The 293T cells were transduced by viruses and the media were changed after overnight. After 2 days of culture, the cells were passaged at 1:10. After 2 days of culture, the media were replaced with fresh media containing 10 mM L-tyrosine. The supernatants were collected after being cultured until the next morning and centrifuged at 4500 rpm for 5 min. The clear supernatants were transferred to 1.5 mL centrifuge tubes and stored in freezer at 80 C. before testing.

[0064] The levels of dopamine in the samples were measured by mass spectrometry as follows. 500 L of cell culture medium was collected and an appropriate amount of internal standard was added. The solution was diluted and mixed with 1 mL of 50 mM ammonium acetate serving as the sample loading solution. After methanol activation, the cartridge was subsequently rinsed with 20 mM ammonium acetate, acetonitrile: isopropanol (1:1), and drained. The sample was eluted with 2% formic acid in acetonitrile and blown dry with nitrogen. The residue was dissolved in 100 L of 0.1% FA and centrifuged at 15000 r/min for 5 min. The supernatant was loaded onto the machine (Angilent 1290UPLC-6470MS/MS detection system) for analysis.

[0065] The results are shown in FIG. 5. Transduction of 293T cells and SH-SY5Y cells with KL-PD2 lentiviral vector greatly increased the production of dopamine in the cell culture supernatants due to the effective and balanced expression of the three enzymes in dopamine synthesis.

[0066] In addition, it should be noted that the specific examples described in this specification may bear different names for various substances or carriers. Any equivalent or simple variation made according to the structure configuration and principles described in the patent conception of the present invention all belong to the scope of the present patent protection. Those skilled in the art to which the present invention pertains can make various modifications or additions to the described specific examples or substitute in a similar manner, as long as they do not depart from the structures of the present invention or go beyond the scope defined by the claims, all should belong to the scope of protection of the present invention.