TRANSGENIC MOUSE EXPRESSING INACTIVATED HUMAN IDURONATE-2-SULPHATASE AND METHOD FOR IMPROVING A HUNTER SYNDROME TREATING AGENT USING SAME

Abstract

A transgenic mouse expressing inactivated human iduronate-2-sulphatase (IDS) and a method for improving agents for treating Hunter syndrome using the transgenic mouse are provided. The transgenic mouse expressing the inactivated human IDS, which has immune tolerance to IDS, does not cause an immune response to IDS which is an active component of the agents for treating Hunter syndrome, and thus the transgenic mouse may be effectively used to identify immunogenic factors other than the immunogenicity of the protein components of the agents.

Claims

1. A targeting vector comprising a nucleotide sequence encoding inactivated human iduronate-2-sulphatase (IDS) in which the cysteine at amino acid position 84 is substituted with threonine.

2. The targeting vector of claim 1, wherein the nucleotide sequence encoding the inactivated human iduronate-2-sulphatase is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3.

3. The targeting vector of claim 1, wherein the nucleotide sequence encoding the inactivated human iduronate-2-sulphatase is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4.

4. A transgenic zygote obtained by delivering the targeting vector of claim 1 to a zygote of a mouse.

5. A transgenic mouse expressing inactivated human iduronate-2-sulphatase in which the cysteine at amino acid position 84 is substituted with threonine.

6. The transgenic mouse of claim 5, wherein the transgenic mouse is obtained by implanting the transgenic zygote of claim 4 into the uterus of a surrogate mother.

7. A transgenic mouse with immune tolerance to iduronate-2-sulphatase (IDS) produced by mating the transgenic mouse of claim 5 with an IDS heterozygous mouse, wherein the IDS protein moiety of the mouse is removed.

8. A method for providing information on the immunogenic factors of a Hunter syndrome treating agent, comprising: i) injecting two or more agents for treating Hunter syndrome to the transgenic mouse with immune tolerance to IDS of claim 7; ii) confirming whether or not an anti-drug antibody (ADA) is produced in the transgenic mouse with immune tolerance to IDS; and iii) comparing the results of step ii) above obtained by two or more agents for treating Hunter syndrome with each other.

9. The method of claim 8, wherein the ADA is an antibody to IDS.

10. The method of claim 8, wherein the agent for treating Hunter syndrome comprises IDS.

11. The method of claim 9, wherein the IDS is an IDS protein represented by the amino acid sequence of SEQ ID NO: 1.

12. The method of claim 8, wherein the agent for treating Hunter syndrome comprises a pharmaceutically acceptable additive.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0016] FIG. 1 shows a schematic diagram illustrating the targeting vector (pcDNA3.1 (+)-IDS.sup.C84T) for preparing an inactivated human IDS transgenic mouse in which the cysteine at amino acid position 84 is substituted with threonine.

[0017] FIG. 2 shows an image illustrating the electrophoresis results of a targeting vector after the treatment with NheI and XhoI.

[0018] FIG. 3 shows a graph illustrating the quantification of the amount of the inactivated human IDS protein produced by culturing the 293FT cells in which a targeting vector was introduced.

[0019] FIG. 4 shows an image of the inactivated human IDS protein produced by culturing the 293FT cells, into which a targeting vector was introduced, and confirmed by western blot.

[0020] FIGS. 5a to 5e show images illustrating the results of genomic DNA of mice (10 mice secured out of 96 mice, NO: no template, N/C: normal mouse genomic DNA, P/C: positive control (plasmid DNA)) for the screening of the transgenic mice (IDS.sup.C84T) expressing the inactivated human IDS, confirmed by PCR amplification.

[0021] FIG. 6 shows images illustrating the results of genomic DNA of mice (selection of #1, #2, #3 and #8 mice having both characteristics of a transgenic mouse and an IDS KO mouse among male mice KO: knock-out, WT: wild-type, Tg: transgenic) for the screening of the transgenic mouse, (IDS.sup./C84T), which has immune tolerance to IDS, where only the inactivated human IDS is expressed and the IDS protein of the mice is removed, confirmed by PCR amplification.

[0022] FIG. 7 shows a schematic diagram illustrating the IDS drug administration time-point and the sample collection time-point in an immunogenicity measurement experiment using transgenic mice (IDS.sup./C84T) which have immune tolerance to IDS.

[0023] FIGS. 8a and 8b show images confirming the ADA production through ELISA using the plasma samples collected from the IDS KO mice (IDS.sup./) at the 20.sup.th week after IDS drug administration and the transgenic mice (IDS.sup./C84T) which have immune tolerance to IDS.

[0024] FIG. 9 shows a graph illustrating the level of ADA production in the IDS KO mice (IDS.sup./) and the transgenic mice (IDS.sup./C84T), which have immune tolerance to IDS, up to the 28.sup.th week after IDS drug administration.

[0025] FIG. 10 shows a schematic diagram illustrating the time-point for the administration of an adjuvant-containing IDS drug and the time-point for sample collection in an immunogenicity measurement experiment using transgenic mice (IDS.sup./C84T) which have immune tolerance to IDS.

[0026] FIGS. 11a and 11b show images confirming the ADA production through ELISA with respect to the samples of day 48 in the IDS KO mice (IDS.sup./) and the transgenic mice (IDS.sup./C84T) which have immune tolerance to IDS, after the administration of two kinds of adjuvant-containing IDS drugs.

[0027] FIG. 12 shows a graph illustrating the level of ADA production in the IDS KO mice (IDS.sup./) and the transgenic mice (IDS.sup./C84T), which have immune tolerance to IDS, up to day 54 after the administration of two kinds of adjuvant-containing IDS drugs.

BEST MODE FOR CARRYING OUT THE INVENTION

[0028] The present invention provides a targeting vector, which includes a nucleotide sequence encoding inactivated human iduronate-2-sulphatase (IDS) in which the cysteine at amino acid position 84 is substituted with threonine.

[0029] The human IDS is an enzyme involved in the degradation of heparan sulfate and dermatan sulfate and it may cause Hunter syndrome or type II mucopolysaccharidosis when deficient. The human IDS may have the amino acid sequence of SEQ ID NO: 1. In addition, the human IDS may be encoded by the nucleotide sequence of SEQ ID NO: 2.

[0030] When a point mutation of converting the cysteine at position 84 to threonine occurs in the human IDS, the formyl glycosylation may not proceed properly and thus the human IDS may not exhibit its activity.

[0031] Additionally, the nucleotide sequence included in a targeting vector comprising the nucleotide sequence encoding the inactivated human IDS may be a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3. Specifically, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 may be a nucleotide sequence represented by SEQ ID NO: 4.

[0032] The targeting vector has multiple cloning sites (MCS) for cDNA cloning, is capable of replication in host cells, and the vector is not particularly limited as long as the vector has a reporter gene or antibiotic-resistant gene for the selection of the targeting vector. In a specific embodiment of the present invention, the vector used was pcDNA3.1(+) vector, but is not limited thereto.

[0033] Additionally, the present invention provides a transgenic zygote obtained by delivering the targeting vector to a zygote of a mouse.

[0034] The transgenic zygote may be obtained by injecting the DNA of a targeting vector into zygote of a mouse by microinjection. Specifically, the transgenic zygote may be obtained by inducing superovulation in a female mouse, mating it with a male mouse, confirming the mating, collecting zygotes from the fallopian tube of the female mouse with vaginal plug, loading the target vector DNA solution into a microinjection injection pipette and injecting it into the male pro-nucleus under a microscope.

[0035] Additionally, the present invention provides a transgenic (IDS.sup.C84T) mouse capable of expressing inactivated human IDS, in which the cysteine at amino acid position 84 is substituted with threonine.

[0036] The transgenic mouse may be obtained by implanting a transgenic zygote of a mouse into the uterus of a surrogate mother. It may be confirmed whether the inactivated human IDS gene has been inserted, by extracting the genomic DNA from the tail of the mouse born 19 days after the implantation.

[0037] Specifically, the transgenic mouse may be screened by confirming the inactivated human IDS amplified by PCR using primers having the nucleotide sequences of SEQ ID NOS: 5 and 6, which specifically bind to the inactivated human IDS gene.

[0038] Additionally, the present invention provides a transgenic (IDS.sup./C84T) mouse with immune tolerance to IDS, which is prepared by mating the transgenic (IDS.sup.C84T) mouse with the IDS heterozygous (IDS.sup./+) mouse, and expresses human IDS in which the cysteine at amino acid position 84 is substituted with threonine, and from which the IDS protein moiety is removed.

[0039] The IDS heterozygous (IDS.sup./+) mouse may be obtained by mating a normal mouse with a chimeric mouse which is obtained by introducing a targeting vector in which the IDS gene is deleted into an embryonic stem cell and injecting the transgenic embryonic stem cell into a blastocyst of a zygote.

[0040] Additionally, the IDS heterozygous (IDS.sup./+) mouse may be prepared referring to the technology disclosed in KR Patent No. 10-0884564, but is not limited thereto.

[0041] As used herein, the term transgenic mouse with immune tolerance to IDS refers to a transgenic mouse, where part of the human IDS gene is substituted, part of the mouse IDS gene is deleted, and which expresses inactivated human IDS but does not express mouse IDS.

[0042] Additionally, the present invention provides a method for providing information on the immunogenic factors of a Hunter syndrome treating agent, which includes: i) injecting two or more agents for treating Hunter syndrome to the transgenic mouse with immune tolerance to IDS; ii) confirming whether an anti-drug antibody (ADA) is produced in the transgenic mouse with immune tolerance to IDS; and iii) comparing the results of step ii) above obtained by two or more agents for treating Hunter syndrome with each other.

[0043] The ADA may be an antibody to IDS.

[0044] The Hunter syndrome treating agent may contain IDS. IDS is an active ingredient of a Hunter syndrome treating agent, and it may be ELAPRASE or HUNTERASE. Specifically, the above two agents for treating Hunter syndrome have the same amino acid sequence for the IDS protein, but show a difference with respect to the polymer ratio (HUNTERASE (1.4%) and ELAPRASE (3.2%)) and purity (HUNTERASE (98.9%) and ELAPRASE (96.9%)) of the IDS protein (Chihwa Kim et al, Journal of Human Genetics, 8:1-8, 2016). The ELAPRASE and HUNTERASE as agents for treating Hunter syndrome may be used as a sample for a method for investigating conditions more suitable for immunological inducing action.

[0045] The IDS may be a protein in which the amino acid at position 84 of the amino acid sequence of SEQ ID NO: 1 is N-glycosylated.

[0046] Additionally, the agents for treating Hunter syndrome may contain a pharmaceutically acceptable additive.

Mode for the Invention

[0047] Hereinafter, the present invention is explained in detail by Examples. The following Examples are intended to further illustrate the present invention without limiting its scope.

[0048] I. Preparation of IDS.sup./C84T Transgenic Mouse

EXAMPLE 1

Preparation of pcDNA3.1(+)-IDS.SUP.C84T .Targeting Vector

[0049] For the preparation of a targeting vector expressing inactivated human IDS, the nucleotide sequence of SEQ ID NO: 4 in which a cysteine-encoding DNA sequence (i.e., tgc) was substituted with a threonine-encoding DNA sequence (i.e., acc) in the human normal IDS nucleotide sequence of SEQ ID NO: 2, was cloned into the pcDNA3.1(+) vector.

[0050] For stable expression of cloned IDS.sup.C84T cDNA in mammalian 293FT cells, the pcDNA3.1(+)-IDS.sup.C84T vector was prepared by subcloning the cloned IDS.sup.C84T cDNA between the Nhel and Xhol restriction enzyme sites of the pcDNA3.1(+) vector (Invitrogen) including the mammalian CMV promoter (FIGS. 1 and 2).

EXAMPLE 2

Confirmation of Expression of pcDNA3.1(+)-IDS.SUP.C84T .Targeting Vector

[0051] The nucleotide sequence of the pcDNA3.1(+)-IDS.sup.C84T vector was analyzed, and as a result, it was confirmed that a point mutation from cysteine at amino acid position 84 to threonine has occurred. Then, the vector was transfected into the 293FT cells, and the cells were cultured and inactivated human IDS was produced. The inactivated human IDS obtained from the cultured 293FT cells were confirmed to be normally expressed, by ELISA and western blotting (FIGS. 3 and 4).

EXAMPLE 3

Preparation of Transgenic Zygotes

[0052] The pcDNA3.1(+)-IDS.sup.C84T targeting vector was degraded with suitable restriction enzymes to prepare linear DNA fragments, electrophoresed on an agarose gel, and the resulting DNA was purified. The purified DNA was prepared to a concentration of about 4 ng/L.

[0053] For the inducement of superovulation, a female mouse was injected with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) hormones at 48 hour intervals. After inducing a mating with a male mouse, the female mouse was checked of its vaginal plug the next morning to confirm the result of the mating and zygotes were collected from the fallopian tube with the vaginal plug. Then, the DNA solution was loaded into a microinjection injection pipette and injected into the male pro-nucleus under a microscope.

EXAMPLE 4

Preparation of Transgenic (IDS.SUP.C84T .) Mouse Expressing Inactivated Human IDS

[0054] The zygotes into which the targeting DNA prepared in Example 3 was injected was selected and implanted into the surrogate mother's fallopian tube. Transgenic mice were delivered on day 19 after the implantation.

[0055] Then, genomic DNA of 4- to 5 week-old mice was extracted from the tails to confirm whether the gene was inserted. PCR was performed using primers having the nucleotide sequences of SEQ ID NOS: 5 and 6, and the transgenic (IDS.sup.C84T) mouse expressing the inactivated human IDS were screened and 10 mice were obtained (FIGS. 5A to 5E). The sperm of the transgenic (IDS.sup.C84T) mouse was deposited on Nov. 28, 2016, at the Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, as an international depository authority (Deposit No.: AZ00001310).

EXAMPLE 5

Screening of Transgenic (IDS.SUP./C84T.) Mouse with Immune Tolerance to IDS

[0056] Progenies were obtained by a mating between the transgenic (IDS.sup.C84T) mouse and an IDS heterozygous (IDS.sup./+) mouse. The IDS.sup./C84T mouse with immune tolerance to IDS, which has both the IDS deleted gene and the inactivated human IDS gene, was screened by PCR using the tail genomic DNA of 4-5 week old mouse (FIG. 6).

[0057] II. Experiment of Measuring Immunogenicity

EXAMPLE 6

Confirmation of ADA Production in Accordance with IDS

EXAMPLE 6-1

Collection of Samples After IDS Treatment

[0058] Three kinds of mice (wild type (WT), IDS Knock-out (KO), and transgenic mice with immune tolerance to IDS (IDS.sup./C84T )) were divided into 5 groups and drugs were administered at 1 mg/kg once a week for 6 months (Table 1 and FIG. 7). Plasma samples were collected at 3 days before administration and at week 4, 8, 12, 16, 20, 24, and 28 after administration at intervals of 4 weeks. All of the samples were stored at 70 C. until analysis to be analyzed in one batch.

TABLE-US-00001 TABLE 1 Duration of Group Drug Mouse Administration Analysis G1 Saline WT Every Week Collection of for 6 Months Plasma Sample (once per month) - Confirmation of Formation of Anti- Drug-Antibody, by ELISA G2 Saline IDS KO G3 IDS 1 mg/kg G4 Saline IDS.sup./C84T G5 IDS 1 mg/kg

EXAMPLE 6-2

Confirmation of Occurrence of ADA Production After IDS Treatment

[0059] The plasma samples were diluted to 1:10, 1:25, 1:50, 1:100, 1:500, 1:1000, 1:5000 and 1:10,000 with PBS and subjected to ELISA (FIGS. 8a and 8b). Specifically, 10 of the IDS protein diluted in PBS was respectively added into a 96-well plate in an amount of 100 and placed overnight at 2 C. to 8 C. After discarding the solution in the plate, 300 of 1 vol % BSA/PBS was added thereto and allowed to react at room temperature for 1 hour. The diluted plasma samples were injected in an amount of 100 into two wells per concentration. The plate was covered with a plate sealer and the samples were allowed to react at room temperature for 2 hours. 300 of wash solution (0.1 vol % PBST) was added to each well and washed 3 times. The remaining wash solution was completely removed by hitting against several layers of paper towels.

[0060] Anti-mouse-IgG labeled with horseradish peroxidase (HRP) was diluted to 1:2000 with PBS and added to all of the wells in an amount of 100 , respectively. The plate was covered with a plate sealer and the samples were allowed to react at room temperature for 1 hour. 300 of wash solution (0.1 vol % PBST) was added to each well and washed 3 times. The remaining wash solution was completely removed by hitting against several layers of paper towels.

[0061] 100 l of TMB peroxidase substrate solution was added to all of the wells and were allowed to react at room temperature for 15 minutes. 100 l N sulfuric acid was added to all of the wells to stop the reaction, and the absorbance was measured at a wavelength of 450 nm.

[0062] The plasma sample collected at the 20th week was diluted to 1:100 and the occurrence of ADA production was confirmed by ELISA. As a result, the IDS KO mice showed high antibody responses but the transgenic mice with immune tolerance (IDS.sup./C84T) did not show any antibody response thereby to confirm that they have immune tolerance to IDS protein (FIGS. 8a and 8b).

[0063] In the case of the IDS KO mice, ADA production was observed from about 8 weeks after administration of the IDS drug, whereas the transgenic mice with immune tolerance (IDS.sup./C84T) showed no ADA production by the administration of IDS drug (FIG. 9).

EXAMPLE 7

Confirmation of ADA Production in Accordance with IDS Types and Adjuvants

EXAMPLE 7-1

Sample Collection After Treatment with Adjuvant-Containing IDS

[0064] Three kinds of mice (wild type (WT), IDS Knock-out (KO), and transgenic mice with immune tolerance to IDS (IDS.sup./C84T)) were divided into 7 groups and intraperitoneally administered with two kinds of adjuvant-containing drugs, HUNTERASE and ELAPRASE, and the administration was performed 3 times at intervals of 3 weeks.

[0065] Plasma samples were collected at 3 days before administration, at week 1 (the 7.sup.th day) and week 2 (the 14.sup.th day) after the first administration (the 0.sup.th day), at week 1 (the 28.sup.th day) and week 2 (the 35.sup.th day) after the second administration (the 21.sup.st day), and at week 1 (the 48.sup.th day) and week 2 (the 54.sup.th day) after the third administration (the 42.sup.nd day) (Table 2 and FIG. 10). All of the samples were stored at 70 C. until analysis to be analyzed in one batch.

TABLE-US-00002 TABLE 2 Duration of Group Drug Mouse Administration Analysis G1 PBS WT 54 Days Serum Collection - Day 3, 7, 14, 28, 35, 48, and 54 G2 PBS IDS KO G3 IDS-1 50 g Quil A 5 g MPL 5 g G4 IDS-2 50 g Quil A 5 g MPL 5 g G5 PBS IDS.sup./C84T G6 IDS-1 50 g Quil A 5 g MPL 5 g G7 IDS-2 50 g Quil A 5 g MPL 5 g

EXAMPLE 7-2

Confirmation of Occurrence of ADA Production After Treatment with Adjuvant-Containing IDS

[0066] The plasma samples were diluted to 1:10, 1:25, 1:50, 1:100, 1:500, 1:1000, 1:5000, and 1:10,000 with PBS and subjected to ELISA. 10 of the IDS protein diluted in PBS was respectively added into a 96-well plate in an amount of 100 and was allowed to react overnight at 2 C. to 8 C. After discarding the solution in the plate, 300 of 1 vol % BSA/PBS was added thereto and allowed to react at room temperature for 1 hour. The diluted plasma samples were injected in an amount of 100 into two wells per concentration. The plate was covered with a plate sealer and the samples were allowed to react at room temperature for 2 hours. 300 of wash solution (0.1 vol % PBST) was added to each well and washed 3 times. The remaining wash solution was completely removed by hitting against several layers of paper towels.

[0067] Anti-mouse-IgG labeled with horseradish peroxidase (HRP) was diluted to 1:2000 with PBS and added to all of the wells in an amount of 100 . The plate was covered with a plate sealer and the samples were allowed to react at room temperature for 1 hour. 300 of wash solution (0.1 vol % PBST) was added to each well and washed 3 times. The remaining wash solution was completely removed by hitting against several layers of paper towels.

[0068] 100 of TMB peroxidase substrate solution was added to all of the wells and was allowed to react at room temperature for 15 minutes. 100 of 1 N sulfuric acid was added to all of the wells to stop the reaction, and the absorbance was measured at a wavelength of 450 nm.

[0069] As a result, it was confirmed that the transgenic mice with immune tolerance (IDS.sup./C84T) showed that no ADA was produced by two kinds of adjuvant-containing IDS drugs (FIGS. 11a and 11b).

[0070] All of the plasma samples collected 7 times were diluted to 1:100 and the occurrence of ADA production was checked by ELISA. It was confirmed that the transgenic mice with immune tolerance (IDS.sup./C84T) have immune tolerance to the two kinds of adjuvant-containing IDS drugs (FIG. 12).