HPAH mutant as well as coding gene and application thereof
By providing the hPAH mutant and its encoding gene and base editing system, replacing the CGC mutation with TTT, and generating an active hPAH R413F mutant, the problem of the inability to correct the hPAH R413P mutation in the prior art is solved. This enables the reduction of phenylalanine concentration in cell and animal models and the restoration of phenylalanine metabolic function in patients with phenylketonuria.
Patent Information
- Application Number
- CN202511154837.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-18
- Publication Date
- 2025-11-07
AI Technical Summary
There is no suitable base editing scheme in the current technology that can directly correct the hPAH R413P mutation, which prevents patients with phenylketonuria from effectively restoring their phenylalanine metabolism function.
The hPAH mutant and its encoding gene are provided. Gene editing is performed in cell and animal models using base editor systems such as BE3-NRCH, BE3-xCas9-NRCH, BE3-xCas9-RY and sgRNA1/2 to replace the CGC mutation with TTT, generating an active hPAH R413F mutant.
In HEK293T cell and PKU mouse models, the hPAH mutants R413F and R413L significantly reduced the concentration of phenylalanine in the blood, restored the activity of PAH enzyme, and effectively treated phenylketonuria.
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Figure CN120905168A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of genetic engineering, and particularly relates to an hPAH mutant, a coding gene thereof and application. BACKGROUND
[0002] Phenylketonuria (PKU) is an autosomal recessive genetic disease. The supernatant of liver tissue of a PKU patient cannot convert phenylalanine into tyrosine as a normal person does. The onset of the PKU patient is related to phenylalanine metabolic disorder in the liver. The disease is a metabolic disease caused by deficiency of phenylalanine hydroxylase or its coenzyme tetrahyrobipperin (BH4) in the liver. If a PKU child is not treated in time after birth, it will lead to high phenylalanine blood, and high concentration of phenylalanine in the blood will cause irreversible damage to the central nervous system. Generally, mental retardation, hypopigmentation, autism, epilepsy, and about 90% of children also show that the body development and speech are lagging behind normal people. In addition, women with PKU must strictly control Phe levels before pregnancy and during pregnancy to prevent fetal exposure to a high Phe environment, otherwise it will cause microcephaly, mental retardation, behavioral problems and congenital heart defects, i.e. the so-called maternal PKU syndrome. The incidence of PKU varies around the world. Since this disease is an autosomal recessive genetic disease, consanguineous marriage will increase the probability of disease in offspring, and it has been listed as one of the diseases for neonatal screening.
[0003] A study showed that among 1592 chromosomes from a total of 796 patients, 1555 kinds of PAH mutations of genes were identified, and other mutations may be located in non-coding regions or in unknown regions of introns. The gene mutations of PAH deficiency patients mainly include p.R243Q, p.R241C, p.Y204C, p.Y356*, p.R111*, p.R413P, p.R408Q, c.442-1G>A and p.V399V, and there is no suitable base editing scheme to directly correct and restore the hPAH R413P mutation. SUMMARY
[0004] In view of the above prior art, the application provides an hPAH mutant, a coding gene thereof and application, which solves the problem that there is no suitable base editing scheme to directly correct and restore the hPAH R413P mutation in the prior art.
[0005] In order to achieve the above purpose, the technical scheme adopted by the application is to provide an hPAH mutant, and the amino acid sequence of the hPAH mutant is shown in SEQ ID NO. 1 or SEQ ID NO. 2.
[0006] On the basis of the above technical scheme, the application can be further improved as follows.
[0007] Further, the nucleotide sequence of the coding gene of the hPAH mutant is shown in SEQ ID NO. 3 or SEQ ID NO. 4.
[0008] Further, the hPAH mutant is used in the preparation of a medicine for reducing the content of phenylalanine in blood or a medicine for treating phenylketonuria.
[0009] Further, the medicine contains the amino acid sequence of the hPAH mutant shown in SEQ ID NO. 1 or SEQ ID NO. 2.
[0010] Further, the medicine contains the nucleotide sequence of the coding hPAH mutant shown in SEQ ID NO. 3 or SEQ ID NO. 4.
[0011] Further, the medicine contains a base editor for editing the nucleotide sequence shown in SEQ ID NO. 6 into the nucleotide sequence shown in SEQ ID NO. 3.
[0012] Further, the base editor is a plasmid containing BE3-NRCH and sgRNA1 sequences, the nucleotide sequence of BE3-NRCH is shown in SEQ ID NO. 7, and the nucleotide sequence of sgRNA1 is shown in SEQ ID NO. 8.
[0013] Further, the base editor is a plasmid containing BE3-xCas9-NRCH and sgRNA1 sequences, the nucleotide sequence of BE3-xCas9-NRCH is shown in SEQ ID NO. 9, and the nucleotide sequence of sgRNA1 is shown in SEQ ID NO. 8.
[0014] Further, the base editor is a plasmid containing BE3-xCas9-RY and sgRNA2 sequences, the nucleotide sequence of BE3-xCas9-RY is shown in SEQ ID NO. 10, and the nucleotide sequence of sgRNA2 is any one of SEQ ID NO. 11-SEQ ID NO. 15.
[0015] The hPAH mutants (R413F and R413L) can effectively reduce the Phe concentration in the blood of mice, and in HEK293T cells, the gene editing scheme for the PAH (R413P) site realizes effective editing of the inactive hPAH R413P to the active hPAH R413F. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1Results of activity detection of hPAH mutants (R413F and R413L); Figure 2 Results of gene editing using BE3-NRCH and sgRNA1; Figure 3 Results of gene editing using BE3-xCas9-NRCH and sgRNA1; Figure 4 Results of gene editing using BE3-xCas9-RY and sgRNA2 shown in SEQ ID NO. 11; Figure 5 Results of gene editing using BE3-xCas9-RY and sgRNA2 shown in SEQ ID NO. 12; Figure 6 Results of gene editing using BE3-xCas9-RY and sgRNA2 shown in SEQ ID NO. 13; Figure 7 Results of gene editing using BE3-xCas9-RY and sgRNA2 shown in SEQ ID NO. 14; Figure 8 Results of gene editing using BE3-xCas9-RY and sgRNA2 shown in SEQ ID NO. 15. DETAILED DESCRIPTION
[0017] The specific embodiments of the present application will be described in detail below with reference to the examples.
[0018] Example 1 Activity detection of hPAH mutants 1. Preparation of plasmids to be tested: three PAH plasmids (wild-type PAH and two hPAH mutant endotoxin-free plasmids), physiological saline.
[0019] The endotoxin-free plasmids of wild-type PAH and two hPAH mutants are respectively obtained by inserting the nucleotide sequences shown in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5 into pVAX1 plasmid.
[0020] 2. Preparation of experimental mice: PKU homozygous mice, divided into 4 groups. The PKU animal model is Pah exon 7 F263S mutant mouse B6.BTBR-Pah enu2 , purchased from Jackson Laboratory; since the female model mice suffer from PKU maternal syndrome, they cannot successfully give birth to offspring, so hybrid mice are used for mating to obtain homozygous offspring mice with PKU.
[0021] 3. Experimental steps: (1) Zero-point blood samples were obtained from mice by orbital blood collection before injection. All blood samples were stored at 4°C in the form of dried blood spots. (2) The three PAH plasmids were diluted with physiological saline and injected into PKU homozygous mice via tail vein high pressure at a dose of 4 mg / kg. The negative control mice were injected with an equal volume of physiological saline. (3) Blood samples were taken again about 24 hours after injection, and the blood Phe concentration was measured by fluorescence spectrophotometry; (4) Analyze the changes in serum Phe concentration in mice before and after PAH injection.
[0022] This method can only qualitatively determine PAH enzyme activity and cannot perform precise enzyme activity measurement. If PAH is active, the blood Phe concentration in PKU homozygous mice significantly decreases approximately 24 hours after high-pressure injection via the tail vein. The activity detection results of the hPAH mutant are as follows: Figure 1 As shown, the serum Phe content in mice injected with wild-type PAH decreased from an average of 3297 μM to 1931 μM, in mice injected with mutant PAH (R413F) it decreased from an average of 3288 μM to 1124 μM, and in mice injected with mutant PAH (R413L) it decreased from an average of 3934 μM to 2491 μM. In contrast, the serum Phe content in the control group, i.e., mice injected with saline, did not decrease. This indicates that both hPAH mutants (R413F and R413L) can effectively reduce the serum Phe concentration in mice.
[0023] Example 2 Base editing scheme to restore hPAH R413P activity 1. Analysis of the hPAH R413P mutation revealed that it was caused by a CGC>CCC mutation. This invention provides an alternative: using the BE3 editing system, the CCC base encoding P is mutated to TTT encoding F, generating the active mutant hPAH R413F.
[0024] 2. Experimental steps: (1) HEK293T cell plating: After passage, the remaining cells were diluted to an appropriate number of cells and seeded into appropriate well plates. The cells were cultured in a 5% CO2, 37℃ incubator until the cell confluence was about 80%. (2) The base editing system plasmid and the target plasmid with the hPAH (R413P) mutant sequence were transfected into cells using LipoMAX; the nucleotide sequence of hPAH (R413P) is shown in SEQ ID NO.6; Base editing system: BE3-NRCH and sgRNA1 are used, and their nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively; (3) After 24h transfection, cells were collected after 48h selection with puromycin-containing medium; (4) The extracted genome was used as a template for PCR amplification according to the system and conditions described in Table 1; the PCR product was outsourced to Shengong Bioengineering Co., Ltd. for Sanger sequencing, and the sequencing results were analyzed for editing efficiency by software EditR (https: / / moriaritylab.shinyapps.io / editr_v10).
[0025] Table 1 PCR amplification system for base editing efficiency verification
[0026] Primer Primer F: 5'-CCTTGGGGAGTCATACCTCA-3' (SEQ ID NO. 16); Primer Primer R: 5'-ATAAAGCAGGCAGTGGATCA-3' (SEQ ID NO. 17).
[0027] Example 3 Base editing scheme for restoring the activity of hPAH R413P Base editing system: BE3-xCas9-NRCH and sgRNA1 were used, and the nucleotide sequences were shown in SEQ ID NO. 9 and SEQ ID NO. 8, respectively; the remaining steps were the same as in Example 2.
[0028] Example 4 Base editing scheme for restoring the activity of hPAH R413P Base editing system: BE3-xCas9-RY and sgRNA2 were used, and the nucleotide sequences were shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively; the remaining steps were the same as in Example 2.
[0029] Example 5 Base editing scheme for restoring the activity of hPAH R413P Base editing system: BE3-xCas9-RY and sgRNA2 were used, and the nucleotide sequences were shown in SEQ ID NO. 10 and SEQ ID NO. 12, respectively; the remaining steps were the same as in Example 2.
[0030] Example 6 Base editing scheme for restoring the activity of hPAH R413P Base editing system: BE3-xCas9-RY and sgRNA2 were used, the nucleotide sequences were shown as SEQ ID NO. 10 and SEQ ID NO. 13, respectively; the remaining steps were the same as those in Example 2.
[0031] Example 7 Base editing scheme for restoring the activity of hPAH R413P Base editing system: BE3-xCas9-RY and sgRNA2 were used, the nucleotide sequences were shown as SEQ ID NO. 10 and SEQ ID NO. 14, respectively; the remaining steps were the same as those in Example 2.
[0032] Example 8 Base editing scheme for restoring the activity of hPAH R413P Base editing system: BE3-xCas9-RY and sgRNA2 were used, the nucleotide sequences were shown as SEQ ID NO. 10 and SEQ ID NO. 15, respectively; the remaining steps were the same as those in Example 2.
[0033] The results of the BE3 base editing scheme used in Examples 2-8 are shown in Table 1, respectively. In HEK293T cells, two editing schemes for PAH (R413P) site achieved editing efficiencies of 17% and 15%, respectively. Figures 2-8 Figure 2 and Figure 4 ).
[0034] Although the specific embodiments of the present application are described in detail in combination with the examples, it should not be understood as limiting the protection scope of the patent. Various modifications and variations made by those skilled in the art within the scope described in the claims are still within the protection scope of the patent.
Claims
1. A hPAH mutant characterized in that: The amino acid sequence of the hPAH mutant is shown in SEQ ID NO. 1 or SEQ ID NO.
2.
2. The coding gene of the hPAH mutant according to claim 1, characterized by: The nucleotide sequence of the coding gene of the hPAH mutant is shown in SEQ ID NO. 3 or SEQ ID NO.
4.
3. Use of the hPAH mutant of claim 1 in the preparation of a medicament for reducing the content of phenylalanine in blood or treating phenylketonuria.
4. Use of a mutant hPAH according to claim 3, characterized in that: The medicament contains the amino acid sequence of the hPAH mutant shown in SEQ ID NO. 1 or SEQ ID NO.
2.
5. Use of a mutant hPAH according to claim 3, characterized in that: The medicament contains the nucleotide sequence of the coding hPAH mutant shown in SEQ ID NO. 3 or SEQ ID NO.
4.
6. Use of a mutant hPAH according to claim 3, characterized in that: The medicament contains a base editor that edits the nucleotide sequence shown in SEQ ID NO. 6 into the nucleotide sequence shown in SEQ ID NO.
3.
7. Use of a mutant hPAH according to claim 6, characterized in that: The base editor is a plasmid containing BE3-NRCH and sgRNA1 sequences, the nucleotide sequence of BE3-NRCH is shown in SEQ ID NO. 7, and the nucleotide sequence of sgRNA1 is shown in SEQ ID NO.
8.
8. Use of a mutant hPAH according to claim 6, characterized in that: The base editor is a plasmid containing BE3-xCas9-NRCH and sgRNA1 sequences, the nucleotide sequence of BE3-xCas9-NRCH is shown in SEQ ID NO. 9, and the nucleotide sequence of sgRNA1 is shown in SEQ ID NO.
8.
9. Use of a mutant hPAH according to claim 6, characterized in that: The base editor is a plasmid containing BE3-xCas9-RY and sgRNA2 sequences, the nucleotide sequence of BE3-xCas9-RY is shown in SEQ ID NO. 10, and the nucleotide sequence of sgRNA2 is any one of SEQ ID NO. 11-SEQ ID NO. 15.