WO2014067038A1 - Target point, preparation and method for treating human adsl deficiency - Google Patents

Abstract

Disclosed are target points for treating human ADSL deficiency, the target points comprising PAICS gene, mRNA of PAICS gene or PAICS protein. Use of PAICS gene, mRNA of PAICS gene or PAICS protein for treating human ADSL deficiency is also disclosed.

Description

 Target, preparation and method for treating human ADSL deficiency

 Technical field

 The invention relates to a drug target of a gene defect disease, a preparation thereof and a treatment method.

 Background technique

 嘌呤Analysis is a ubiquitous and important biological metabolism of organisms, and its products AMP and GMP are not only DNA and The biosynthesis of RNA provides the raw material, but also many key coenzymes (NAD, NADP, FAD, and CoA), signaling molecules (such as cAMP), and important energy molecules in the body. ATP provides the purine base necessary for its synthesis. It can be seen that hydrazine anabolism is at the core of the entire metabolic network. Strontium synthesis includes de novo purine synthesis And salvage pathways. Two synthetic pathways.

Adenylosuccinate lyase deficiency ( ADSL ) is a metabolic disorder in which adenine de novo synthesis and purine nucleotide metabolism pathways produce deletions and disorders. The cause of the disease is mainly due to mutation or deletion of adenyl succinate lyase in the patient, resulting in excessive accumulation of the enzyme substrate SAICAR in the cell and no timely clearance [Jaeken J, Van den Berghe G. (1984). An infantile autistic syndrome characterized by the presence of succinylpurines in body fluids. Lancet 8411: 1058-1061.]. In 1984, Jaeken and Van den Berghe detected the accumulation of this metabolite for the first time in the body fluids of several patients with bradykinesia and autism. Patients with adenosine succinate lyase deficiency usually develop severe dysplasia, bradykinesia, gaze, epilepsy, autism, etc. [Spiegel, EK, Colman, RF, and Patterson, D. (2006) Adenylosuccinate lyase deficiency. Mol Genet Metab 89, 19-31. Clamadieu, C., Cottin, X., Rousselle, C., and Claris, O. (2008). Adenylosuccinate lyase deficiency: an unusual cause of neonatal seizure. Arch Pediatr 15, 135-138. Castro, M., Perez-Cerda, C., Merinero, B., Garcia, MJ, Bernar, J., Gil Nagel, A., Torres, J., Bermudez, M., Garavito , P., Marie, S., et al. (2002). Screening for adenylosuccinate lyase deficiency: clinical, biochemical and molecular findings in four patients. Neuropediatrics 33, 186-189. Jurecka, A., Zikanova, M., Tylki -Szymanska, A., Krijt, J., Bogdanska, A., Gradowska, W., Mullerova, K., Sykut-Cegielska, J., Kmoch, S., and Pronicka, E. (2008b). Clinical, biochemical And molecular findings in seven Polish patients with adenylosuccinate lyase deficiency. Mol Genet Metab 94, 435-442.].

 Among the metabolic pathways de novo synthesis of adenine, adenyl succinate lyase (hereinafter referred to as ADSL enzyme) is mainly involved in SAICAR. Pyrolysis catalyzes the formation of AICAR and S-AMP to form AMP [Spiegel, E.K., Colman, R.F., and Patterson, D. (2006). Adenylosuccinate lyase deficiency. Mol Genet Metab 89, 19-31. Clamadieu, C., Cottin, X., Rousselle, C., and Claris, O. (2008). Adenylosuccinate lyase deficiency: an unusual cause of neonatal seizure. Arch Pediatr 15, 135-138. Castro, M., Perez-Cerda, C., Merinero, B., Garcia, M.J., Bernar, J., Gil Nagel, A., Torres, J., Bermudez, M., Garavito, P., Marie, S., Et al. (2002). Screening for adenylosuccinate lyase deficiency: clinical, Biochemical and molecular findings in four patients. Neuropediatrics 33, 186-189.]. Adenosine succinate lyase deficiency in patients with a mutation or deletion of ADSL enzyme, resulting in harmful metabolites SAICAR Without timely clearance, there are usually very serious neurological and physiological symptoms such as epilepsy, brain development disorders, and exercise sluggishness [Ciardo, F., Salerno, C., and Curatolo, P. (2001). Neurologic aspects of adenylosuccinate lyase deficiency. J Child Neurol 16, 301-308. Gitiaux, C., Ceballos-Picot, I., Marie, S., Valayannopoulos, V., Rio, M., Verrieres, S., Benoist, J.F., Vincent, M.F., Desguerre, I., and Bahi-Buisson, N. (2009). Misleading behavioural phenotype With adenylosuccinate lyase deficiency. Eur J Hum Genet 17, 133-136. Mierzewska, H., Schmidt-Sidor, B., Jurkiewicz, E., Bogdanska, A., Kusmierska, K., and Stepien, T. (2009). Severe encephalopathy with brain atrophy and Hypomyelination due to adenylosuccinate lyase deficiency--MRI, clinical, Biochemical and neuropathological findings of Polish patients. Folia Neuropathol 47, 314-320.]. In the patient's cerebrospinal fluid and body fluids, the intermediate metabolites usually accumulate SAICAr and S-Ado ( SAICAr is SAICAR dephosphoric acid product, S-Ado is the product of S-AMP dephosphorylation) [Spiegel, E.K., Colman, R.F., and Patterson, D. (2006). Adenylosuccinate lyase deficiency. Mol Genet Metab 89, 19-31. Mierzewska, H., Schmidt-Sidor, B., Jurkiewicz, E., Bogdanska, A., Kusmierska, K., and Stepien, T. (2009). Severe encephalopathy with brain Atrophy and hypomyelination due to adenylosuccinate lyase deficiency--MRI, Clinical, biochemical and neuropathological findings of Polish patients. Folia Neuropathol 47, 314-320.]. Van den Berghe et al. found S-do and SAICAr in body fluids The ratio has a certain correlation with the severity of the patient's condition [Van den Bergh F, Vincent MF, Jaeken J, Van den Berghe G. (1993). Residual adenylosuccinase activities in fibroblasts of Adenylosuccinase-deficient children: parallel deficiency with adenylosuccinate And succinyl-AICAR in profoundly retarded patients and non-parallel deficiency In a mildly retarded girl, J. Inherit. Metab.Dis. 16 (2) 415-424.].

In the human body, the gene ADSL exists in the form of an essential gene, and its complete absence will lead to symptoms of congenital lethality. In all patients with ADSL enzyme deficiency, the activity of ADSL enzymes was not completely lost [Van den Bergh F, Vincent MF, Jaeken J, Van den Berghe G. (1993). Residual adenylosuccinase activities in fibroblasts of adenylosuccinase-deficient children: parallel Deficiency with adenylosuccinate and succinyl-AICAR in profoundly retarded patients and non-parallel deficiency in a mildly retarded girl, J. Inherit. Metab.Dis. 16 (2) 415-424. Van den Bergh F, Vincent MF, Jaeken J, Van Den Berghe G,. (1993). Functional studies in fibroblasts of adenylosuccinase-deficient children, J. Inherit. Metab. Dis. 16 (2) 425-434.]. At present, 38 different ADSL gene mutation sites have been discovered [Spiegel, EK, Colman, RF, and Patterson, D. (2006). Adenylosuccinate lyase deficiency. Mol Genet Metab 89, 19-31.] As the number of patients diagnosed increases, more mutation sites will be discovered. These mutation sites weaken the activity of ADSL enzymes in patients to varying degrees, leading to the accumulation of the harmful metabolite SAICAr in the body.

 Clinically, the diagnosis of patients with ADSL deficiency is mainly through the detection of cerebrospinal fluid (ceebrospinal fluid And the amount of metabolites in the body fluid, especially the accumulation of the harmful metabolite SAICAr. The earliest people used Bratton-Marshall Method of analysis [Laikind PK, Seegmiller JE, Gruber HE, (1986). Detection of 5'-phosphoribosyl-4- (N-succinylcarboxamide) -5-aminoimidazole in urine by use Of the Bratton-Marshall reaction: identification of patients deficient in Adenylosuccinate lyase activity, Anal. Biochem. 156. (1) 81-90.] This method mainly uses the analysis method of diazotized amine, but since the patient usually produces a false positive after taking the relevant medicine, the method is gradually abandoned. The most commonly used detection method is by high performance liquid chromatography (HPLC). ) to detect the levels of SAICAr and S-Ado in the cerebrospinal fluid and body fluids of patients [Marie S, Flipsen JW, Duran M, Poll-The BT, Beemer FA, Bosschaart AN, Vincent MF, Van den Berghe G, (2000a). Prenatal Diagnosis in adenylosuccinate lyase deficiency. Prenat Diagn 20, 33-36. Domkin VD, Lazebnik TA, Roudneff A, Smirnov MN, (1995). A new diagnostic technique for Adenylosuccinate lyase deficiency. J Inherit Metab Dis 18, 291-294.] . At present, there is no effective clinical treatment for ADSL deficiency treatment.

In the de novo synthesis pathway, 10 enzymatic reactions are required from PRPP to synthetic IMP [Hartman, SC & Buchanan, JM (1959). Biosynthesis of the purines. XXVI. The identification of the formyl donors of the transformylation reactions. J Biol. Chem. 234, 1812-1816. Lukens, LN & Buchanan, JM (1959). Biosynthesis of the purines. XXIV. The enzymatic synthesis of 5-amino-1-ribosyl-4-imidazolecarboxylic acid 5 ′ -phosphate from 5-amino-1-ribosylimidazole 5 '-phosphate and carbon dioxide. J. Biol. Chem. 234, 1799-1805.]. The study found that the growth of tumor cells is mainly dependent on (partial tumor cells are completely dependent on) the nucleotides produced by the de novo purine synthesis synthesis, while the normal cells of the human body are more inclined to use the nucleosides produced by the salvage pathway. Acid [Jackson, R. & Harkrader, R. (1981). Nucleosides and Cancer Treatment. Academic Press, Sydney.]. It has been found that many cancers, such as approximately 30% of acute T-lymphocytic leukemias lack an alternative pathway for the synthesis of adenosine, require complete de novo synthesis of synthetic 嘌呤 [Batova, A., Diccianni, MB, Omura-Minamisawa, M., Yu , J., Carrera, CJ, Bridgeman, LJ et al. (1999). Use of alanosine as a methylthioadenosine phosphorylase selective therapy for T-cell acute lymphoblastic leukemia in vitro. Cancer Res. 59, 1492-1497. Aminoimidazole succinyl carbamoyl nucleotide synthetase/aminoimidazole nucleotide carboxylase, PAICS (phosphoribosylaminoimidazole succinocarboxamide synthetase /phosphoribosylaminoimidazole carboxylase) is an important bifunctional enzyme in the de novo synthesis pathway, which has SAICAR synthesis. The functions of the enzymes (4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase, SAICARs) and 5-aminoimidazole ribonucleotide carboxylase (AIRc) catalyze the sixth and seventh steps of de novo anabolism. In 1990, the E.coli gene-deficient strain was used for the first time to clone the chicken-derived PAICS gene using functional complementation methods, and it was confirmed that SAICARs and AIRc are located at the N-terminus and C-terminus of PAICS, respectively [Chen, ZD, Dixon, JE & Zalkin , H. (1990). Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants. Proc. Natl Acad. Sci. USA, 87 , 3097-3101.]. In 2007, it was first reported that human PAICS existed as an octamer. The flower-like octamer structure consisted of an octameric AIRc flower and four dimerized SAICARs petals. In addition, the study also found that there are four independent channel systems in the octamer of PAICS. Each channel system is connected to two AIRc and two SAICARs active sites [Li SX., Tong YP., Xie XC., Wang QH., Zhou HN., Han Y, et al. (2007). Octameric structure of The human bifunctional enzyme PAICS in purine biosynthesis. J Mol Biol. 366(5): 1603-1614.]. In 2009, it was reported that PAICS is very important for embryonic development of vertebrates [Ng A., Uribe RA., Yieh L., Nuckels R., Gross JM. (2009). Zebrafish mutations in gart and paics identify crucial roles for de Novo purine synthesis in vertebrate pigmentation and ocular development. Development. 136(15):2601-2611.]. Until now, PAICS has not been found to have the effect of reducing SAICAR synthesis or accumulation.

 Summary of the invention

 It is an object of the present invention to provide a therapeutic target for human ADSL deficiency.

 Another object of the present invention is to provide a preparation for treating human ADSL deficiency.

 Another object of the present invention is to provide a method of treating human ADSL deficiency.

The invention establishes an ADSL defect rescue model in the nematode through RNA interference technology. The efficiency of RNA interference in nematodes can be well verified by RT-PCR. The study found that the RNA interference gene PAICS alone will increase the expression of the ADSL gene.

The phenotypic observation of nematode growth and development indicates that RNA interference with the nematode gene ADSL alone will lead to serious phenotypes of nematodes, such as delayed growth, sluggish movement, decreased number of eggs, and phenotypes after ADSL deficiency. The growth rate is only about 73.94% of the negative control; the RNA interference gene PAICS alone has little effect on the growth and development of the nematode, which is related to the gene PAICS is a non-essential gene; while the nematode can interfere with the gene PAICS , it can be very Good to save the serious phenotype of ADSL deficiency, the growth and development of nematodes tend to be normal, which can reach 92.4% of the negative control.

Analysis of adenine metabolites by LC-MS revealed that the loss of ADSL in the nematode will cause a large accumulation of the intermediate metabolite SAICAr in the nematode. According to the KEGG metabolic network, the nematode ADSL gene is deleted and the gland is deleted. The de novo synthesis of the intermediate SAICAR is not effectively eliminated. In the nematode, SAICAR is abundantly dephosphorylated in the form of SAICAr, which is compared with the clinical diagnosis of a large amount of SAICAr accumulated in the body fluid of patients with ADSL deficiency in the human body. Consistent. It is verified that SAICAr in the living body, if accumulated in a large amount and not effectively cleared, will have serious toxic effects on cells. The simultaneous RNA interference gene PAICS (synthetic enzyme encoding SAICAR) and gene ADSL in the nematode can well rescue the phenotype of growth and development disorders caused by ADSL, a necessary gene deletion. By simultaneously interfering with the expression of the PAICS gene, RNA can effectively reduce the synthesis of SAICAR, thereby saving the phenotype of ADSL deficiency. In the analysis of RNA double-interfering nematode metabolites, the results also confirmed the hypothesis that SAICAR in the nematode no longer accumulates (LC-MS does not detect SAICAR and SAICAr accumulation), and the growth and development of nematodes tends to be normal. The experiment analyzed the toxic effects of the accumulation of harmful metabolites on cells from the perspective of metabolites, and the feasibility of the ADSL defect rescue model established in the nematode.

Based on the results of the nematode model, it is foreseeable that by interfering with the expression of PAICS gene in human ADSL deficiency patients, it can reduce the accumulation of SAICAR in the body fluids of patients, reduce their toxicity to cells, achieve the purpose of treating human ADSL deficiency, or alleviate humans. ADSL deficiency symptoms.

 DRAWINGS

 Figure 1 shows the total RNA electrophoresis pattern of F3 generation nematodes;

Figure 2 shows the relative expression levels of gene PAICS and gene ADSL in different treatment groups;

 Figure 3 shows the relative growth rate of nematode RNA interference (0 days, 3 days, 6 days);

 Figure 4 shows the relative increase in length of nematode RNA interference.

 detailed description

 A target for treating human ADSL deficiency, the target being at least one of the following:

 PAICS gene;

 mRNA of PAICS gene ;

 And PAICS protein.

 A formulation for treating human ADSL deficiency, the preparation comprising at least one of the following:

 Reagents and/or oligonucleotides that can interfere with PAICS gene expression;

 An agent and/or an oligonucleotide that can interfere with the normal functioning of the PAICS gene mRNA;

 An agent that specifically inactivates the PAICS protein.

 Agents that can interfere with PAICS gene expression are selected from the group consisting of peptide hormones, enzymes, interferons, interleukins, colony-stimulating factors, and PAICS A nucleic acid sequence in which a gene hybridizes or a modified nucleic acid sequence, a recombinant protein, or the like.

 Oligonucleotides that can interfere with PAICS gene expression or the normal functioning of PAICS gene mRNA contain at least humans The nucleotide sequence of the hybridization of the PAICS gene cDNA (GenBank ID 30582814).

 As a general knowledge in the art, to ensure the stability and specificity of hybridization, and PAICS gene or its cDNA, mRNA The length of the hybridized nucleic acid sequence or modified nucleic acid sequence should be no less than 5 bases, preferably no less than 8 bases, 10 bases, 20 bases, 30 Bases. Considering the difficulty of synthesis and the ease of use, the bases in the sequence should be no longer than 2000 bases, 1500 bases, 1000 bases, 800 bases, 500 bases, 400 bases, 300 bases, 200 bases, 100 bases.

 Can interfere with PAICS gene mRNA The normally functioning agent is selected from the group consisting of a polypeptide hormone, an enzyme, an interferon, an interleukin, a colony stimulating factor, a nucleic acid sequence which can hybridize with the PAICS gene, or a modified nucleic acid sequence, a recombinant protein.

 An agent that specifically inactivates the PAICS protein is selected from the group consisting of a polypeptide hormone, an enzyme, an interferon, an interleukin, a colony stimulating factor, a recombinant protein, PAICS protein antibody.

 A method of treating human ADSL deficiency comprising administering to a patient a formulation comprising at least one of the following:

 Reagents and/or oligonucleotides that can interfere with PAICS gene expression;

 An agent and/or an oligonucleotide that can interfere with the normal functioning of the PAICS gene mRNA;

 An agent that specifically inactivates the PAICS protein.

 RNA interference in C. elegans PAICS gene can rescue the deleted phenotype of ADSL gene

 In the nematode, many genes play an important role in embryonic development and growth of the worm. About 60% of the genes in the nematode The gene is homologous to human genes, and therefore, nematodes are also widely used in the study of human genetic metabolic diseases [Kuwabara, P.E., and O'Neil, N. (2001). The Use of functional genomics in C. elegans for studying human development and Journal of Inherited Metabolic Disease 24, 127-138.] . At present, people mainly use nematodes to study apoptosis, neurodevelopment, behavioral biology, etc., but the use of nematodes as model organisms to study human ADSL deficiency has not been reported.

In C. elegans, the gene ADSL (gene number R06C7.5a) mainly encodes adenyl succinate lyase, which is mainly involved in the adenosine de novo cleavage of SAICAR into AICAR and S-Amp to produce AMP; gene PIACS (gene No. B0286.3) is the upstream gene of this reaction, mainly encoding SAICAR synthetase, which is mainly involved in the synthesis of the metabolic intermediate SAICAR in adenine de novo synthesis, and is a non essential gene. The gene ADSL is a necessary gene in the nematode. When it is completely deleted, it will lead to nematode development, larval death and other lethal phenotypes [Sonnichsen, B., Koski, LB, Walsh, A., Marschall, P., Neumann, B., Brehm, M., Alleaume, AM, Artelt, J., Bettencourt, P., Cassin, E., et al. (2005). Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434 , 462-469. Julian Ceron, Jean-François Rual, Abha Chandra, Denis Dupuy, Marc Vidal and Sander van den Heuvel. (2007). Large-scale RNAi screens identify novel genes that interact with the C. elegans retinoblastoma pathway as well As splicing-related components with synMuv B activity.BMC Developmental Biology 7-30.]. The nematode gene ADSL ( R06C7.5a ) is homologous to the human gene ADSL . As a multicellular organism, nematodes can be used to study ADSL deficiency, and study the phenotype of nematode growth and development after genetic ADSL deficiency. influences.

 This study used a feeding RNA interference method for the nematode gene ADSL and the gene PAICS Silent expression is used to study changes in the nematode phenotype. The efficiency of RNA interference can be well verified by RT-PCR to ensure that the gene of interest is interfered with by RNA.

 First, the cultivation and synchronization of nematodes

 (1) Cultivation of nematodes

 Preparation of NGM-OP50 plate: Pick OP50 monoclonal from LB plate to 10ml LB liquid medium Incubate overnight at 37 °C to log phase. Collect 1 ml of the bacterial solution in a 1.5 ml centrifuge tube, centrifuge, and wash twice with M9 Buffer, then resuspend with an appropriate amount of M9 Buffer (approximately After 150 ul), the bacteria solution was pipetted into the center of the NGM plate, and the NGM-OP50 plate was placed in a 37 °C incubator overnight to be transferred to the terminal insect.

 Transfer culture of nematodes: from a piece of NGM-OP50 containing a large number of nematodes A small amount of agar medium was cut out from the plate with a sterile blade, transferred to a new NGM-OP50 plate, and cultured in a 16 °C incubator.

 (2) Synchronization of nematodes

 Cut the agar medium containing the adult nematodes from the NGM-OP50 plate with a sterile blade and transfer to a new one. On the NGM-OP50 plate, under the inverted microscope, the excess nematode was killed with a red-burning sterile inoculating silk, leaving only a hermaphroditic nematode as the F1 female parent to produce a large amount of F2. For the larvae, the plate was placed in a 16 °C incubator, and a large number of L1-L2 larvae were produced in about 4-5 days. The F1 progeny was killed with a red-sterilized sterile inoculum, and the appropriate amount of M9 was used. Buffer Wash the larvae on the plate and centrifuge at 0.4g for 2mins to wash away the remaining E. coli OP50. Synchronize the nematodes to 4-5 nematodes per plate to RNAi On the tablet.

 Second, the nematode RNAi feeding method

 (1) Preparation of RNAi plates

 1. Pick HT115-L4440 from the LB-tetra+ plate. HT115-L4440-1100, HT115 monoclonal to 2ml LB-Cb+ liquid medium cultured overnight at 37 °C shaker.

2, the next day, the above bacterial solution was transferred 1:100 to 20ml fresh LB-Cb+ liquid medium and cultured at 37 °C to the logarithmic phase (D ₆₀₀ ≈ 0.5 ), adding 200 μl of 0.1M IPTG The final concentration is 1 mM.

 3. Transfer the above culture flask to a 16 °C 120 rpm shaker overnight.

 On the third day, the appeal liquid is mixed in the following proportions:

Negative control	500 μ l HT115-L4440-VAC	500 μ l HT115-L4440-VAC
RNAi ADSL /vac	500 μ l HT115-L4440-VAC	500 μ l HT115-L4440-1100
RANi PAICS /vac	500 μ l HT115-L4440-VAC	500 μ l HT115-L4440-1600
RANi ADSL / PAICS	500 μ l HT115-L4440-1100	500 μ l HT115-L4440-1600

 4. Centrifuge the above 4 kinds of bacteria solution at 12000 rpm for 2 mins, remove the supernatant, and use 1 mL of M9. Wash the Buffer twice, resuspend the bacteria solution with an appropriate amount of M9 Buffer, and drop the bacteria into the center of the NGM-IPTG-Cb+ plate. Place the plate at 16 Incubate overnight in a °C incubator.

 (2) Transfer the synchronized F2 generation nematode to the RNAi plate

1.  1) Transfer the synchronized F2 larvae to the RNAi plate according to 4-5, and place the RNAi plate at 16 After 4-5 days of incubation in a constant temperature incubator, the F2 generation has developed into a mature individual under an inverted microscope and begins to produce large numbers of eggs and larvae (F3 generation);

1.  2) Transfer F3 larvae (approximately 10-20) produced on RNAi plates to the corresponding RNAi On the plate, it facilitates the observation and analysis of the next developmental phenotype;

1.  3) Continue to culture the remaining F3 generation on the F2 generation plate. After 5-6 days, rinse off the plate with M9 Buffer and use M9. After washing twice, the Buffer is divided into two equal parts, one for extracting RNA for RT-PCR and one for later metabolite extraction. After freezing the liquid with liquid nitrogen, store it in -80 °C refrigerator.

 (3) Phenotypic observation of growth and development of F3 larvae

 Observations of developmental growth phenotypes of F3 larvae grown on new RNAi plates. In 3 days, 6th Days (already adult) photographed the worm with an inverted microscope, observed the growth phenotype, and used ImageJ software to measure the size of the worm to obtain data on the growth and development phenotype of the nematode after RNA silencing.

 Third, RT-PCR verification RNA interference efficiency

 (1) Nematode RNA extraction (TRIzol reagent description, Invitrogen)

1.  1) Rinse the nematodes from the plate with M9 Buffer, collect the worms with a 1.5ml centrifuge tube, and centrifuge at 400g. 2mins, washed twice with M9 Buffer to remove residual E. coli;

1.  2) Quickly freeze the collected nematode worms with liquid nitrogen and add 1mL of TRIzol Reagent Adding liquid nitrogen to grind the worm with a grinding rod to a powder;

1.  3) After standing at room temperature for 5 mins, add 0.2 ml of chloroform, cover the tube cap tightly, mix upside down for about 16 s, and let stand at room temperature. After 2-3 mins , centrifuge at 15 ° C for 1 min at 4 ° C for 15 mins ;

1.  4) Aspirate the clear aqueous phase (approx. 0.5 ml), add to a new 1.5 ml centrifuge tube, and add 0.5 ml. The isoamyl alcohol was mixed upside down and allowed to stand at room temperature for 10 mins;

1.  5) Centrifuge the above tube at 12000g at 4 °C for 10mins, at which point a white flocculent precipitate can be seen;

1.  6) Carefully pour off the supernatant and add 1 ml of 75% absolute ethanol (diluted with DEPC water);

1.  7) Centrifuge at 12000g for 4mins at 4 °C for 5mins, carefully pour off the supernatant, and let the RNA precipitate dry at room temperature for 10mins;

1.  8) Add a certain amount of RNA free water to dissolve the RNA precipitate, and test the RNA extraction results with formaldehyde-denatured gel.

 ( 2 ) RNA reverse transcription into cDNA ( PrimerScript RT reagent Kit , Takara

 The reverse transcription reaction system is as follows:

Reagent name	Volume (μl)
5 ×PrimerScript Nuffer	4 μl
PrimerScript RT Enzyme Kit I	1 μl
Oligo dT Primer	1 μl
Random 6 mers	1 μl
Total RNA	≤500ng
RNase Free H 2 O	Up to 20 μl

 The reaction conditions for reverse transcription were: 37 ° C, 45 min; 85 ° C, 5 s.

 (3) RT-PCR reaction system (SYBR Premix Ex Taq II , Takara )

 RT-PCR Add the appropriate substance according to the SYBR Premix Ex Taq II instructions for reaction:

Reagent name	volume
SYBR Premix	5 μl
Sence Primer	0.4 μl
Anti-sense primer	0.4 μl
cDNA	1 μl
H 2 O	Up to 10 μl

 The RT-PCR primer sequences are shown in the following table:

Primer name	Primer sequence	SEQ ID NO :
RT-PAICS-S		ACAGTCTTATCGGGACCTCAAA	1
RT-PAICS-A		AGAGCCCATCAACACTACAACC	2
RT-ADSL-S		CACCTTGGTGCTACTTCTTGCT	3
RT-ADSL-A		GGGTAGACTGGCTCGTTCCTT	4
RT-actin-S	ACCGAGCGTGGTTACTCTTTCA	5
RT-actin-A		TCCGACGGTGATGACTTGTCC	6

 Three biological replicates were performed for each sample.

 RT-PCR reaction program setup (two-step method):

 Pre-denaturation: 95 °C 30s, the number of cycles is 1.

 PCR reaction: 95 °C 10s, 60 °C 20s, cycle number 40.

 (4) RT-PCR data analysis

In this experiment, the β-actin gene of nematodes was used as an internal reference to measure the relative expression of genes ADSL and PAICS. The data were analyzed using the commonly used Ct value method 2 ^{-△△ CT} (calculated as follows), and the calculated result is relative to The treatment group, the change in the expression of the target gene in the experimental group relative to the internal reference.

△△ CT= (Ct _{target gene} - Ct _{internal reference} ) _{experimental group} - (Ct _{target gene} - Ct _{internal reference} ) _untreated

Fold change = 2 ^{-△△ CT}

 (5) Total RNA electrophoresis pattern of F3 generation nematodes

 The total RNA electrophoresis pattern of F3 line nematodes is shown in Figure 1. According to Figure 1, we can know the total RNA of nematodes. The extraction quality is good, the band is clear, the 28S brightness is about twice that of 18S, and there is no degradation tailing, which can be used in the next RT-PCR experiment.

 (6) RT-PCR analysis of the efficiency of RNA interference genes ADSL and PAICS in nematodes

 Table 1 RT-PCR test gene ADSL and gene PAICS relative expression

	Gene relative Expression level
Sample name	Gene PAICS	Gene ADSL
Negative Control	100.00%	100.00%
RNAi PAICS	37.89%	105.95%
RNAi ADSL	132.87%	35.60%
RNAi PAICS	41.75%	38.87%

RT-PCR technology can be used to measure the relative expression of a target gene, thus verifying the efficiency of RNA interference in nematodes. In this experiment, the housekeeping gene β-actin was used as an internal reference gene to compare the relative expression levels of the target genes ADSL and PAICS in different treated samples. The relative expression levels of gene PAICS and gene ADSL in different treatment groups are shown in Figure 2. In RT-PCR, the amount of gene expression is calculated by the Cp value, which is proportional to the initial number of templates. At a given cDNA concentration, the initial concentration of the template for a gene is linear with the Cp value.

From Table 1, it can be found that the expression level of the nematode gene PAICS of the single RNA interference gene PAICS is only 37.89% of the negative control, while the expression level of the ADSL gene is basically unchanged, that is, the individual interfering gene PAICS does not affect the gene ADSL expression. The change. While nematode gene expression by RNA interference separate ADSL ADSL only 35.6% of the negative control, the expression of gene silencing of approximately 64.4%, while the amount of gene expression is up-regulated PAICS. In the nematodes of the RNA interference genes ADSL and PAICS , the expression levels of gene ADSL and gene PAICS were only 38.87% and 41.75% of the negative control, respectively.

 The results in Table 1 and Figure 2 indicate that the RNA interference gene ADSL and PAICS in nematodes The efficiency is relatively high, in line with the expected experimental results, the next step of observation of nematode growth and development phenotype and metabolite analysis experiments.

 (7) Phenotypic observation of RNA interference of different genes in nematodes

 In the previous step, the feasibility of RNA interference in a specific gene expression in nematodes was well verified by RT-PCR. By observing The growth and development of F3 larvae can better analyze the corresponding phenotype of elegans after RNA interference (phenotype) )Variety. Thus to study the effect of a gene on the growth and development of nematodes and their role in the cell.

 In this experiment, there was no significant phenotypic change in F2 generation larvae grown on RNA interference plates by observing F3 The phenotype of the nematode can more accurately reflect the changes in the growth and development of the nematode after specific RNA interference. Due to the incubator in a constant temperature incubator at 16 °C, nematodes develop into adult bodies in about 6 days. (adulthood), phenotypic observation of F3 larvae on day 3 and day 6 respectively. Photographing nematode worms under an upright microscope (number of nematodes in each experimental group ≥ 10 ), using the software Image J to measure the length of the worms in order to obtain raw data on the growth and development of F3 larvae.

The relative growth rate (0 days, 3 days, 6 days) after nematode RNA interference is shown in Figure 3. The results show that the nematode growth and development of the RNA interference gene ADSL alone is severely delayed, while the nematode that interferes with the gene PAICS alone is growing. During development, it is relatively normal, and the growth rate of the negative control is relatively close. In the nematodes of the RNA interference gene ADSL and the gene PAICS , the growth and development of the F3 larvae tend to be normal, which is basically consistent with the growth rate of the negative control.

(8) Interfering gene PAICS in nematode can rescue the lethal phenotype of ADSL deficiency

The relative increase in length after nematode RNA interference is shown in Figure 4. It can be seen that between day 6 and day 3, the length of relative growth in the different experimental groups changed significantly. The nematode growth of the RNA interference ADSL gene alone is significantly slower than that of the negative control, while the nematode interfering with the gene PAICS can well alleviate the growth and development delay caused by ADSL defects.

 Table 2 Relative growth rate of F3 line nematode RNA interference

Sample	Growth rate
Negative Control	100%
RNAi ADSL	73.94%
RNAi PAICS	94.51%
RNAi ADSL/PAICS	92.40%

From Table 2, the experimental results can be analyzed more intuitively. In the nematode, the PAICS gene is a non-essential gene, and its deletion or mutation does not cause significant phenotypic changes. The F3 line nematode, which interferes with the gene PAICS alone, is relatively normal in growth and development, and has no phenotype such as developmental delay and slow movement (94.51 which can reach the negative control).

The F3 line nematode, which interferes with the ADSL gene alone, grows slowly (only 73.94% of the negative control), which is slow-moving. This is related to the previously reported ADSL as a necessary gene, and its deletion or mutation will lead to serious growth and development of nematodes. The result of the obstacle is consistent [Sonnichsen, B., Koski, LB, Walsh, A., Marschall, P., Neumann, B., Brehm, M., Alleaume, AM, Artelt, J., Bettencourt, P., Cassin , E., et al. (2005). Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434, 462-469. Julian Ceron, Jean-François Rual, Abha Chandra, Denis Dupuy, Marc Vidal and Sander van den Heuvel. (2007). Large-scale RNAi screens identify novel genes that interact with the C. elegans retinoblastoma pathway as well as splicing-related components with synMuv B activity. BMC Developmental Biology 7-30.] ; and interfere with the ADSL gene F2 alone The number of eggs laid is much less than the negative control (this data is not shown).

The simultaneous RNA interference gene PAICS in C. elegans can well rescue the phenotype of gene ADSL deficiency, and the growth and development of F3 generation tends to be normal. The growth rate of C. elegans is closer to the negative control (up to 92.40% of the negative control).

Claims (7)

1.  A target for treating human ADSL deficiency, the target being at least one of the following:

   PAICS gene;

   mRNA of the PAICS gene;

   And PAICS protein.
2. A formulation for treating human ADSL deficiency, the preparation comprising at least one of the following:

   An agent and/or an oligonucleotide that can interfere with PAICS gene expression;

   An agent and/or an oligonucleotide that can interfere with the normal functioning of the PAICS gene mRNA;

   An agent that can specifically inactivate a PAICS protein.
3. The preparation according to claim 2, characterized in that the agent which can interfere with the expression of the PAICS gene is selected from the group consisting of a polypeptide hormone, an enzyme, an interferon, an interleukin, a colony stimulating factor, a nucleic acid sequence which can hybridize with the PAICS gene or a modified nucleic acid sequence. ,Recombinant protein.
4. The preparation according to claim 2, wherein the oligonucleotide which can interfere with the PAICS gene expression or the normal function of the PAICS gene mRNA contains at least a nucleotide sequence which can hybridize to the cDNA sequence of the human PAICS gene.
5. The preparation according to claim 2, wherein the agent capable of interfering with the normal function of the PAICS gene mRNA is selected from the group consisting of a polypeptide hormone, an enzyme, an interferon, an interleukin, a colony stimulating factor, a nucleic acid sequence which can hybridize with the PAICS gene, or a modification. Nucleic acid sequence, recombinant protein.
6. The preparation according to claim 2, wherein the agent which specifically inactivates the PAICS protein is selected from the group consisting of a polypeptide hormone, an enzyme, an interferon, an interleukin, a colony stimulating factor, a recombinant protein, and a PAICS protein antibody.
7. A method of treating human ADSL deficiency comprising administering a formulation to a patient, the formulation comprising at least one of the following:

   An agent and/or an oligonucleotide that can interfere with PAICS gene expression;

   An agent and/or an oligonucleotide that can interfere with the normal functioning of the PAICS gene mRNA;

   An agent that can specifically inactivate a PAICS protein.

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