WO2002020782A1 - Oligonucleotides sens et antisens de v-erbb et ses applications - Google Patents

Oligonucleotides sens et antisens de v-erbb et ses applications Download PDF

Info

Publication number
WO2002020782A1
WO2002020782A1 PCT/CN2001/000888 CN0100888W WO0220782A1 WO 2002020782 A1 WO2002020782 A1 WO 2002020782A1 CN 0100888 W CN0100888 W CN 0100888W WO 0220782 A1 WO0220782 A1 WO 0220782A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
oligonucleotide
erbb
seq
sequence
Prior art date
Application number
PCT/CN2001/000888
Other languages
English (en)
Chinese (zh)
Inventor
Baozhang Feng
Original Assignee
Baozhang Feng
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baozhang Feng filed Critical Baozhang Feng
Publication of WO2002020782A1 publication Critical patent/WO2002020782A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • V-erbB oligonucleotides Positive and negative genes V-erbB oligonucleotides and applications thereof
  • the present invention relates to a pair of PCR archers specific to the V-erbB gene, a V-erbB sequence defined by the pair of primers, a transcribed mRNA and a translated polypeptide of the V-erbB sequence, comprising the V-erbB sequence and Polypeptide vaccines, plus positive and negative gene oligonucleotides designed for this V-erbB sequence.
  • the present invention also relates to a diagnostic kit containing the primer or positive gene oligonucleotide, a pharmaceutical composition containing the antigenic oligonucleotide, and the above-mentioned substances in the diagnosis and treatment of myelodysplastic syndrome, leukemia and Many other tumors and their early application.
  • Gene therapy is a new disease treatment technology developed in the past ten years. It achieves the purpose of treating diseases by correcting the abnormality of the pathogenic gene or replacing it with a new gene.
  • people have high expectations for tumor gene therapy.
  • the method of introducing a normal gene into a body using a viral vector in the prior art although having some curative effects, faces a series of difficulties.
  • the use of antisense oligonucleotide phosphorothioate as a gene therapy avoids a series of problems caused by the use of viral vectors.
  • Phosphorothioate and some of the toxicity caused by the degradation of oligonucleotides are acceptable and surmountable by the human body and are therefore very safe.
  • antisense oligonucleotides are designed to target the mRNA of cancer cell target genes, and cancer cells are constantly generating m A from the nucleus, it is unlikely to have a solid curative effect.
  • Antigene oligonucleotides developed on this basis are designed for specific mutation positions of oncogenes, so that after entering the cell, they can form a solid triple-stranded DNA structure in the nucleus and inhibit the expression and amplification of the gene This brings new hope for tumor gene therapy.
  • MDS Myelodysplastic syndrome
  • white Preleukemia is a group of malignant bone marrow hematopoietic stem cell diseases. Most patients have excessive bone marrow hyperplasia but have difficulty in differentiation and maturation. As a result, their peripheral blood has reduced formation, and anemia, bleeding, decreased blood cells, dizziness, and fatigue are present. symptom. Most patients develop acute leukemia months or years after diagnosis, and no effective treatment is available.
  • V-erbB and V-erbA are chicken oncogenes of Avian Erythroblastosis virus.
  • the former is homologous to the epidermal growth factor receptor (EGFR) gene and has the function of transforming erythroid progenitor cells; the latter has the effect of blocking the differentiation and maturation of the aforementioned erythroid progenitor cells.
  • EGFR epidermal growth factor receptor
  • Myelodysplastic syndrome has five different subtypes, of which Refractory Anemia 5 RA is its basic type. It can be transformed into refractory anaemia (ie, RAEB) and transformed RAEB (ie, RAEBT) and acute leukemia (Acute Leukemia, AL). RAEBT patients with bone marrow in the primary and early erythrocytes can also be re-diagnosed as red leukemia, which is the same as the above chicken red leukemia. RA patients can also be diagnosed with red blood disease when the bone marrow has a single hypertrophy of the red system. And red blood disease can develop into red leukemia.
  • RAEB refractory anaemia
  • RAEBT transformed RAEB
  • AL acute leukemia
  • RAEBT patients with bone marrow in the primary and early erythrocytes can also be re-diagnosed as red leukemia, which is the same as the above chicken red leukemia.
  • RA patients can also be diagnosed with red
  • FS Fragile Site
  • V-erbB Southern blot hybridization with V-erbA proved that MDS is associated with C-erbB rearrangement / amplification and C-erbA deletion / inactivation.
  • the hybridization results have diagnostic and pathological significance.
  • the inventors applied the V-erbB PCR and positive gene oligonucleotide in situ hybridization method to determine the C-erbB rearrangement position. Surprisingly, both the PCR product and the in situ hybridization result were found. Diagnostic and pathological significance. On this basis, DNA sequencing was performed on the above PCR products. It was processed by Gene Runner software.
  • an object of the present invention is to provide a pair of PCR primers specific to the V-erbB gene and a V-erbB sequence defined by the primer, the mRNA transcribed from the sequence and the translated polypeptide, comprising the V-erbB sequence and Polypeptide vaccine.
  • the V-erbB sequence can be used as a target sequence for designing drugs for treating MDS, leukemia, and many other tumors and their early stages.
  • a second object of the present invention is to provide anti-gene oligonucleotides and positive-gene oligonucleotides directed to the V-erbB sequence defined by the above primers, and a pharmaceutical composition comprising these oligonucleotides and the above-mentioned primers of the present invention or Diagnostic kit.
  • a third object of the present invention is the application of the above-mentioned substances of the present invention in the diagnosis and treatment of myelodysplastic syndromes, leukemias, and various other tumors and their early stages.
  • a pair of PCR primers specific to the V-erbB gene is provided, which can be used for highly sensitive diagnosis related to C_erbB rearrangement / amplification and C-erbA deletion / inactivation
  • MDS myelodysplastic syndrome
  • the nucleotide sequence of the primer pair is as follows: 5, ATG AAA TGT GCC CAT TTT ATA 3 '(SEQ ID NO. 1) and 5, CAA AAC TTT GAC CTT TTT 3' (SEQ ID NO.
  • the present invention also relates to the V-erbB sequence defined by the above primers
  • the sequence can be used as a target sequence for designing drugs for treating MDS, leukemia and other various tumors and their early stages, and can be used for designing any oligonucleotide primers other than the above PCR primer pairs.
  • the sequence is as follows:
  • V-erbB sequence or its polypeptide is used to prevent various cancers associated with C-erbB rearrangement / amplification and C-erbA deletion / inactivation and their early stages, especially myelodysplastic syndrome ( MDS), DNA vaccines or polypeptide vaccines such as leukemia are also part of the present invention.
  • MDS myelodysplastic syndrome
  • two anti-gene oligonucleotides are provided for the V-erbB sequence (SEQ ID N0.3) defined by the primers of the present invention, and the sequences of the anti-gene oligonucleotides are as follows:
  • the antigenic oligonucleotide of the present invention can be used to treat and prevent various cancers associated with C-erbB rearrangement / amplification and C-erbA deletion / inactivation and their early stages, especially abnormal bone marrow hyperplasia, through anti-gene technology. Syndrome (MDS), leukemia, etc.
  • MDS anti-gene technology. Syndrome
  • Antigenic oligo Nucleotides can also be used in combination with antigenic oligonucleotides of genes related to cancer pathogenesis such as thymidine synthase (TS) and thymidine kinase (TK) genes to treat and prevent the above diseases.
  • TS thymidine synthase
  • TK thymidine kinase
  • the present invention also provides a positive gene oligonucleotide contained in the V-erbB sequence (SEQ ID N0.3) of the first aspect of the present invention, and the positive gene oligonucleotide can be used for diagnosis and C-erbB recombination. Exclusion / amplification and C-erbA deletion / inactivation are related to a variety of cancers and their early stages, especially myelodysplastic syndrome (MDS), leukemia, etc.
  • MDS myelodysplastic syndrome
  • the two preferred positive gene oligonucleotide sequences are:
  • CAG GCC CAC CTG AGA ATT 3 '(SEQ ID N0.7).
  • the positive gene oligonucleotides for diagnostic purposes of the present invention also include any oligonucleus of 10-30 bp in length derived from SEQ ID N0.3 Glycylic acid.
  • the present invention also relates to a diagnostic kit containing these positive gene oligonucleotides for diagnosing various cancers associated with C-erbB rearrangement / amplification and C-erbA deletion / inactivation and their early stages, particularly bone marrow Hyperplasia syndrome (MDS), leukemia, etc.
  • MDS bone marrow Hyperplasia syndrome
  • the anti-gene V-erbB oligonucleotide of the present invention is designed and synthesized toward the rearrangement site of C-erbB, and because it is complementary to the base of the positive gene C-erbB rearrangement site, it can specifically bind and inhibit the positive Abnormal expression and amplification of the gene C-erbB, or binding to the gene C-erbB mRNA to block its translation into oncogenic proteins. Therefore, using anti-gene technology known to those skilled in the art, the anti-gene oligonucleotide of the present invention can be used to treat and prevent C-erbB rearrangement / amplification and C-erbA deletion / inactivation-related MDS, leukemia, and many others.
  • the anti-gene oligonucleotide SEQ ID No. 4 has an important therapeutic effect, and SEQ ID N0.5 has an adjuvant therapeutic effect (see the following examples). This It should be understood by those skilled in the art that anti-gene oligonucleotides designed for other sites of the V-erbB sequence of the PCR primer interval described above will have the same auxiliary effect.
  • the antigenic oligonucleotide of the present invention is modified with phosphorothioate, and the modified product has the effect of inhibiting nuclease and bringing it to a target position in the cell.
  • the increase in the proportion of primordial and promyelocytic cells in the bone marrow is not only the conversion of MDS to leukemia, but also the central manifestation of the acute transformation of chronic myelogenous leukemia, as well as the central manifestation of the etiology and recurrence of leukemia. It can be seen from the following examples that the use of the anti-gene V-erbB oligonucleotide of the present invention can quickly and effectively reduce the primordial and promyelocytic cells in the bone marrow of MDS model rats to normal levels (5%). Preventing malignant tumor diseases such as MDS provides a way.
  • the V - er B PCR primer interval sequence and the positive gene oligonucleotide are labeled, for example, they can be used as diagnostic probes and in situ hybridization with bone marrow cell samples to Determine whether an individual has MDS and precancerous lesions or malignancies.
  • the ortho-oligonucleotide can be labeled by any method known in the art, such as biotin, digoxin, and the like.
  • the C-erbB gene abnormality of the patient's bone marrow or precancerous tissue can also be detected by the V-erbB PCR method. Whether or not, to diagnose malignant tumors such as MDS.
  • the use of the antigenic oligonucleotide of the present invention for gene therapy and prevention has many advantages.
  • the biggest advantage is that it does not introduce a series of adverse consequences that are difficult to evaluate due to the introduction of vector (such as retrovirus or adenovirus) DNA or RNA into the cell.
  • vector such as retrovirus or adenovirus
  • the anti-gene oligonucleotide of the present invention is characterized by the in situ repair of oncogene rearrangement positions, rather than the introduction of foreign genes, so it is different from general antisense gene therapy.
  • the latter is designed to synthesize anti-oncogene mRNA Sense oligonucleotide, and because cancer cells are constantly producing rnRNA from the nucleus, the therapeutic effect of limited antisense oligonucleotides is limited and transient.
  • the V-erbB PCR primer interval sequence of the present invention and the positive gene oligonucleotide can be used, for example, as diagnostic probes and in situ hybridization with bone marrow cell samples.
  • the ortho-oligonucleotide may be labeled by any method known in the art, such as biotin, digoxin, and the like.
  • the V-erbB gene-specific PCR primer of the present invention or other primers designed according to the sequence of SEQ ID NO. 3 of the present invention can also be used to detect C-erbB gene abnormalities in patients' bone marrow or precancerous tissues by the V-erbB PCR method. Whether or not, to diagnose malignant tumors such as MDS.
  • Figure 1 shows the effect of rat MDS after treatment with the anti-gene V-erbB oligonucleotide of the present invention in Example 2.
  • Fig. 2 shows that after treatment with the anti-gene V-erbB oligonucleotide of the present invention in Example 2, the rat MDS hemogram returned to normal.
  • Figure 3 shows the increase in rat MDS weight during treatment with the anti-gene V-erbB oligonucleotide of the present invention in Example 2.
  • oligonucleotides were automatically synthesized on a DNA synthesizer (model 394, source PE company)-
  • MDS occurred in 9/14 of rats injected 3 times with DMBA. Symptoms occurred 2 months after the injection, and there was an MDS period of 3-4 months. Wistar rats injected 3 times with DMBA were selected as gene therapy models.
  • the two anti-gene V-erbB oligonucleotides synthesized in Example 1 were modified with phosphorothioate. Dissolve in normal saline at a concentration of 10.0 OD / ml before use. MDS rats are injected once a day by the tail vein for 3 consecutive days. The dose was 0.56 mg / kg of body weight.
  • WR1 primary cytoplasmic RA
  • WR2 primary cytoplasmic RA
  • the experimental mice were injected with V-erbB antigene oligonucleotide through the tail vein once a day for 3 consecutive days as a course of treatment. Then observe.
  • the effect of different administration times and schedules of the anti-gene V-erbB oligonucleotide of the present invention on the bone marrow of rat MDS is shown in Table 1. It can be seen from Table 1 that the primordial and promyelocytic cells of the 16 rats treated were significantly decreased, and the bone marrow of 15 rats returned to normal. And the two oligonucleotides of the present invention have a curative effect when administered alone or in combination. Control mice (Table 2) showed an increase in the percentage of bone marrow primitive and promyelocytic cells, suggesting that the disease was progressing. Subsequently, 8 MDS-RAEB rats were orally treated with the same dose and 1-3 courses of treatment, and the same results as above were obtained.
  • the inventors also used local medication to treat 27 patients with esophageal cancer and 2 patients with early cervical cancer, with an effective rate of 74.1%, which reversed the severe or moderate hyperplasia of the esophagus to normal.
  • V-erbB anti-gene oligonucleotide of the present invention has good curative effect on MDS and curative effect on other tumors.
  • the application of the V-erbB anti-gene oligonucleotide of the present invention in treating early lesions may have similar curative effects.
  • V-erbB positive gene oligonucleotide SEQ ID N0.6 corresponding to the antigenic oligonucleotide SEQ ID NO: 4 was synthesized as described in Example 1, and the oligonucleotide was labeled with digoxin (DIG), Using the labeled oligonucleotide as a probe, the amplification of the C-erbB gene in 33 cases of MDS and 19 other hematological diseases and 3 suspected MDS was detected by in situ hybridization. The results showed that all 33 cases of MDS In situ hybridization was positive, 1 of 19 other hematological diseases was positive, and 3 suspected MDS were positive.
  • DIG digoxin
  • Bone marrow cell DNA was extracted as a template according to a conventional method, and the specific PCR primer pairs 5, ATG AAA TGT GCC CAT TTT ATA (SEQ ID NO. 1) and 5, CAA AAC TTT GAC CTT TTT (SEQ ID NO. 2) of the present invention were used as a template.
  • the primers were used for PCR amplification.
  • the amplification conditions were 94 ⁇ 2 minutes for template denaturation, and then 35 cycles. Each cycle was 94 ° C for 0.5 minutes, annealing conditions were 56 ° C for 1 minute, and extension conditions were 72. ° C for 1.5 minutes. After 5 minutes at 72 ° C after the last cycle.
  • the PCR products were detected by electrophoresis on a 1.8% agarose gel.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une paire d'amorces PCR ayant un effet positif sur les oligonucléotides de V-erbB, une séquence de V-erbB limitée par ladite paire d'amorces, un ARNm de transcription de ladite séquence, un vaccin comprenant ladite séquence V-erbB ou un polypeptide et un oligonucléotide sens et antisens s'adaptant à la séquence de V-erbB. L'invention concerne également un agent de diagnostic renfermant lesdites amorces ou lesdits oligonucléotides sens et antisens, un composé pharmaceutique renfermant ledit oligonucléotide antisens, ainsi que les applications des substances précitées en vue du diagnostic et du traitement du syndrome myélodysplasique, de la leucémie, d'autres tumeurs et des étapes précédant ces pathologies.
PCT/CN2001/000888 2000-06-01 2001-06-01 Oligonucleotides sens et antisens de v-erbb et ses applications WO2002020782A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNB001090232A CN1204142C (zh) 2000-06-01 2000-06-01 反基因V－erbB寡核苷酸及其应用
CN00109023.2 2000-06-01

Publications (1)

Publication Number Publication Date
WO2002020782A1 true WO2002020782A1 (fr) 2002-03-14

Family

ID=4579371

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2001/000888 WO2002020782A1 (fr) 2000-06-01 2001-06-01 Oligonucleotides sens et antisens de v-erbb et ses applications

Country Status (2)

Country Link
CN (1) CN1204142C (fr)
WO (1) WO2002020782A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104928358A (zh) * 2015-02-13 2015-09-23 冯宝章 一种癌症早期基因诊断试剂盒及诊断方法
CN104928296A (zh) * 2015-02-13 2015-09-23 冯宝章 一种CpG寡核苷酸及其应用
CN104928359A (zh) * 2015-02-13 2015-09-23 冯宝章 一种癌干细胞的鉴定方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5183884A (en) * 1989-12-01 1993-02-02 United States Of America Dna segment encoding a gene for a receptor related to the epidermal growth factor receptor
US5985553A (en) * 1986-03-05 1999-11-16 The United States Of America As Represented By The Department Of Health And Human Services erbB-2 gene segments, probes, recombinant DNA and kits for detection

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5985553A (en) * 1986-03-05 1999-11-16 The United States Of America As Represented By The Department Of Health And Human Services erbB-2 gene segments, probes, recombinant DNA and kits for detection
US5183884A (en) * 1989-12-01 1993-02-02 United States Of America Dna segment encoding a gene for a receptor related to the epidermal growth factor receptor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 27 April 1993 (1993-04-27), YAMAMOTO Y. ET AL., retrieved from GI:209676 accession no. NCBI Database accession no. K01216 *

Also Published As

Publication number Publication date
CN1204142C (zh) 2005-06-01
CN1326938A (zh) 2001-12-19

Similar Documents

Publication Publication Date Title
JP6404219B2 (ja) 筋強直性ジストロフィーを治療するためのペプチド結合モルホリノアンチセンスオリゴヌクレオチド
Frederick et al. Analysis of genomic rearrangements associated with EGFRvIII expression suggests involvement of Alu repeat elements
De Simone et al. Thalassemias: From gene to therapy
PT89050B (pt) Meios e metodo para intensificar a hibridacao de acidos nucleicos
Zhao et al. p53 inhibition protects against neuronal ischemia/reperfusion injury by the p53/PRAS40/mTOR pathway
Ni et al. Evolution of hepatitis C virus quasispecies in mothers and infants infected through mother-to-infant transmission
Hernandez-Encinas et al. CCAAT/enhancer binding protein β directly regulates the expression of the complement component 3 gene in neural cells: implications for the pro-inflammatory effects of this transcription factor
WO2002020782A1 (fr) Oligonucleotides sens et antisens de v-erbb et ses applications
JP2001506586A (ja) ケラチノサイト増殖因子―２の使用
CN114409733B (zh) 靶向抑制mlkl乙酰化的多肽和/或其衍生物及其用途
TWI703977B (zh) 化合物在製備用於治療腦膠質瘤的藥物中的用途
US20210008164A1 (en) Erythropoietin for Gastrointestinal Dysfunction
CA3223109A1 (fr) Arn guides modifies et polynucleotides
Lu et al. Molar loss further exacerbates 2-VO-induced cognitive impairment associated with the activation of p38MAPK/NFκB pathway
CN111358791A (zh) Jak/stat信号通路磷酸盐抑制剂在制备治疗dba疾病的药物中的应用
BR122023024088A2 (pt) Uso de um conjugado de toxina de difteria-interleucina-3 humana (dtil3) e um ou mais inibidores de jak e/ou um ou mais agentes hipometilantes
CN112933215A (zh) Sirt1在制备卵巢功能保护药物方面中的应用
WO2019174610A1 (fr) Virus oncolytique, séquence d'adn synthétique et utilisation associée
CN111705061A (zh) 和心脏疾病相关的piRNA-P1与piRNA-P1反义核苷酸、应用和药物
US20210145783A1 (en) Compositions and Methods for Treating Sickle Cell Disease
CN115252787B (zh) Zip7在抑制阿片类药物诱发的痛觉过敏中的应用
CN109718365B (zh) 重组人角质细胞生长因子的应用
WO2002058722A1 (fr) Methode de diagnostic et de traitement de la dentinogenese imparfaite de type ii au moyen du gene de la sialophosphoproteine dentinaire (dspp) et son produit de codage
CN112877431B (zh) snoRNA-U41在检测以及治疗胰腺癌中的用途
WO2022253340A1 (fr) Peptide traduit par l'arn circulaire circ-ace2 et son application

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP