32 results about "Nonsense mutation" patented technology

Analyze gene mutations including missense mutations and / or nonsense mutations and silent mutations with high precision. 【Solution means】Detect signals from a first probe corresponding to a missense mutation or nonsense mutation in a gene mutation, a second probe corresponding to a silent mutation in the gene mutation, and a wild-type probe corresponding to the wild type, and determine the signal from the second probe as the wild type in the gene mutation.

The invention belongs to the technical field of biology, and particularly relates to ANK1 gene nonsense mutation and application. The invention firstly provides an ANK1 gene non-sense mutation c.2230 Cgt and an ANK1 gene non-sense mutation c.2230 Cgt. The invention relates to the field of genetic engineering, and in particular relates to nonsense mutation T (p.Q744X), the nonsense mutation significantly reduces the expression of ankyrin, and further causes phenotypic changes such as abnormal cell morphology and increased osmotic fragility after K562 erythroid differentiation, which indicates that the K562 is related to the pathological process of hereditary polycythemia spheroides (HS), and indicates that the mutation has pathogenicity; secondly, a treatment evaluation system taking drug-induced translation readthrough and adenine base editing as a core is constructed around the mutation, a new strategy and a technical platform are provided for precise molecular treatment of the ANK1 nonsense mutant HS, and important clinical transformation prospects and application values are achieved.

The invention provides sup-tRNA for treating Duchenne muscular dystrophy, a recombinant plasmid and a construction method of the sup-tRNA, and relates to the technical field of biological medicines. The sequence of the sup-tRNA for treating the Duchenne muscular dystrophy is as shown in SEQ ID No. 6. According to the application disclosed by the invention, the sup-tRNA is delivered by utilizing MyoAAV of targeted muscle, PTC (Positive Temperature Coefficient) can be recognized in an mdx mouse body, and the expression of dystrophin protein is increased, so that the muscular dystrophin phenotype of the mdx mouse is effectively relieved, and Ribo-seq also proves that the sup-tRNA has relatively high safety. Meanwhile, the strategy also successfully increases the dystrophin protein expression quantity of skeletal muscle precursor cells and myocardial cells from the DMD patient. In addition, the inventor explores the combined use of three small molecule compounds, namely PTC124, CC-90009 and SRI-41315, and the sup-tRNA, so that the read-through efficiency of the sup-tRNA can be improved. In conclusion, the sup-tRNA readthrough therapy is expected to become a new choice for treating DMD and other nonsense mutation diseases.

CD39 (CAG>TAG) is one of the most common β0-thalassemic mutation in the Mediterranean area and Latin America, representing >40% of β-thalassemic mutations in Tunisia, Argentina and Italy3. This is a nonsense mutation within the codon of amino acid 39, thus it causes premature translation termination and absence of β-globin4. Here, the inventors exploited adenine base-editors (ABEs) to correct the CD39 (CAG>TAG) mutation in HSPCs from β-thalassemia patients and demonstrated the potential of this strategy to correct the pathological phenotype observed during erythroid differentiation. In particular the inventors demonstrated that reverting the CD39 (CAG>TAG) mutation using base editing corrected in vitro the β-thalassemic cell phenotype in terms of erythroid differentiation, enucleation, RBC size and apoptosis. The present invention thus relates to base editing approaches for the treatment of β-thalassemia, including sickle β-thalassemia.

The invention discloses a DNA shuffling method based on flanking sequence segmented amplification and application of the DNA shuffling method in screening herbicide-resistant hybrid enzymes, on the basis of a single-stranded DNA shuffling technology, the success rate of single-stranded preparation is increased by introducing flanking sequences and segmented amplification recombinant products into single-stranded amplification primers, and the yield of herbicide-resistant hybrid enzymes is increased. Meanwhile, the proportion of nonsense mutation in the mutation library is reduced. The method is expected to provide a new method for establishing a protein qualitative evolution mutation library. The method is used for shuffling the rice and corn hppd genes and screening to obtain the hybrid enzyme of which the resistance is stronger than that of parent rice and corn HPPD under the treatment of 300 mu M mesotrione.

The present disclosure relates to nucleic acids, compositions, and methods for treating a disease or disorder associated with premature termination codon. Certain aspects of the disclosure relate to polynucleotides, vectors, and host cells, and uses thereof.

The invention provides a PMFBP1 mutant gene related to azoospermia syndrome and an application of the PMFBP1 mutant gene. The mutant gene is a PMFBP1 gene c.2641Cgt; the protein p.Arg881Ter is subjected to non-sense mutation, so that the encoded protein p.Arg881Ter is truncated, and the function of a sperm head-tail connection device is damaged. The invention also provides a specific Sanger sequencing detection method and a kit of the mutation, which can be used for rapid molecular diagnosis, genetic counseling and assisted reproduction guidance of the disease, and provides a basis for precise medical treatment.

A site-specifically-modified engineered tRNA and the use thereof. Said modification refers to replacement, with one or more modified nucleotides, of one or more native unmodified nucleotides selected from sites 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58 and 72 of a tRNA molecule. Compared with unmodified tRNAs, the site-specifically-modified tRNA has higher structural stability and / or higher nuclease degradation resistance, the aminoacylation efficiency thereof is not remarkably reduced, and immunogenicity thereof is not remarkably increased. The modified tRNA can be used for eliminating premature translation terminations caused by nonsense mutations, and thus has great application potentials in treating nonsense mutation-related genetic diseases, tumors, etc. The modification of the engineered tRNA also provides valuable insights for drug development of tRNAs and thus can guide the modification design of native tRNAs.

The invention discloses an editing system for specifically repairing a pathogenic mutant gene by combining Sup-tRNA with gsnoRNA. According to the method, the Sup-tRNA and the gsnoRNA are combined to repair the pathogenic nonsense mutation for the first time, so that the mutation read-through efficiency is improved, and off-target read-through is avoided. In addition, the invention also provides a recombinant vector and a cell comprising the editing system, and provides a pharmaceutical composition. The invention provides a new strategy for treating pathogenic nonsense mutation related diseases, and has a wide application prospect clinically.

The present invention relates to a purine derivative for use in the treatment of a disease due to a nonsense mutation in a gene leading to the premature introduction of a UGA stop codon.

The present disclosure relates to compositions and methods for treating genetic disorders caused by nonsense mutations using anticodon-engineered transfer RNA (ACE-tRNA) constructs. These DNA-based ACE-tRNA constructs are designed to suppress premature termination codons (PTCs) and restore the expression of full-length, functional proteins. The disclosure further provides formulations of ACE-tRNA constructs with poly(amine-co-ester) (PACE) polymeric nanoparticles to improve stability, protect nucleic acids, and enhance delivery to airway epithelial cells. Also described are functionalized ACE-tRNA Picovectors (sLPVs) incorporating targeting elements such as nuclear localization signals (NLSs), nucleolar localization sequences (NoLSs), and DNA nuclear targeting sequences (DTSs) to improve nuclear import and localization.

This invention relates to a method for constructing a mouse model of autosomal dominant polycystic kidney disease and its applications, particularly to a method for preparing a non-human animal model carrying a nonsense mutation in the Pkd1 gene c.616_618GAG>TAG based on CRISPR / Cas9 gene editing technology. The method involves co-injecting mouse zygotes with gRNA targeting exon 5 of the Pkd1 gene, homologous recombinant donor oligonucleotides containing the c.616_618GAG>TAG mutation, and Cas9 nuclease. Mutant mice are obtained via embryo transfer. The mutation site is verified by PCR combined with sequencing, and a stable mutant line is established through breeding. This invention also includes the application of this model animal as a research tool for autosomal dominant polycystic kidney disease, particularly in the areas of pathogenesis analysis, drug screening, and therapeutic target validation. This model can stably simulate the typical clinical manifestations and pathological features of human autosomal dominant polycystic kidney disease, providing an important tool for basic research and translational medicine of this disease.

The application belongs to the technical field of molecular markers, and provides a Hu sheep 5K low-density SNP chip and application thereof.The chip comprises 5000 SNP molecular markers, and the SNP molecular markers are shown in Table 1.Based on resequencing data of Hu sheep, the application screens SNP markers with rich polymorphic information content of Hu sheep breeds, integrates functional variation sites such as nonsense mutations and splicing mutations, and customizes the 5K low-density chip of Hu sheep, which can be used for Hu sheep kinship identification and functional harmful mutation screening.

The invention provides a glycine oxidase mutant strain as well as a construction method and application thereof in nucleoside production. A new microorganism capable of efficiently producing nucleoside is successfully constructed by performing point mutation on a thiO gene (enabling the 143rd amino acid of a ThiO protein coded by the thiO gene to be subjected to nonsense mutation) or directly knocking out the gene in wild type or bacillus amyloliquefaciens or bacillus subtilis with nucleoside production capacity by utilizing a genetic engineering technology.

The invention provides a detection method of XPV / POLH single base mutation and application of the detection method. The detection method comprises the following steps: by taking nucleic acid of a sample to be detected as template DNA, carrying out real-time fluorescence collection through PCR reaction, drawing an amplification curve and a melting curve, and carrying out result interpretation; the interpretation of the result comprises interpretation of mutation sites of four missense mutations and three nonsense mutations of the POLH gene. On the basis of a nucleic acid extraction method, detection of missense mutation and nonsense mutation of the POLH gene by using a melting curve is established, and the method has the advantages that operation is simple, operation can be carried out on the basis of PCR, consumed time is short, the whole process only needs about 2-3 hours, special equipment is not needed, and the method is suitable for large-scale popularization and application. A conventional fluorescent quantitative PCR instrument can realize interpretation of a detection result, the detection method is simple, the cost is low, the real-time fluorescent quantitative PCR method is equivalent, and a reliable and accurate basis can be quickly provided for auxiliary diagnosis of XP diseases.

The invention relates to the technical field of biology, and discloses a recombinant Zika virus and application thereof. According to the invention, point mutation is carried out on wild-type Zika virus, motifs conforming to a DRACH rule in a genome are increased, and a recombinant mutant strain with obvious attenuated characteristics is obtained. As nonsense mutation is introduced into the recombinant mutant strain, the amino acid sequence, the secondary structure and the like of coded protein are not influenced, so that the antigenicity of a wild type virus is reserved, and the recombinant mutant strain has the potential of serving as an alternative strain of an attenuated live vaccine.

This invention relates to a gene encoding coagulation factor IX. F9 This study investigates animal models of nonsense mutations, methods for screening hemophilia drugs using these models, and the application of the screened stRNAs in the preparation of hemophilia drugs. Based on an analysis of the probability of nonsense mutations in coagulation factor IX in hemophilia patients, the study verifies that stRNAs can read the coagulation factor IX encoding gene. F9 The function and efficiency of the premature termination codon (PTC) were investigated. Seven Arg-stRNAs were designed to target the coagulation factor IX R75* mutation, and readthrough efficiency was tested at the cellular level. A mouse model of the coagulation factor IX R75* mutation was constructed, and Arg-stRNA was delivered via AAV, restoring the expression of endogenous coagulation factor IX and reducing clotting time, demonstrating the effectiveness of stRNA in treating hemophilia B.

The invention discloses application of single-base nonsense mutation of a sheep FUT2 gene in diarrhea resistance, and belongs to the technical field of diarrhea-resistant breeding of sheep. The invention provides a single base mutation related to diarrhea resistance of sheep, which is located at the 55082566th nucleotide from the 5'end of chromosome 14 of a sheep genome AR-UIRamv2.0 version, and when the site is T, the sheep shows the ability to resist infectious diarrhea; the provided single base mutation is linked with sheep anti-diarrhea, can be used as a molecular marker, is used for breeding infectious diarrhea resistant sheep varieties and / or screening natural anti-diarrhea individuals, is extremely high in accuracy, strong in universality and simple and convenient to operate, can realize fundamental transformation from post phenotype selection to beforehand genotype selection, and has a wide application prospect. The method has important significance in simplifying the breeding process, shortening the breeding period and workload and improving the breeding efficiency.

The present invention provides for inhibitory transporter ribonucleic acids (sup-tRNAs) that inhibit premature termination codons (PTCs) during translation and promote full length readthrough of cancer suppressor gene nonsense mutation transcripts, as well as the use of the sup-tRNAs to treat disease conditions of cancer that contain cancer suppressor gene nonsense mutations. In particular, Arg-tRNAUGA and Gln-tRNAUAG can restore the tumor suppressor gene function of tumor cells and indirectly regulate and control the expression of downstream target genes of the tumor suppressor genes, so that the clone number, the total clone area and / or the single clone area of in-vitro cultured tumor cells are reduced, and the volume and / or the weight of in-vivo tumors are reduced.

The invention relates to a construction method and application of an autosomal dominant polycystic kidney disease mouse model, in particular to preparation of a Pkd1 gene carrying c.616618GAGgt gene based on a CRISPR / Cas9 gene editing technology. The invention relates to a method for a non-human animal model of TAG nonsense mutation. The construction method comprises the following steps: co-injecting gRNA (guide Ribonucleic Acid), containing c.616618GAGgt, of a No. 5 exon of a targeted Pkd1 gene into a mouse fertilized egg; carrying out embryo transplantation on TAG mutated homologous recombination donor oligonucleotide and Cas9 nuclease to obtain a mutated mouse; a mutation site is verified by combining PCR (Polymerase Chain Reaction) with sequencing, and then a stably inherited mutation line is established by breeding. The invention also comprises application of the model animal as an autosomal dominant polycystic kidney disease research tool, especially application in the fields of pathogenesis analysis, drug screening and therapeutic target verification. The model can stably simulate typical clinical manifestation and pathological characteristics of human autosomal dominant polycystic kidney disease, and provides an important tool for fundamental research and transformation medicine of the disease.

The present disclosure relates to compositions and methods for treating genetic disorders caused by nonsense mutations using anticodon-engineered transfer RNA (ACE-tRNA) constructs. These DNA-based ACE-tRNA constructs are designed to suppress premature termination codons (PTCs) and restore the expression of full-length, functional proteins. The disclosure further provides formulations of ACE-tRNA constructs with poly(amine-co-ester) (PACE) polymeric nanoparticles to improve stability, protect nucleic acids, and enhance delivery to airway epithelial cells. Also described are functionalized ACE-tRNA Picovectors (sLPVs) incorporating targeting elements such as nuclear localization signals (NLSs), nucleolar localization sequences (NoLSs), and DNA nuclear targeting sequences (DTSs) to improve nuclear import and localization.

The invention belongs to the field of molecular biology, and provides a functional molecular marker of a major gene AhNAC2 for regulating and controlling the oil content of peanut seed kernels and application of the functional molecular marker. The invention discloses a major site qSOCA08-1 for regulating and controlling the oil content of peanut seed kernels, and the phenotypic variation of 11.41-20.97% is explained. The qSOCA08-1 is finely positioned in a 352.2 kb physical interval between a No.8 chromosome marker Tif2. A08.38582892 and a No.8 chromosome marker Tif2. A08.38935104, and a candidate gene of the qSOCA08-1 is confirmed to be AhNAC2. The invention also discloses a method for identifying the qSOCA08-1. The 285th basic group of the coding sequence of the AhNAC2 shows C-A nonsense mutation between parents Jihuansweet No.1 and W191, and the early termination can cause the function loss of the AhNAC2. A competitive allele specific PCR molecular marker is developed on the basis of a C-A mutation SNP site, the association between the molecular marker and the oil content of the seed kernel is successfully verified in a peanut germplasm resource material and a Yuhua 15 * ST001 recombinant inbred line population, and the important value of the marker in marker-assisted selection breeding of the high oil content of the seed kernel of the peanut is verified.

The invention discloses application of a PTCHD1 gene mutant and a detection reagent. Belongs to the technical field of gene mutation detection. According to the mutation, compared with a wild type PTCHD1 gene, cytosine C is replaced with adenine A at the 1644th nucleotide of a coding sequence, so that protein translation is terminated in advance at the 548th amino acid, and a truncated protein is formed. And the c.1644Cgt of the PTCHD1 gene is confirmed; a is pathogenic nonsense mutation causing AUTS4, a new insight is provided for the genetic basis of the disease, and the blank of the mutation spectrum of the disease is filled.

The invention belongs to the technical field of biological medicine and gene therapy, and particularly relates to a tRNA binding protein assisted termination codon readthrough system and application thereof. The tRNA binding protein assisted termination codon reading-through system disclosed by the invention comprises inhibitory tRNA and binding protein thereof, wherein the inhibitory tRNA contains 70 pairs of G3: U, can be recognized by an endogenous alanyl-tRNA synthetase (AlaRS) of a cell, and is loaded with alanine; the tRNA binding protein is specifically bound with any structure, except for a receptor arm, of the corresponding tRNA, and can be specifically bound with a tRNA binding structural domain of pyrrole lysyl-tRNA synthetase. According to the system, alanine can be introduced in the translation process of a target gene carrying nonsense mutation, so that full-length translation of protein is recovered. The system can be applied to functional recovery and treatment research of various genetic diseases caused by the early termination codon.

The present invention discloses a ZmWRKY60 gene mutant and its application in regulating the plant height of maize, which relates to the technical fields of functional genes and plant gene breeding. The ZmWRKY60 gene mutant is obtained by a nonsense mutation caused by the substitution of base C with base T at the 736th position in the coding region of the wild-type ZmWRKY60 gene. The nucleotide sequence of the mutant is shown in SEQ ID NO.3. The gene-edited plant KOwryk60 uses a single-gene double-target method to inactivate the ZmWRKY60 gene. The results show that both the mutant Zmwrky60 and the gene-edited plant KOwryk60 can reduce the plant height of maize, indicating that the ZmWRKY60 gene is involved in the regulation of maize plant height. This provides important theoretical significance and application value for creating new maize germplasms and cultivating ideal maize plant types.

The invention discloses an SEC23B gene mutant capable of reducing the content of SEC23B protein and application of the SEC23B gene mutant. The nucleotide sequence of the SEC23B gene mutant capable of reducing the content of SEC23B protein is shown as SEQ ID NO.3. Compared with an SEC23B gene with the nucleotide sequence shown as SEQ ID NO.1, the SEC23B gene mutant capable of reducing the content of SEC23B protein has nonsense mutations (c.181Tgt; c and c.1832 Ggt; according to the SEC23B gene capable of reducing the SEC23B protein content, the SEC23B gene is (A), cysteine at the 61th site of an SEC23B gene coding protein sequence is changed into arginine (p.C61R), and arginine at the 611th site of the SEC23B gene coding protein sequence is changed into glutamine (p.R611Q) through mutation. According to the SEC23B gene capable of reducing the SEC23B protein content, an SEC23B gene mutant with the SEC23B protein content is provided for the first time through systematic research, and the SEC23B protein content is reduced by 50% compared with a control group.

Provided herein are methods and compositions for modulating CNOT3 recruitment in a cell ribosome. The methods and compositions can modulate a target mRNA stability and / or half-life. Also provided are pharmaceutical compositions for use in the treatment of an mRNA-decay associated disease, nonsense mutation associated disease, obesity, a mitochondrial- associated disorder, or a metabolic disorder.

The invention relates to the skipping of the CEP290 exon 36 in an individual suffering from a retinal dystrophy accounted for by a nonsense mutation or a premature termination codon generated by a frameshift mutation in exon 36 or an upstream exon, including the c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG (35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A mutations, to bypass protein truncation and lessen retinal damages. Here, studying fibroblasts from control individuals, and two patients carrying the CEP290 c.4723A>T nonsense mutation, they show low levels of spontaneous skipping of exon 36 arising from both endogenous basal skipping and mutation-induced skipping. The minimally shortened and mutation-free CEP290 mRNA produced by skipping of exon 36 in the fibroblasts of the two patients is translated into a protein isoform that localizes at the centrosome and allows the formation of primary cilia, yet with elongated axonemes. Using an AON consisting of a sequence set forth as SEQ ID NO: 1, complementary to a nucleic acid sequence of CEP290 pre-mRNA, wherein said AON targeting an mRNA encoding the donor splice site (H36D) is capable to alter splicing by blocking the recognition of exon 36 and bypass protein truncation while maintaining the open reading frame, leading to the production of near full-length CEP290 protein, they were able to increase the abundance of the alternatively spliced mRNA and shortened protein and to reduce axonemal length in patient cells.

The invention discloses a construction method and application of an angel syndrome mouse model. The construction method comprises the following steps: preparing c.1435Agt carrying a maternal Ube3a gene on the basis of combination of a pilot editing technology and a tetraploid compensation technology; the invention relates to a non-human animal model of T nonsense mutation. The method comprises the following steps: co-delivering pegRNA (Ribonucleic Acid) and PEmax of a No.4 exon of a targeted Ube3a gene to a mouse embryonic stem cell, and introducing c.1435Agt at a target site; carrying out T nonsense mutation, and after tetraploid compensation technology treatment, transplanting the edited recombinant embryo to a pregnant female mouse to obtain an F0-generation mutant mouse; the accuracy of a mutation site is verified through combination of PCR amplification and gene sequencing, and a mutant strain with genetic stability is established through two generations of breeding and screening by using maternal genetic characteristics of the UBE3A gene. The invention further comprises a genotype identification method of the model animal, Ube3a gene mRNA expression quantity evaluation, behavioral evaluation and the like, and the Ube3a: c.1435 Agt is verified; the T mutant mouse model accords with the pathological characteristics of the angel syndrome, and can stably simulate the typical clinical manifestation and pathological characteristics of the angel syndrome.

The present invention relates to compositions and methods for editing mutations to allow transcription or expression, characterized by compositions and methods for editing a gene associated with Schwashan Syndrome, SDS, using a programmable nucleobase editor, such that the gene allows transcription and generates a functional gene product (e.g., a mutant or a mutant). Splicing sites and / or altered nonsense mutations are provided).