43 results about "Globin gene" patented technology

This invention provides vectors (e.g., lentiviral vectors) and expression cassettes for incorporation into vectors for expressing an alpha-globin (α-globin) gene. In certain embodiments method of treatment using such vectors are provided.

The invention provides a preparation method and application of a genetically modified non-human mammal for producing a humanized antibody. The preparation method comprises the following steps: (1) destroying an endogenous heavy chain immune globulin locus of a non-human mammal; and (2) introducing a human IGHV gene, a human IGHD gene, a human IGHJ gene, and an endogenous IgHG gene, an endogenous IgHE gene, an endogenous IgHA gene and an endogenous LCR region of the non-human mammal into the non-human mammal obtained in the step (1). The non-human mammal prepared by the preparation method disclosed by the invention can efficiently generate a humanized complete antibody or a single-chain antibody or a nano antibody after being immunized by an antigen, and has relatively high immune titer, and moreover, the preparation method disclosed by the invention can be used for obtaining a positive animal with relatively high efficiency, so that the non-human mammal can be used for preparing the non-human mammal. Therefore, humanized mice with different antibody diversity can be easily prepared, and the nanometer antibody sequence diversity can be realized by using various strains of mice.

Genetically modified animals and cells with humanized light chain immunoglobulin locus and / or humanized heavy chain immunoglobulin locus. The animals and cells can also express a human or chimeric (e.g., humanized) major histocompatibility complex (MHC) protein complex.

In certain embodiments, a lentiviral vector for the treatment of sickle cell disease (SCD) is provided. In certain embodiments, the vector comprises an expression cassette that encodes that an anti-sickling β-globin gene and an shRNA that inhibits expression of a BCL11A gene (BCL11A shRNA) wherein said expression cassette is in reverse orientation in the vector; a β-globin locus control region (LCR) comprising a reduced length hypersensitive site 1 (HS1) sequence, a reduced length hypersensitive site 2 (HS2) sequence, a reduced length hypersensitive site 3 (HS3) sequence, and a reduced length hypersensitive site 4 (HS4) sequence, where said anti-sickling β-globin gene is operably linked to the human β-globin locus control region.

The present invention provides an application of thyroid hormones and its analogues in preparation of drugs for treating alpha-thalassemia, especially in preparation of drugs for regulating expression of zeta-globin gene. During differentiation of K562 cells, thyroid hormone analogue (Triac) can significantly up-regulate the expression of zeta-globin gene (HBZ) by 50 folds or above. The expression of zeta-globin gene (hbae5) can also be up-regulated by 30-70 folds in zebrafish treated with thyroid hormones and thyroid hormones analogues. Therefore, according to the present invention, the expression of zeta-globin gene can be significantly activated by thyroid hormones and its analogues, which may develop new potential therapies for patients with alpha-thalassemia. It provides an economical, safe and effective method for treating alpha-thalassemia and can be widely used.

The invention relates to a construction method, an evaluation method and application of an inflammatory nephropathy animal model, and relates to the technical field of nephropathy animal model construction. The construction method comprises the steps that a CRIPSR / Cas9 system is adopted to shear a first targeting site on an Smu region of a mouse immune globulin gene, and a second targeting site on an Sgamma2c region of the mouse immune globulin gene is sheared, so that a base sequence between the first targeting site and the second targeting site is knocked out, and the inflammatory nephropathy animal model is obtained. The application comprises an application of the inflammatory nephropathy animal model constructed by the construction method in screening drugs for treating inflammatory nephropathy and an application in researching a molecular mechanism of occurrence and development of inflammatory nephropathy.

The invention relates to a method for predicting an alpha globin gene triad by analyzing high-throughput sequencing data, and belongs to the technical field of gene detection.The method comprises the steps that multiple samples are collected and subjected to high-throughput sequencing, read counts A1 and A2 of non-homologous regions of alpha globin genes HBA1 and HBA2 are obtained, and the A2 / A1 ratio is calculated; the method comprises the following steps: analyzing A2 / A1 ratio data to find that the data is in normal distribution, making a histogram for the A2 / A1 ratio data, determining data in an area greater than u + 2sigma as abnormal data in combination with a normal distribution data distribution rule, and judging that an alpha globin gene triad exists in a sample corresponding to the abnormal data. In the subsequent detection sample inspection, whether the alpha globin gene triad exists in the detection sample can be directly judged according to the A2 / A1 ratio, and compared with the verification of a polymerase chain reaction method, the judgment result of the method is consistent with the result of the polymerase chain reaction method; therefore, the method provided by the invention can be directly used for judging the existence of the alpha globin gene triad.

The invention relates to a method for creating a rice material with low absorbable protein content and remarkably thickened endosperm cell walls and application of the rice material. Specifically, the invention provides a method for improving rice, and improved rice with unique characters is obtained by down-regulating the expression of glutelin genes and down-regulating the expression of globulin genes. In the improved rice, the content of absorbable protein is low, and endosperm cell walls are remarkably thickened. Besides, the improved rice is low in in-vitro digestion rate, has the potential postprandial blood sugar control effect and is suitable for preparing auxiliary food for treating chronic kidney disease patients, diabetic patients and the like.

Disclosed herein are gene therapy vectors used for efficiently transducing cells to express a human β-globin gene. Specifically disclosed is an expression vector which comprises: an expression cassette for a β-globin gene, which comprises exons and introns of human β-globin gene, as well as cis-acting elements including one or more of WPRE, SV40 polyadenylation signal and / or SV40 ori. The disclosed expression vectors have significantly enhanced viral vector packaging efficiency in viral vector packaging cell lines, which leads to effective integration of lentiviral vectors and high expression level of β-globin gene in target cells. Also disclosed are pharmaceutical compositions and therapeutic methods utilizing such expression vectors.

The present invention provides the use of thyroid hormone and its analogs in the preparation of a drug for treating α-thalassemia. Specifically, the drug can be used to regulate ζ-globin gene expression. During K562 cell differentiation, the thyroid hormone analogs can specifically and significantly upregulate the expression of the ζ-globin gene (HBZ) by as much as 50 times. After treatment with thyroid hormone and its analogs in model animal zebrafish embryos, the expression of the ζ-globin gene (hbae5) can also be specifically upregulated by as much as 30-70 times. Therefore, the present invention uses thyroid hormone and its analogs to specifically activate the expression of the ζ-globin gene, that is, to reactivate the silenced ζ-globin gene in α-thalassemia patients to inhibit the destruction of red blood cells. This method provides a method for preparing a drug for treating α-thalassemia, provides an economical, safe and effective method for the treatment of α-thalassemia, and can be widely promoted and used.

The invention relates to a probe, a primer, a kit and a method for detecting an alpha-globin gene. The probe provided by the invention is selected from one or more of the following probes P1 to P4: the probe P1 has a sequence as shown in SEQ ID NO.10; the sequence of the probe P2 is as shown in SEQ ID NO. 11; the sequence of the probe P3 is as shown in SEQ ID NO. 12; the sequence of the probe P4 is as shown in SEQ ID NO. 13. The technical scheme for detecting the genotype of the alpha-globin gene is simple and low in cost.

The invention provides corynebacterium glutamicum for synthesizing high-activity heme protein and application, and belongs to the technical field of genetic engineering. By enhancing globin gene transcription and optimizing heme prothetic group supply, the synthetic ability of hemoglobin and myoglobin is improved. The corynebacterium glutamicum constructed by the invention can enable the titers of soybean hemoglobin, clover hemoglobin, bovine myoglobin and porcine myoglobin to reach 1.10 g / L, 1.12 g / L, 0.56 g / L and 1.14 g / L respectively during shake flask fermentation, and enables the heme binding rates of produced S-Hb, C-Hb, B-Mb and P-Mb to be improved by 3.64 times, 2.38 times, 1.60 times and 1.49 times respectively, which can be similar to those of naturally extracted standard substances H-Mb and B-Hb.

The invention belongs to the technical field of gene editing, and particularly relates to a method for reducing cell gene editing large fragment deletion. The invention provides a method for reducing cell gene editing large fragment deletion, which comprises the following steps: introducing a gene editing system into a cell, and carrying out gene editing on a gamma-globin gene promoter region so as to reduce HBG gene large fragment deletion, the gene editing system comprises Cas nuclease, gRNA and ssODN, the ssODN comprises a 5'homologous arm, a replacement sequence and a 3 'homologous arm, the length of the 5' homologous arm and the length of the 3 'homologous arm are respectively and independently 30-60 nt, and HBG gene large fragment deletion caused by multiple DSBs can be reduced by adjusting the length of the ssODN.

Methods of using one or more fumaric acid esters or pharmacologically active salts, derivatives, analogues, or prodrugs thereof to increase expression of fetal hemoglobin (HbF) are disclosed. The methods typically include administering to a subject an effective amount of one or more fumaric acid esters optionally in combination or alternation with hydroxyurea to induce HbF expression in the subject in an effective amount to reduce one or more symptoms of a sickle cell disorder, a hemoglobinopathy, or a beta-thalassemia, or to compensate for a genetic mutation is the human beta-globin gene (HBB) or an expression control sequence thereof. Pharmaceutical dosage units and dosage regimes for use in the disclosed methods are also provided.

Provided are a method for preparing a genetically modified non-human mammal for producing a humanized antibody and the use thereof. The preparation method comprises: (1) performing disruption of an endogenous heavy chain immunoglobulin locus on a non-human mammal; and (2) introducing into the non-human mammal obtained in the step (1) a human IGHV gene, a human IGHD gene, a human IGHJ gene, and an endogenous IgHG gene, an IgHE gene, an IgHA gene and an LCR region of a non-human mammal. The non-human mammal prepared according to the preparation method can efficiently produce a humanized whole antibody or a heavy-chain-only antibody or a nano antibody after antigen immunization, and has a relatively high immunological potency. In addition, the preparation method can be used to obtain positive animals with a relatively high efficiency, so that humanized mice with different antibody diversities can be easily prepared, and the various strains of mice can be used to realize the sequence diversity of nano antibodies.

The invention belongs to the field of food testing and discloses a method for preparing a hazelnut ultrafine powder standard sample for detecting characteristic quantitative values ​​of food allergens. The standard sample adopts hazelnut kernels as raw materials and is ground at low temperature by an ultrafine grinder to obtain hazelnut ultrafine powder. The obtained hazelnut ultrafine powder standard sample retains the characteristic quantitative values ​​of hazelnut component characteristic 2S globulin gene nucleic acid and allergen protein Cora9 to the greatest extent, and the preparation process does not add any auxiliary materials and preservatives. It is purely green production and highly purified, can simultaneously meet the requirements of allergen characteristic 2S globulin gene detection and allergen protein Cora9 detection, and its uniformity and stability meet the requirements of national standard samples, filling the gap of international standard samples of this type, improving the detection level of food allergen safety testing laboratories, and can be used for food allergen hazelnut detection method verification, detection kit evaluation, laboratory detection process quality control, inter-laboratory proficiency verification and comparison, and personnel detection ability assessment.

The invention discloses a method and a composition for activating zeta-globin gene expression. The method comprises the step of artificially forming an enhancer element containing an NTG-N (7-8)-WGATAR sequence or an NAA-N (7-8)-WGATAR sequence in a DNA (deoxyribonucleic acid) sense strand or an antisense strand in a promoter region of a zeta-globin gene through homologous recombination repair by using a CRISPR-Cas9 system. The invention also discloses gRNA, ssODN, a composition and a cell, and application of the gRNA, the ssODN, the composition and the cell in preparation of a medicine for treating alpha-thalassemia. According to the method, overexpression of exogenous globin genes is not needed, and the safety risk of gene therapy is reduced. Moreover, expression of the zeta-globin gene is activated, so that the method can be suitable for various types of alpha-thalassemia patients and alpha-gene deletion or mutation patients, and is not limited to alpha-thalassemia caused by a certain mutation site.

The present application relates to a genetically modified non-human animal comprising a humanized heavy chain immunoglobulin locus and / or a humanized light chain immunoglobulin locus and methods of making the same. The present application also provides methods of producing antibodies by the non-human animal.

The invention belongs to the technical field of gene editing, and discloses a method for inducing high expression of a delta-globin gene, a composition and application. The invention provides a method for inducing high expression of a delta-globin gene. The method comprises the following steps: artificially introducing a regulation motif into a positive-sense strand or an antisense strand of an HBD gene regulation region through a gene editing technology; the regulation and control motif is at least one of CCNCACCCCT, NTG-N (7-8)-WGATAR and CCAAT (C-CAAT); or at least one of the GGGTGNGG, the YTATCW-N (7-8)-CAN and the ATTGG. The HBD gene is edited by using a gene editing technology to induce high expression of the delta globin. And overexpression of exogenous globin genes is not needed, so that the safety risk of gene therapy is reduced. And the method can be suitable for diseases caused by various types of beta-hemoglobin deletion or mutation, and is not limited to diseases caused by a certain mutation site.

The present invention relates to a method for predicting α-globin gene triplets by analyzing high-throughput sequencing data, belonging to the field of gene detection technology. The present invention collects multiple samples and performs high-throughput sequencing on the multiple samples to obtain read counts A1 and A2 of the non-homologous regions of the α-globin genes HBA1 and HBA2, and calculates the A2 / A1 ratio. By analyzing the A2 / A1 ratio data, it is found that the data is normally distributed. Then, a histogram of the A2 / A1 ratio data is made. In combination with the distribution law of normally distributed data, data within the region greater than u+2σ is identified as abnormal data, and the presence of α-globin gene triplets in the samples corresponding to the abnormal data is determined. In subsequent sample testing, the presence of α-globin gene triplets in the test samples can be directly determined based on the A2 / A1 ratio. The method of the present invention has been verified and compared with the polymerase chain reaction method. The determination results of the present invention are consistent with the results of the polymerase chain reaction method, indicating that the method of the present invention can be directly used to determine the presence of α-globin gene triplets.

A method and device for detecting alpha-globin gene copy number variation, the method comprising: a primary detection step, comprising predicting whether the sample to be detected exists alpha-globin gene deletion according to the overall feature matrix of the sequencing data of the sample to be detected; a secondary detection step, comprising analyzing the alpha-globin gene copy number of the sample to be detected according to the prediction result of the primary detection step; and a tertiary detection step, comprising analyzing the accurate type of the alpha-globin gene copy number variation of the sample according to the prediction result of the secondary detection step. The present application constructs a combined model for identifying alpha-globin copy number variation, which can quickly and accurately identify the type of alpha-globin copy number variation including triplets using high-throughput sequencing data.

One aspect of the invention is a method for amplifying alpha globin genes HBA1, HBA2 and HBA12 in a single PCR tube to determine an HBA genotype of a subject. This method employs five primers selected to accurate and sensitively identify the HBA1, HBA2, and HBA12, a gene found at a higher frequency in citizens of Saudi Arabia, by accurately annealing to nucleic acids in a biological sample and simultaneously amplifying sequences encoding the alpha globin genes. This invention includes a procedure and required reagents for the amplification of alpha globin genes in a single PCR tube.

The invention discloses a method and a composition for activating zeta-globin gene expression. The method comprises artificially forming an enhancer element comprising an NTG-N (7-8)-WGATAR sequence, an NAA-N (7-8)-WGATAR sequence, a YTATCW-N (7-8)-CAN sequence and / or a YTATCW-N (7-8)-CTN sequence on a sense strand or an antisense strand of a non-coding region of a zeta-globin gene by using a gene editing technique. The invention also discloses gRNA, ssODN, a composition and a cell, and application of the gRNA, the ssODN, the composition and the cell in preparation of a medicine for treating alpha-thalassemia. According to the method, overexpression of exogenous globin genes is not needed, and the safety risk of gene therapy is reduced. Moreover, expression of the zeta-globin gene is activated, so that the method can be suitable for various types of alpha-thalassemia patients and alpha-gene deletion or mutation patients, and is not limited to alpha-thalassemia caused by a certain mutation site.

To provide genetically modified animals and cells with a humanized heavy chain immunoglobulin locus and / or a humanized light chain immunoglobulin locus.SOLUTION: A genetically modified, non-human animal comprises, at an endogenous heavy chain immunoglobulin gene locus, one or more human IGHV genes, one or more human IGHD genes and one or more human IGHJ genes, where the human IGHV genes, the human IGHD genes and the human IGHJ genes are operably linked and can undergo VDJ rearrangement.SELECTED DRAWING: Figure 1A

The present invention relates to a polynucleotide construct comprising a hemoglobin subunit alpha-hemoglobin subunit beta (HBA-HBB) hybrid gene construct for enhancing the potency of gene therapy for the β-hemoglobinopathies, particularly β-thalassemia and sickle cell disease (SCD). The polynucleotide construct comprising elements that are operatively connected to each other in the following sequence: HBB locus control region (HBB-LCR), HBA promoter, and β-like globin gene. Additionally, the HBA-HBB gene construct further comprises a microRNA-based short hairpin RNA (shRNAmir) targeting one of the endogenous HBA or HBB genes to increase the ratio of vector-encoded HBBT87Q to endogenous HBA and β-like globin chains while maintaining high vector titers.

The invention discloses a method and a kit for detecting copy number and tiny variation of an alpha-globin gene and a beta-globin gene and application of the method and the kit. An alpha-globin gene and a beta-globin gene are amplified at the same time in a single-tube PCR reaction, and as many as 61 deletion or repetition types such as normal types, 8 heterozygous deletion types, 36 homozygous deletion types, 2 repetition types, 14 composite heterozygous deletion / repetition types and the like can be detected at a time. Meanwhile, three types of non-deletion type alpha-thalassemia and 18 types of non-deletion type beta-thalassemia can be respectively detected, and normal, heterozygous and homozygous mutations can be distinguished. According to the method, deletion, repetition and tiny variation detection of alpha and beta-thalassemia can be achieved only through one-time PCR, the process is simple, standardization is easy to achieve, meanwhile, the detection time is greatly shortened, and integrity detection is provided for thalassemia.

The present disclosure relates to genetically modified animals and cells having a humanized lambda light chain immunoglobulin locus and / or a humanized heavy chain immunoglobulin locus. The disclosure also relates to genetically modified animals and cells having a modified locus comprising a human lambda light chain immunoglobulin gene and / or a humanized heavy chain immunoglobulin locus. In some embodiments, the animal also has a humanized kappa light chain immunoglobulin locus.

The invention discloses a method for detecting a gene segment through immunoglobulin gene rearrangement NGS. The method comprises the following steps: S1, extracting DNA of a sample; s2, performing a first round of multiple PCR amplification; s3, carrying out first round of magnetic bead purification; s4, carrying out a second round of Index connection PCR amplification reaction; s5, performing second round of magnetic bead purification to obtain a final library; s6, controlling the quality of the final library; and S7, sequencing on a machine, analyzing data, and judging a result. According to the method, the amplification cycle number of multiple PCR and Index connection PCR and the magnetic bead proportion of secondary magnetic bead purification are optimized, and non-specific amplification products caused in the PCR amplification process can be effectively reduced. Meanwhile, exhaustion of part of specific primers in multiplex PCR amplification can be prevented, and the purpose of improving the accuracy of a detection result is achieved.

The invention discloses a construction method and application of a globin gene humanized mouse beta-thalassemia CD17 point mutation animal model, by utilizing a guide editing technology, an epegRNA sequence (independently designed sequences mainly comprise spacer, PBS, RT and 1inner sequences) of a targeted HBB gene CD17 site, a candidate ngRNA sequence and an mRNA combination of an enzyme of PE are autonomously constructed, and by utilizing a micro-injection mode, a CD17 point mutation animal model of the HBB gene humanized mouse beta-thalassemia CD17 point mutation animal model is constructed. Injecting the combined system to 1-2 cell stage fertilized eggs of Townes mice, transplanting the fertilized eggs to receptor pseudopregnant mice, after the mice are delivered to F0-generation mice, quantitatively analyzing targeted site point mutation mice by using an NGS sequencing technology, and hybridizing to obtain hybrid site mutation F1-generation mice from CD17AAG to TAG. The mouse is used as a classical animal model for in-depth study of a beta-thalassemia pathogenic mechanism, beta-thalassemia targeted drug screening and gene therapy study of point mutation precise repair.

Provided herein are kits and methods for determining a thalassemia genotype of a subject. The present kit includes at least primers that target ζ2, Ψζ1, Ψα2, Ψα1, α2, α1 and θ1 genes in α-globin gene cluster, and primers that target variations in α2-globin gene and β-globin gene. Also encompasses herein is a method for determining a thalassemia genotype of a subject, in which gene copy number of each target sites in ζ2, Ψζ1, Ψα2, Ψα1, α2, α1 and θ1 genes in α-globin gene cluster are determined by use of the present kit, and compared with those of normal or known thalassemia subject to arrive at the thalassemia genotype of the subject.