30 results about "Globin genes" patented technology

The disclosure features systems and methods for correcting a mutation in the human beta-globin (HBB) gene in a cell or population of cells. The disclosure also features methods of increasing repair of a DNA double stranded break (DSB) in an HBB gene by the homology-directed repair (HDR) pathway. The disclosure also features compositions for use in the methods.

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.

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 present disclosure provides improved compositions for the homology directed repair of the human globin locus for the prevention, treatment, or amelioration of at least one symptom of a hemoglobinopathy.

Disclosed herein are new compounds and compositions and their application as pharmaceuticals for the treatment of diseases. Methods of inhibition of KDM1A, methods of increasing gamma globin gene expression, and methods to induce differentiation of cancer cells in a human or animal subject are also provided for the treatment of diseases such as acute myelogenous leukemia.

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 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.

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 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 disclosure provides methods and compositions for genetically modifying hematopoietic stem and progenitor cells (HSPCs), in particular by replacing the HBA1 or HBA2 locus in the HSPCs with a transgene encoding a therapeutic protein.

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.

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.

Disclosed herein are gene therapy vectors for efficient transduction of cells to express human beta globin genes. Specifically disclosed is an expression vector comprising: an expression cassette of a beta globin gene comprising an exon and an intron of a human beta globin gene, and a cis-acting element comprising one or more of WPRE, an 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 results in efficient integration of lentiviral vectors in target cells and high expression levels of beta globin genes. Pharmaceutical compositions and methods of treatment utilizing such expression vectors are also disclosed.

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.

Materials and methods for treating a patient with hemoglobinopathy, both ex vivo and in vivo and materials and methods for deleting at least a portion of a human beta globin locus on chromosome 11 in a human cell by genome editing and thereby increasing the production of fetal hemoglobin (HbF).

The invention discloses a repair method for alpha thalassemia large-fragment gene deletion, and belongs to the field of biological gene engineering. According to the method, HBA2 genes are successfully placed in gamma-globin gene loci of the iPSCs sourced from the severe alpha-thalassemia Bart's patients through the CRISPR / Cas9 technology, the gamma-globin genes are replaced with the alpha-globin genes, alpha-globin expression of the iPSCs sourced from the severe alpha-thalassemia Bart's patients is recovered, the proportion unbalance degree of alpha- / beta-globin peptide chains is remarkably relieved, and the expression of the gamma-globin gene loci of the iPSCs sourced from the severe alpha-thalassemia Bart's patients is improved. A new treatment direction and thought are provided for effectively treating patients with severe alpha-thalassemia Bart's; according to the repairing method, HBA2 gene expression of severe alpha-thalassemia cells is achieved by placing HBA2 gene in gamma-globin gene loci, the repairing method can be used for repairing the defect of alpha-thalassemia large-fragment gene deletion, effective treatment of severe alpha-thalassemia is achieved, and a more effective and safer treatment scheme can be provided for patients.

The presently disclosed subject matter provides for expression cassettes that allow for expression of a globin gene or a functional portion thereof, vectors comprising thereof, and cells transduced with such expression cassettes and vectors. The presently disclosed subject matter further provides methods for treating a hemoglobinopathy in a subject comprising administering an effective amount of such transduced cells to the subject.

The invention discloses construction and application of bacillus subtilis for synthesizing high-activity heme protein, and belongs to the technical field of genetic engineering. By enhancing globin gene transcription and optimizing heme prothetic group supply, the synthesis capability of heme proteins is improved, so that the titers of soybean hemoglobin, clover hemoglobin, bovine myoglobin, porcine myoglobin and P450-BM3 of the constructed bacillus subtilis respectively reach 0.81 g / L, 0.82 g / L, 1.11 g / L and 1.01 g / L under the condition of fed-batch fermentation, and the bacillus subtilis can be used for preparing the recombinant bacillus subtilis. The heme binding rate and peroxidase activity of Hb and Mb produced by fed-batch fermentation can be similar to those of naturally extracted standard substances H-Mb and B-Hb.

To provide a novel method for activating transcription of a γ-globin gene.MEANS: The present invention uses a single-stranded oligonucleotide (ssODN) containing GATA or an antisense complementary sequence TATC thereof as guidance information, and performs gene editing in a γ-globin gene regulatory region to form a GATA-containing enhancer element, which can promote the expression of the γ-globin gene in mature red blood cells. Hematopoietic stem cells genetically edited by the technique according to the present invention have normal functions, can significantly improve the expression of fetal hemoglobin after being differentiated into red blood cells, and therefore can be used in clinical treatment of β-thalassemia and sickle cell anemia.SELECTED DRAWING: Figure 1

The invention relates to the technical field of animal breeding, in particular to an expression vector based on a bovine-derived K5 gene and application of the expression vector. The expression vector comprises an expression cassette, wherein the expression cassette comprises a 5'end regulation region of a bovine-derived K5 gene, an intron of a beta-globin gene, a target gene and a 3 'end regulation region of the bovine-derived K5 gene. An expression vector based on a bovine-derived K5 gene regulatory region and a beta-globin gene intron is obtained through research and used for specific expression in pig skin cells, and then a pig variety with specific characters is obtained through cultivation. Based on the expression vector, the pig with the black skin character is successfully prepared.

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

The invention is applicable to the technical field of biology, and provides a primer probe combination, a kit and a method for detecting high-risk human papilloma virus nucleic acid, a specific detection system is designed aiming at 18 high-risk HPVs, the system comprises a specific primer pair, a fluorescent probe, a nucleic acid extraction reagent and a PCR reaction reagent, a Beta-Globin gene is used as a reference gene, and the specific primer pair and the fluorescent probe are used for detecting the high-risk human papilloma virus nucleic acid. According to the invention, the number of primers is reduced through base merging design, primer dimer, mismatch and nonspecific binding are avoided, and specific typing of HPV16 and HPV18 and qualitative detection of other 16 high-risk HPVs are realized in combination with a multiplex fluorescent PCR technology; meanwhile, the reagent concentration and the PCR amplification procedure are optimized, and the kit has the advantages of being high in detection specificity, high in sensitivity, easy and convenient to operate, low in cost and high in clinical accuracy rate, can be effectively used for in-vitro qualitative detection of the high-risk HPV in a cervical exfoliated epithelial cell sample, and provides a reliable basis for early screening of cervical cancer.

Peptide nucleic acid (PNA) oligomers that target the β-globin gene and can increase the frequency of recombination of donor oligonucleotide at the site of a Sickle Cell Disease mutation are provided. Nanoparticle formulations for delivering the PNA oligomers and donor oligonucleotides, and potentiating agents for increase the frequency of recombination of the donor oligonucleotide are also provided. Methods of using the PNA oligomers, donor oligonucleotides, nanoparticles, and potentiating agents for treating Sickle Cell Disease are also provided.

The embodiments of the present application relate to the field of biological detection technologies, specifically an anemia screening kit for targeted capture of long - fragment DNA by CRISPR and Cas9, including sgRNA for targeted capture of thalassemia - related genes, Y - type adapters for circular amplification, and primers for circular amplification; the targeted capture range of the sgRNA is: chr11:5166361 - 5296578 (hg38), chr16:48994 - 210817 (hg38). This anemia screening kit for targeted capture of long - fragment DNA by CRISPR and Cas9 and its method target and capture large - fragment regions with thalassemia mutations through the CRISPR and Cas9 systems, covering five types of thalassemia mutation regions, namely α, β, δ, δβ, and εγδβ. By using third - generation or fourth - generation long - read sequencing technologies, it can effectively detect pathogenic mutations, complex structural variations, and large - fragment deletions that may cause thalassemia in the population, covering non - deletion and deletion mutations of the β - globin gene and related regulatory genes, including HS - 40 and the βLCR gene.