36 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 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.

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.

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.

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.

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.

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.

Provided herein are compounds of Formula (I). The compounds described herein are useful in treating a disease associated with the expression of endogenous embryonic / fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and / or sickle cell disease through at least the induction of the globin gene expression in erythrocytes), and / or anemia (β-thalassemia and / or sickle cell anemia). The compounds described herein are useful in treating, delaying, and / or preventing the adverse effects of β-thalassemia and / or sickle cell disease, inducing γ globin production, and / or inducing the expression of embryonic / fetal globin genes in a subject, cell, tissue, or biological sample. Also provided in the present disclosure are pharmaceutical compositions, kits, and methods of using the compounds for inducing γ globin production described herein and for treating any of the target diseases described herein.

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.