CN118086341A - Expression cassette for high expression of human glucocerebrosidase gene in liver - Google Patents
Expression cassette for high expression of human glucocerebrosidase gene in liver Download PDFInfo
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present disclosure relates to expression cassettes for high expression of human glucocerebrosidase genes in the liver, and in particular to expression cassettes for GBA1 genes encoding GCase proteins. In addition, the present disclosure also relates to vectors, viral particles and compositions comprising the GBA1 gene expression cassettes of the present disclosure, and to the use in the manufacture of a gene therapy for the treatment of diseases associated with GCase deficiency.
Description
Technical Field
The present disclosure is in the biomedical field, in particular, relates to expression cassettes for the GBA1 gene encoding GCase proteins. In addition, the present disclosure also relates to vectors, viral particles and compositions comprising the GBA1 gene expression cassettes of the present disclosure, and to the use in the manufacture of a gene therapy for the treatment of diseases associated with GCase deficiency.
Background
Gaucher disease (Gaucher disease) is a rare autosomal recessive metabolic disease, mainly due to mutations in the gene encoding Glucocerebrosidase (GBA). In recent years, rAAV has become a hotspot in gene therapy research by using the rAAV as a gene therapy vector, and an expression frame for efficiently expressing a target gene can reduce the dosage required by the therapeutic effect, so that the treatment cost is greatly reduced. In many eukaryotes, introns can increase the expression of a gene of interest by affecting the transcription rate, transcription stability, and translation efficiency of the mRNA.
Disclosure of Invention
The present disclosure provides a GBA1 gene expression cassette comprising a sequence selected from the group consisting of SEQ ID NOs: 2. SEQ ID NO: 4. or SEQ ID NO: 5.
In some embodiments, the intron is located between positions 27-28 bp of the GBA1 gene.
In some embodiments, the GBA1 gene expression cassette further comprises SEQ ID NO:1 and TTRm promoter as set forth in seq id no.
In some embodiments, the GBA1 gene is a code optimized sequence.
In some embodiments, the GBA1 gene expression cassette comprises SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO:9 or SEQ ID NO:10; the GBA1 gene has a nucleic acid sequence shown in SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO:9 or SEQ ID NO: shown at 10.
In another aspect, the present disclosure provides a vector comprising the GBA1 gene expression cassette as described above.
In some embodiments, wherein the vector is a viral vector, preferably the viral vector is an AAV vector, an adenovirus vector, or a lentiviral vector.
In another aspect, the present disclosure provides an adeno-associated virus (AAV) particle comprising a vector and a capsid protein as described above.
In some embodiments, wherein the AAV is selected from the group consisting of serotypes 9, 8, 1,2, 3B, 4,5, 6, 7, 10, 11, 12, 13, rh10, or hu37 and any one AAV serotype isolated from human and non-human mammals or variants thereof.
In another aspect, the present disclosure provides a pharmaceutical composition comprising a vector as described above, or an AAV particle as described above, and a pharmaceutically acceptable excipient.
In another aspect, the present disclosure provides the use of a GBA1 gene expression cassette as described above, a vector as described above, an AAV particle as described above, or a pharmaceutical composition as described above, in the manufacture of a medicament for treating or preventing a disease in a subject.
In some embodiments, an effective amount of a vector as described above, an AAV particle as described above, or a pharmaceutical composition as described above is administered to a subject.
In some embodiments, wherein the disease is gaucher disease.
In some embodiments, wherein the subject is a mammal, preferably the subject is a human.
Drawings
The present disclosure may be more fully understood with reference to the following drawings.
FIG. 1 shows the in vitro transfection results of plasmids containing the GBA1 expression cassette of the different introns.
Detailed Description
The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the particular modifications discussed should not be construed as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are intended to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated by reference in their entirety.
Unless otherwise the skilled artisan generally understand the same meaning.
The term "intron" as used herein means a non-coding DNA fragment. The intron of the present application refers to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2. SEQ ID NO: 4. or SEQ ID NO:5, and a nucleic acid sequence of the group consisting of seq id no.
The term "promoter" as used herein means a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence, such as a gene or a transgene. Promoters may be constitutive, inducible, repressible, or tissue specific.
The term "vector" as used herein refers to a nucleic acid comprising, consisting essentially of, or consisting of an intact replicon such that the vector may be replicated when placed into a cell by, for example, transfection, infection, or transformation procedures. It will be appreciated in the art that once inside the cell, the vector may replicate as an extrachromosomal (episomal) element, or may be integrated into the host cell chromosome. The vector may comprise nucleic acid derived from a retrovirus, adenovirus, herpes virus, baculovirus, modified baculovirus, papilloma virus, AAV viral vector, lentiviral vector, adenovirus vector, alphaviral vector, etc., preferably the vector described herein is selected from AAV vector, adenovirus vector, or lentiviral vector.
The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses associated with that name and belonging to the genus parvoviridae-dependent parvovirus. Adeno-associated virus is a single stranded DNA virus that grows only in cells, with some functions provided by co-infected helper viruses. All AAV serotypes apparently exhibit very similar replication characteristics mediated by homologous rep genes; and all carry three related capsid proteins. At least 13 sequentially numbered naturally occurring AAV serotypes are known in the art. Non-limiting exemplary serotypes for use in the methods disclosed herein include any of these 13 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes such as AAV-DJ and AAV php.b. AAV particles comprise, consist essentially of, or consist of three major viral proteins VP1, VP2, and VP 3. In embodiments, the AAV comprises an AAV capsid protein selected from the group consisting of AAVPHP.B、AAVrh74、AAV 110、AAV 204、AAV 214、AAV 214A、AAV 214e、AAV 214e8、AAV 214e9、AAV 214el 0、AAV ITB102_45 and AAV 214 AB. In embodiments, AAV refers to any of serotypes AAV1, AAV2, AAV3B, AAV, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, AAVrh10, AAVhu37, or AAV serotypes isolated from humans and non-human mammals, or variants thereof. In embodiments, the AAV particle comprises a polypeptide selected from the group consisting of 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AAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhER1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV SM 10-2、AAV Shuffle 100-1、AAV Shuffle 100-3、AAV Shuffle 100-7、AAV Shuffle 10-2、AAV Shuffle 10-6、AAV Shuffle 10-8、AAV Shuffle 100-2、AAV SM 10-1、AAV SM 10-8、AAV SM 100-3、AAV SM 100-10、BNP61 AAV、BNP62 AAV、BNP63 AAV、AAVrh.50、AAVrh.43、AAVrh.62、AAVrh.48、AAVhu.19、AAVhu.11、AAVhu.53、AAV4-8/rh.64、AAVLG-9/hu.39、AAV54.5/hu.23、AAV54.2/hu.22、AAV54.7/hu.24、AAV54.1/hu.21、AAV54.4R/hu.27、AAV46.2/hu.28、AAV46.6/hu.29、AAV128.1/hu.43、 true type AAV (ttAAV), UPENN AAV, japanese AAV10 serotype 、AAV CBr-7.1、AAV CBr-7.10、AAV CBr-7.2、AAV CBr-7.3、AAV CBr-7.4、AAV CBr-7.5、AAV CBr-7.7、AAV CBr-7.8、AAV CBr-B7.3、AAV CBr-B7.4、AAV CBr-E1、AAV CBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-e5、AAV CBr-E6、AAV CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.10、AAV CHt-6.5、AAV CHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-P1、AAV CHt-P2、AAV CHt-P5、AAV CHt-P6、AAV CHt-P8、AAV CHt-P9、AAV CKd-1、AAV CKd-10、AAV CKd-2、AAV CKd-3、AAV CKd-4、AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9、AAV.hu.48R3、AAV.VR-355、AAV3B、AAV4、AAV5、AAVF1/HSC1、AAVF11/HSC11、AAVF12/HSC12、AAVF13/HSC13、AAVF14/HSC14、AAVF15/HSC15、AAVF16/HSC16、AAVF17/HSC17、AAVF2/HSC2、AAVF3/HSC3、AAVF4/HSC4、AAVF5/HSC5、AAVF6/HSC6、AAVF7/HSC7、AAVF8/HSC8、AAVF9/HSC9、AAV-PHP.B（PHP.B）、AAV-PHP.A（PHP.A）、G2B-26、G2B-13、TH1.1-32、TH1.1-35、AAVPHP.B2、AAVPHP.B3、AAVPHP.N/PHP.B-DGT、AAVPHP.B-EST、AAVPHP.B-GGT、AAVPHP.B-ATP、AAVPHP.B-ATT-T、AAVPHP.B-DGT-T、AAVPHP.B-GGT-T、AAVPHP.B-SGS、AAVPHP.B-AQP、AAVPHP.B-QQP、AAVPHP.B-SNP（3）、AAVPHP.B-SNP、AAVPHP.B-QGT、AAVPHP.B-NQT、AAVPHP.B-EGS、AAVPHP.B-SGN、AAVPHP.B-EGT、AAVPHP.B-DST、AAVPHP.B-DST、AAVPHP.B-STP、AAVPHP.B-PQP、AAVPHP.B-SQP、AAVPHP.B-QLP、AAVPHP.B-TMP、AAVPHP.B-TTP、AAVPHP.S/G2A12、AAVG2A15/G2A3、AAVG2B4、AAVG2B5, and variants thereof.
As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, reptiles, and the like. The terms "patient" or "subject" are used interchangeably unless otherwise indicated. In the present disclosure, the preferred subject is a human.
As used herein, "treatment" of a disease in a subject refers to: (1) Preventing symptoms or disease occurrence in a subject susceptible to or not yet exhibiting symptoms of the disease; (2) inhibiting or arresting the development of the disease; or (3) ameliorating a disease or disease symptom or causing their regression. As understood in the art, "treatment" is a method of achieving a beneficial or desired result, including clinical results. For purposes of the present technology, beneficial or desired results can include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of the condition (including a disease), delayed or slowed progression of the condition (including a disease), amelioration or palliation of the condition (including a disease), and remission (whether partial or total), whether detectable or undetectable.
As used herein, the term "effective amount" means an amount sufficient to achieve the desired effect. In the case of use in therapy or prophylaxis, the effective amount will depend on the type and severity of the condition in question and the characteristics of the individual subject, such as general health, age, sex, weight and tolerance to the pharmaceutical composition. In gene therapy, for example, in some embodiments, an effective amount is an amount sufficient to modulate some or all of the functions of the gene of interest. In other embodiments, an effective amount of an AAV viral particle is an amount sufficient to obtain partial or complete function of a defective gene in a subject. One skilled in the art will be able to determine the appropriate amount based on these and other factors.
Examples
The following examples are merely illustrative and are not intended to limit the scope or content of the present invention in any way.
In vitro transfection
The hepatocyte line Huh7 (China academy of sciences typical culture Collection, cat. SCSP-526) was placed in 24 well plates at a cell density of 1.5X10 5 cells/well 24 h before transfection. mu.L of cell culture medium was added to each well. The transfection used was PEI-based transfection reagent, and 0.15. Mu.g of plasmids containing different introns and 0.15. Mu.g of plasmids containing the luciferase reporter gene were co-transfected per well. After transfection of 48 h, 300 μl of fresh whole cell culture medium was added to each well and the cells were re-incubated 24 h. Collecting cell culture supernatant, and detecting GCase enzyme activity by adopting an enzymatic reaction; cell lysis supernatant cells were collected after lysis of cells with cell lysis buffer (Promega) and the fluorescence intensity was read using a Varioskan LUX reader using a Steady-Glo luciferase assay system (Promega). All GCase activity reports were normalized with luciferase intensity.
Statistical analysis
Statistical analysis of t-test was performed using Prism 7 (Graph Pad) software.
Examples
Example 1: GBA1 expression cassette
In order to improve the therapeutic effect of gene therapy on diseases related to GBA1 gene, several expression frame sequences capable of efficiently expressing human glucocerebrosidase in liver are obtained through reasonable design and in vitro screening, wherein the intron 1, the intron 2, the intron 3 and the intron 4 are specifically arranged at the positions of 27-28 bp of GBA1 gene and are driven to be expressed by TTRm promoter (the nucleotide sequences are shown in Table 1).
TABLE 1 nucleotide sequences of the different elements
Example 2: in vitro transfection
The Plasmid backbone used in the invention is derived from Addgene pAAV. PU1a-SpCas9 (Plasmid # 121507), on the basis of the backbone Plasmid, the ampicillin resistance is replaced by kana resistance, the expression frame part is replaced by GBA1 expression frame sequences containing different introns in the patent, first, the intron 1, the intron 2, the intron 3 and the intron 4 are respectively cloned between the positions of 27-28 bp of a human glucocerebrosidase gene (GBA 1), and are driven to be expressed by TTRm promoters, and the information of the Plasmid expression frame is shown in Table 2. The constructed plasmid was then transfected in vitro in Huh7 hepatocytes and the ability of the different GBA1 expression cassette sequences to express glucocerebrosidase in hepatocytes was compared by detecting GCase enzyme activity. As shown in FIG. 1, the GBA1 expression cassette containing the intron 1 (PG 173), the intron 3 (PG 175) and the intron 4 (PG 176) can remarkably enhance the expression level of GCase protein in liver cells compared with the GBA1 expression cassette (PG 172) without the intron, and the hGBA expression cassette containing the intron 2 (PG 174) can remarkably reduce the expression level of GCase protein in liver cells. It was demonstrated that the modified and screened GBA1 expression cassette sequences comprising intron 1 (PG 173), intron 3 (PG 175) and intron 4 (PG 176) are capable of efficiently expressing GCase protein in liver cells.
TABLE 2 plasmid information
Incorporated by reference
The entire contents of each patent and scientific document referred to herein is incorporated by reference for all purposes.
Equivalency of
The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The above embodiments should therefore be regarded as illustrative in all respects, rather than limiting on the invention described herein. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.
Claims (10)
1. A GBA1 gene expression cassette comprising a sequence selected from the group consisting of SEQ ID NOs: 2. SEQ ID NO: 4. or SEQ ID NO:5, said intron being located between positions 27-28 bp of the GBA1 gene.
2. The GBA1 gene expression cassette of claim 1, wherein the GBA1 gene expression cassette comprises the sequence of SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO:9 or SEQ ID NO:10.
3. A vector comprising the GBA1 gene expression cassette of claim 1 or 2.
4. The vector of claim 3, wherein the vector is a viral vector.
5. The vector of claim 4, wherein the viral vector is an AAV vector, an adenovirus vector, or a lentiviral vector.
6. An adeno-associated virus (AAV) particle comprising the vector of any one of claims 3-5 and a capsid protein.
7. The adeno-associated virus (AAV) particle of claim 6, wherein the AAV is selected from the group consisting of serotypes 9, 8, 1,2, 3B, 4,5, 6, 7, 10, 11, 12, 13, rh10 or hu37, and any one AAV serotype isolated from humans and non-human mammals or variants thereof.
8. A pharmaceutical composition comprising the vector of any one of claims 3 to 5, or the adeno-associated virus (AAV) particle of claim 6 or 7, and a pharmaceutically acceptable excipient.
9. Use of the GBA1 gene expression cassette of claim 1 or 2, the vector of any one of claims 3 to 5, the adeno-associated virus (AAV) particle of claim 6 or 7, or the pharmaceutical composition of claim 8 in the manufacture of a medicament for treating or preventing a disease in a subject.
10. The use of claim 9, wherein the disease is gaucher's disease.
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