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Recombinant adeno-associated virus vectors and methods for treating or preventing hemophilia B

A virus particle, construct technology, applied in the field of gene therapy

Active Publication Date: 2022-04-05
SHANGHAI VITALGEN BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application only demonstrated the successful expression of coagulation factor IX in vitro and did not perform any in vivo experiments

Method used

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  • Recombinant adeno-associated virus vectors and methods for treating or preventing hemophilia B
  • Recombinant adeno-associated virus vectors and methods for treating or preventing hemophilia B
  • Recombinant adeno-associated virus vectors and methods for treating or preventing hemophilia B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] Embodiment 1. Selection of highly active FIX mutants

[0193] In the early clinical trials of FIX gene therapy, the wild-type FIX protein sequence was selected, and the codon optimization of the FIX coding sequence was not performed, and the steady-state maintenance level of FIX activity was low and the time was short.

[0194] FIX PADUA is a naturally occurring FIX mutant with a single amino acid mutation (R338L) found in an Italian adolescent patient with venous thrombosis. Compared with wild-type FIX, the coagulation activity (FIX:C) can be increased by 5-10 times under the same expression level (FIX:Ag). Spark's SPK-9001, UniQure's AMT-061 and Freeline's FLT180a all selected highly active FIX PADUA variants as transgenes.

[0195] To confirm FIX PADUA Whether the variant does have improved expression levels and protein activity relative to wild-type FIX, the FIX WT 、FIX PADUA Ligated into scAAV vectors and ssAAV vectors and used to transfect human liver tumo...

Embodiment 2

[0198] Screening of Regulatory Elements in Example 2.FIX Gene Expression Cassette

[0199] The inventors selected different regulatory elements to design and prepare 9 scAAV vector expression cassettes (HB1-HB9) and 8 ssAAV vector expression cassettes (HB10-HB17) ( image 3and Table 1), compared their expression efficiencies in human liver tumor cells HepG2 and Huh7, and expressed in CMV-FIX PADUA As a control (HB0). Considering the difference in the DNA loading capacity of scAAV and ssAAV, the FIX expression efficiency of scAAV and ssAAV vectors were compared separately.

[0200] Table 1. Combinations of regulatory elements in the constructs

[0201]

[0202]

[0203] The transfection conditions were the same as in Example 1. Cell culture supernatant was collected 72 hours after transfection for FIX ELISA detection (abcam, ab168546) and Biophen FIX activity detection (HYPHEN BioMed, 221802).

[0204] The HepG2 cell experiment results of the first batch of 7 scAAV ...

Embodiment 3

[0207] Example 3. Optimization of Coding Sequences

[0208] The inventor designed and synthesized 8 codon-optimized FIX PADUA The coding sequences are respectively named as FIX-PADUA-co1 to FIX-PADUA-co8, and the nucleotide sequences thereof are respectively shown in SEQ ID NOs: 16-23. Using these codon-optimized coding sequences, the corresponding recombinant vectors were constructed and the transduction experiments as described in previous examples were repeated in HepG2 and Huh7 cells.

[0209] Figure 10 The detection results of the expression level and activity of the above 8 different codon optimized sequences delivered by the HB9 vector in HepG2 cells are shown, and are compared with the FIX of SEQ ID NO:12 without codon optimization PADUA Coding sequences were compared. From Figure 10 The results show that FIX-PADUA-co8, FIX-PADUA-co5 and FIX-PADUA-co2 are the three codon-optimized sequences with the best performance, especially the performance of FIX-PADUA-co8 an...

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PUM

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Abstract

The present invention relates to expression constructs and recombinant adeno-associated virus (rAAV) vectors that express coagulation factor IX, viral particles and pharmaceutical compositions comprising said rAAV vectors, and their use for the treatment or prevention of hemophilia B.

Description

technical field [0001] The present invention relates to the field of gene therapy. In particular the present invention relates to expression constructs and recombinant adeno-associated viruses (rAAV) expressing (human) coagulation factor IX, viral particles and compositions comprising said rAAV, and their use in the treatment or prevention of human coagulation factor IX deficiency The use of the hemorrhagic symptoms of hemophilia B (referred to as HB) caused by coagulation dysfunction. Background technique [0002] Hemophilia (hemophilia) is a group of bleeding disorders of hereditary coagulation dysfunction. Gene mutations lead to active thromboplastin production disorders. It is X-chromosomal recessive inheritance. It is divided into hemophilia A (deficiency of coagulation Factor VIII (FVIII) and HB (deficiency of factor IX (FIX). The main clinical manifestations of HB are intra-articular hemorrhage and intra-muscular hemorrhage, and the resulting permanent joint deforma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01C12N15/12A61K38/36A61P7/04
CPCA61K38/36C12N15/867C07K14/435A61P7/04C12N7/00
Inventor 郭璐韩稼葆李伟田幸雪刘宾朱晰赵小平
Owner SHANGHAI VITALGEN BIOPHARMA CO LTD
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