Vectors for treatment of friedreich's ataxia

A carrier and mutual aid technology, applied in the direction of vectors, the use of vectors to introduce foreign genetic material, gene therapy, etc., can solve the problems that there is no effective treatment for FRDA

Pending Publication Date: 2020-08-14
FUNDACIO INST DINVESTIGACIO & CIENCIES DE LA SALUT GERMANS TRIAS I PUJOL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] There is currently no effective therapy for the treatment of FRDA and thus new therapeutic agents for the treatment of FRDA are needed

Method used

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  • Vectors for treatment of friedreich's ataxia
  • Vectors for treatment of friedreich's ataxia
  • Vectors for treatment of friedreich's ataxia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: Plasmid construction

[0098] The coding sequence of isoform 1 of human futaxin (hFXN) was fused to the hemagglutinin tag (HA) and utilized Cloning into the pcDNA3.1 expression vector was performed using the HD cloning kit (Clontech). This fusion system was used for all cloning steps. Several constructs were generated combining CMV or synapsin (phSYN), neuron-specific enolase (phNSE), the 1,255 bp FXN promoter (phFXN1255), or human phosphoglycerate kinase isoform 1 ( The human (h) promoter (p) of phPGK1) was fused to the coding region of the FXN gene. In addition to the woodchuck hepatitis response element (WPRE) sequence at the 3' end, additional regulatory elements such as CMV enhancer and Kozak sequences were added at the 5' end. The constructs generated by all these expression vectors are listed in Table 2.

[0099] Also by replacing the coding sequence of FXN with the firefly luciferase coding sequence (LUC) (amplified from the pGL3-LUC vector (Pr...

Embodiment 2

[0100] Example 2: Construction and preparation of recombinant adeno-associated virus vector.

[0101] The expression cassettes from the pcDNA3.1-phPGK-kFXN-HA-WPRE and pcDNA3.1-phPGK-kLUC-HA-WPRE plasmids were cloned into the SnaBI-MfeI site of the recombinant AAV9 vector (rAAV9-phPGK1-FXN-HA-WPRE vector and rAAV9-phPGK-LUC-WPRE vector). In addition, a control null vector (AAV2 / 9-null) lacking the FXN coding sequence was generated. All three constructs and virus particles were produced by the Vector Production Unit at the Center of Animal Biotechnology and GeneTherapy, Universitat Autònoma de Barcelona. The final titer obtained was 1.4×10 13 vg / ml (AAV9-phPGK-FXN-HA-WPRE), 9.8×10 12 vg / ml (AAV9-phPGK-LUC-WPRE), and 6×10 12 vg / ml (AAV2 / 9-null).

Embodiment 3

[0102] Example 3: Optimizing Musclein Expression

[0103] 1. Cell culture, in vitro expression and luciferase reporter assay

[0104] Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (Sigma), 2 mM glutamine, 50 μg / ml penicillin / streptomycin (Life technologies) at a 70% confluency in a 10 cm dish Mouse neuroblastoma cells (N2a), human neuroblastoma cells (SH-SY5Y) and human embryonic kidney (HEK 293) cells were cultured in medium. Transfection was performed using lipofectamine 2000 (Life technologies) for N2a and SH-SY5Y cells and calcium phosphate for HEK 293, using only 4 μg of plasmid DNA in addition to 0.25 μg of EGFP. After transfection, the medium was replaced with fresh DMEM medium (for HEK cells) and Neurobasal, B27 supplement, 10 μM retinoic acid, 2 mM glutamine, 50 μg / ml penicillin / streptomycin (for N2a and SH- SY5Y cells). Forty-eight hours after changing the cell culture medium, cells transfected with the expression plasmids listed in...

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Abstract

The present invention provides gene therapies for the treatment of Friedreich's ataxia. Specifically, the present invention provides a nucleic acid, cloning vector and transfer vector for the production of an adeno-associated virus (AAV) vector. The nucleic acid comprises (i) a nucleic acid sequence encoding frataxin, (ii) a phospho-glycerate-kinase (PGK) promoter, and (iii) a woodchuck hepatitisvirus posttranscriptional regulatory element (WPRE). The present invention also provides a pharmaceutical composition which comprises the AAV vector or nucleic acid. Also, the AAV vector, nucleic acidor pharmaceutical composition can be used as a medicament, specifically as a medicament for the treatment of Friedreich's ataxia.

Description

technical field [0001] The invention may be included in the field of new therapies for the treatment of Friedreich's ataxia. In particular, the application relates to new products for gene therapy. Said product is capable of treating Friedrich's ataxia. Background technique [0002] Friedrich's ataxia (FRDA; OMIM #229300) is a rare inherited neurodegenerative disorder that causes progressive damage to the nervous system, causing problems ranging from gait disturbance and speech problems to heart disease or myocardial symptoms of illness. The disease is named after the physician Nikolaus Friedreich who first described it in the 1860s. Ataxia, which involves movement coordination problems such as clumsy movements and instability, occurs in Friedrich's ataxia due to degeneration of nerve tissue in the spinal cord and nerves that control muscle movement in the arms and legs. Spinal cord atrophy with nerve cells losing some of their myelin sheath. Although Friedrich's ataxia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86
CPCC12N15/86C12N2750/14143C12N2830/48A61P25/00A61K48/005A61K48/0058A61K48/0066A61P25/14
Inventor 安东尼·马蒂拉·杜埃尼亚斯艾维丽斯·桑切斯·迪亚兹爱德华·巴拉古·卡巴塞斯
Owner FUNDACIO INST DINVESTIGACIO & CIENCIES DE LA SALUT GERMANS TRIAS I PUJOL
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