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Construct

A technology of constructs and viral vectors, applied in the field of constructs, can solve the problem that the titer does not have an inhibitory effect

Active Publication Date: 2014-08-27
OXFORD BIOMEDICA (UK) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Contrary to expectations, for many constructs the improvements brought about by these fusion designs correlated with increases in vector titers No association, indicating that IRES sequences have no inhibitory effect on titers

Method used

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Embodiment 1

[0260] Example 1 - Generation of vector from pONYK-TAiC and generation of catecholamines from integrated vector

[0261] The first fusion construct to be generated and tested for improved titers compared to pONYK1 was pONYK-TAiC( figure 1 ). Lentiviral vector (LV) preparations were generated in triplicate using the pONYK-TAiC or pONYK1 genomes and the titers of the resulting vectors were quantified by DNA integration assays ( figure 2 a). These data surprisingly show that the titer of the vector generated from pONYK-TAiC is the same as pONYK1, ie removal of 1 IRES element does not improve the titer. HPLC analysis was performed on transduced HEK293T cell supernatants to examine the levels of L-DOPA and dopamine produced. HPLC results ( figure 2 b) shows that cells transformed with the pONYK-TAiC vector produced a 2.4-fold increase in total catecholamine levels compared to cells transduced with the pONYK1 vector. L-DOPA levels were comparable between the two genomes, h...

Embodiment 2

[0262] Example 2 - Vector Production and Production of Catecholamines from Integrated Vectors

[0263] Together with pONYK-TAiC and pONYK1, three other dopaminease fusion plasmids (pONYK-ATiC, pONYK-TCiA, pONYK-ATC( figure 1 ). LV preparations were generated in triplicate using each of the different genomic plasmids and the titers of the resulting vectors were quantified by DNA integration analysis ( image 3 a). The results showed that the titers were similar for all vectors, ranging from 1.3E+05TU / ml to 4.0E+05TU / ml. Interestingly, pONYK-ATC lacking both IRES elements showed no increase in titer, suggesting that the fusion construct did not alter vector production and that the presence of the transgenic rearranged GS15 linker did not affect titer. The results of HPLC analysis showed that HEK293T cells transduced with each of the different vectors resulted in different catecholamine production ( image 3 b). Furthermore, the amount of L-DOPA converted to dopamine vari...

Embodiment 3

[0265] Example 3 - Evaluation of Dopaminerase Levels in HEK293T Cells Transfected with Different Fusion Plasmids

[0266] To investigate protein expression levels, western blot analysis of each transgene product (AADC, CH1, and TH) was performed from cell lysates of HEK293T cells that had been transfected with each fusion genome plasmid ( Figure 4 ). The results show that for each of the different fusion constructs, each dopamine synthetase is present and of the predicted size. This indicates that each genomic construct is capable of expressing dopamine synthase with a GS15 linker, including the triple fusion cassette (pONYK-ATC), as a 124 kDa band is visible in all three western blots, which is the dopamine enzyme containing all three links The expected size of the fusion protein of the protein. The levels of each of the different proteins expressed by the different genomic constructs varied widely. The highest levels of all three proteins appeared to be expressed from ...

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Abstract

The present invention provides a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP- cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding TH is linked to the nucleotide sequence encoding CHI such that they encode a fusion protein TH-CH1. The invention also provides a viral vector comprising such a nucleotide sequence and its use in the treatment and / or prevention of Parkinson's disease.

Description

field of invention [0001] The present invention relates to a construct comprising a nucleotide sequence encoding an enzymatic activity involved in the dopamine synthesis pathway. At least two nucleotide sequences are operably linked such that they encode a fusion protein. The present invention also provides viral vector genomes, vector production systems and viral vector particles comprising the nucleotide sequence. Expression of the nucleotide sequence in vivo, e.g. by gene therapy using viral vector particles, results in the synthesis of dopamine, which is useful in the treatment and / or prevention of neurological disorders characterized by a reduction or loss of dopamine-producing neurons, such as Pa It is useful in Parkinson's disease. Background of the invention [0002] Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine-producing neurons in the substantia nigra. This eventually leads to a depletion of dopamine in the striat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/55A61K48/00C12N15/62
CPCA61K48/00C12P13/001C12Y114/16002A61K31/137C12N15/86C12N9/78C12N2799/027C12N9/88C12N15/62C12N9/0071C12Y401/01028C12Y305/04016A61K48/005C07K2319/00A61P25/16A61P25/28C12N2740/15043C12N2830/48C12N2840/203A61K38/54C12N15/867
Inventor K.米特罗范奥斯S.拉尔夫H.斯图尔德A.金斯曼
Owner OXFORD BIOMEDICA (UK) LTD
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