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Plasmid system

A technology of plasmids and vector plasmids, applied in the direction of using vectors to introduce foreign genetic material, viral peptides, biochemical equipment and methods, etc.

Pending Publication Date: 2022-01-04
登高制造有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When rep and cap are present on the same plasmid, there is a risk of producing unacceptable levels of rcAAV via intramolecular or intermolecular (depending on the plasmid carrying the ITR-heterologous nucleic acid-ITR)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0567] Example 1 - Construction ('trans-resolution') of helper and carrier plasmids for a two-plasmid system

[0568] helper plasmid

[0569] Nucleotides 200-4497 of wild-type AAV2 (Genbank Accession No. AF043303; SEQ ID NO: 1 ) containing the rep and cap genes were cloned into pUC19 to prepare a helper plasmid (Yanisch-Perron et al (1985), Gene, 33: 103-119). Next, AAV2 nucleotides 4461-4497 were deleted to minimize sequence homology to the vector plasmid (the construction of which is described below). To prevent Rep 78 expression while maintaining Rep 68 expression, an intron within the cloned rep gene was deleted. To provide expression of Rep52 following intronic deletion, the AAV2 nucleotide corresponding to rep 52 containing the p19 promoter was cloned immediately 3' to the intronless rep 68 gene. Most of the cap gene sequence was then deleted.

[0570] Ablation of the TATA box (T corresponding to AAV2 position 1823 and mutation of AAG corresponding to AAV2 position...

Embodiment 2

[0579] Example 2 - Two-plasmid System: Comparison of Helper Plasmid:Vector Plasmid Ratio

[0580] cell culture

[0581] Under standard conditions at 37°C, 95% relative humidity and 5% v / v CO 2 conditions, supplemented with 10% fetal bovine serum (FBS) and 1% GlutaMax TM HEK293T cells were maintained by adherent culture in Duchenne's modified Eagle's medium (DMEM) (L-alanine-L-glutamine dipeptide). Cell confluency during passaging ranged from 40-95%.

[0582] Transfection of HEK293T cells and preparation of cell lysates

[0583] Different molar plasmid ratios (from helper plasmid:vector plasmid 3:1 to 1:6) were used with helper plasmid and carrier plasmid (the latter containing the engineered cap gene and Factor IX expression cassette; Example 1) while maintaining total plasmid The amount of DNA transfected into HEK293T cells. The day before transfection, 1.5 × 10 per square centimeter of culture area 5 live cells were seeded in a 6 cm dish in a volume of 3 ml DMEM, 10%...

Embodiment 3

[0599] Example 3 - Determination of replication-competent AAV (rcAAV) in rAAV produced by the trans-split two-plasmid system frequency

[0600] rcAAV test

[0601] will use Purified drug substance from rAAV, which was mass-produced in bioreactor cells transfected with the two-plasmid system and purified using a number of downstream processes to remove product impurities, was limit tested for the presence of rcAAV. Using an engineered cap gene and (i) α-galactosidase A expression cassette (as described in Example 1) or (ii) except for a different partial codon-optimized Factor IX coding sequence as in Example The same Factor IX expression cassette vector plasmid as mentioned in 1 was used to produce rAAV batches. The two plasmid systems used to produce this rAAV batch used a shortened vector plasmid backbone and a corrected helper plasmid sequence as mentioned in Examples 1 and 2, respectively. In this limit test, rAAV transduced HEK293 cells in its most concentrated fo...

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Abstract

The present invention relates to two-plasmid systems, helper plasmids, and / or vector plasmids for producing recombinant AAV (rAAV) vectors. The invention further relates to methods using, or uses of, the two-plasmid systems, helper plasmids and / or vector plasmids of the invention.

Description

[0001] field of invention [0002] The present invention relates to two-plasmid systems, helper plasmids and / or vector plasmids for the production of recombinant AAV (rAAV) vectors. The invention further relates to methods of using the two-plasmid system, helper plasmids and / or carrier plasmids of the invention or uses thereof. [0003] Background of the invention [0004] Recombinant adeno-associated virus (rAAV) vectors have considerable potential for gene therapy because of their favorable safety profile and ability to transduce many tissues in vivo. However, production remains rather difficult and complex, and industrial-scale production scale-up has only been achieved to a limited extent. One reason is that rAAV production relies on co-infection with helper viruses to reproduce and establish a productive life cycle. A disadvantage of infecting cells with a replication-competent helper virus (eg, adenovirus) for rAAV production is that the produced rAAV stock is contamina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86
CPCC12N15/86C12N2750/14143C12N2750/14122C12N2750/14151C07K14/005
Inventor M·霍莱尔F·桑塔格R·科伯
Owner 登高制造有限公司
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