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In-vitro preparation method of linear double-chain adeno-associated virus genome

A virus genome and double-stranded gland technology, applied in the field of molecular biology, can solve the problems of slow gene expression and limited virus packaging capacity, and achieve the effects of simple and easy to control production process, high product purity and easy purification

Inactive Publication Date: 2016-11-09
BIOMICS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Conventional single-stranded linear AAV also has disadvantages as a gene delivery vector: 1. Virus packaging capacity is limited, only about 4.4kb heterologous DNA (Grieger JC, Samulski RJ. Advances in Biochemical Engineering / Biotechnology, 2005); 2. AAV Mediated gene expression is slower because the single-stranded AAV genome must be converted to a double-stranded DNA genome prior to heterologous gene expression
The single-stranded adeno-associated virus genome produced by this method can avoid the production of AAV viral proteins, but still requires a lot of separation and purification work, and needs to clean the contamination of recombinant baculovirus (Lina Li, et al., PLOS One, 2013)

Method used

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  • In-vitro preparation method of linear double-chain adeno-associated virus genome
  • In-vitro preparation method of linear double-chain adeno-associated virus genome
  • In-vitro preparation method of linear double-chain adeno-associated virus genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Preparation of linear double-stranded adeno-associated virus genome expression vector

[0065]The left and right ITR gene sequences of AAV2 were synthesized onto the pUC57 vector (pUC57-IT). Add cloning point PvuII or PacI at both ends of the vector to form pAAVeGFP vector. Using the pAAVeGFP vector as a template, the CMV-EGFP-PolyA expression cassette was synthesized by PCR polymerase chain reaction, and subcloned into pUC57-ITR to obtain the pAAVEGFP-ITR expression vector. Digest the pAAVEGFP-ITR vector with PacI or PVUII enzymes to isolate ITR-CMV-EGFP-polyA + -ITR fragment, which is a linear double-stranded adeno-associated virus genome for expressing enhanced green fluorescent protein eGFP, its molecular structure is as attached figure 1 shown.

[0066] Among them, the left and right ITR gene sequences of AAV2, namely L-ITR (SEQ ID No: 5) and R-ITR (SEQ ID No: 6), are:

[0067] 5'-CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC...

Embodiment 2

[0069] Embodiment 2 prepares circular DNA template,

[0070] Using ligase (ligase T4, purchased from NEB Company), according to the instructions of the kit, the ITR-CMV-EGFP-polyA obtained in Example 1 was + -ITR fragments are connected to obtain a circular template (see attached figure 2 ).

Embodiment 3

[0071] Example 3 Rolling Circle Amplification (RCA)

[0072] 3.1 Design of specific paired primers

[0073] Design and synthesize specific DNA amplification primers complementary to the adeno-associated virus inverted terminal repeat (AAV2ITR), as shown in Table 1:

[0074] Table 1 Specific DNA amplification primers

[0075]

[0076] 3.2 DNA polymerase selection

[0077] Any commercially available strand-displacing DNA polymerase is suitable for use in the methods of the invention. phi29 DNA polymerase is preferred in the methods of the invention. The full-length phi29DNA polymerase gene is 1719bp, encoding a total of 572 amino acids, and the molecular weight of the enzyme protein is 66kD, which is a single subunit enzyme. It has high-efficiency strand displacement activity, 3'-5' nucleotide correction activity, high fidelity, and its optimal reaction temperature is 30°C. The phi29 DNA polymerase used in this experiment was purchased from NEB Company.

[0078] 3.3 Rol...

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Abstract

The invention discloses a linear double-chain adeno-associated virus genome. The linear double-chain adeno-associated virus genome comprises an adeno-associated virus left inverted terminal repeat L-ITR, a nucleic acid polymerase promoter sequence, a target gene sequence, a polyadenylic acid signal sequence and an adeno-associated virus right inverted terminal repeat R-ITR and does not contain virus replication protein genes Rep, virus structural protein genes Cap, a bacterial plasmid DNA sequence and bacterial DNA methylated modification. The linear double-chain adeno-associated virus genome has the advantages that the limitation that a single-chain adeno-associated virus genome needs a rate-limiting step for converting the single-chain genome into a double-chain genome during gene expression is broke through, and the toxic and side effect of triggering congenital immunity response is avoided. The invention further discloses an in-vitro preparation method of the linear double-chain adeno-associated virus genome.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a linear double-strand adeno-associated virus genome, its in vitro preparation method, and its use in the preparation of gene therapy drugs. Background technique [0002] Gene therapy has been widely used in the treatment of genetic diseases, malignant tumors, cardiovascular diseases, infectious diseases and so on. As of February 2016, 2,356 gene therapy drugs have entered clinical trials in the world (Gene therapy Clinicaltrials worldwide. Feb. 2016). Gene therapy vectors include bacterial plasmid expression vectors and viral expression vectors. Prokaryotic DNA such as bacterial plasmid vectors contain bacterial replication sequences, resistance genes, and CpG sequences for bacterial DNA methylation. These may raise serious safety concerns during gene therapy. Bacterial plasmid vectors also often contain endotoxins, which can reduce gene transfer efficiency and tri...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/35C12N15/54C12N15/10A61K48/00A61K31/713A61P31/20A61P35/00
CPCC12N15/11A61K31/713A61K48/005A61K48/0091C07K14/00C07K14/005C12N2750/14122
Inventor 仇建萍吉怡周宋峰李铁军朱远源
Owner BIOMICS BIOTECH
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