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Nucleic acid construct for AIDS gene therapy

A nucleic acid construct, anti-AIDS technology, applied in genetic engineering, virus/phage, plant genetic improvement, etc., can solve the problems of high development and use cost, difficult clinical use, and lack of advantages in combination drugs

Pending Publication Date: 2022-06-21
KANGLIN BIOTECHNOLOGY (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the same as the high-efficiency anti-HIV small-molecule drugs: the specific broad-spectrum neutralizing antibody only targets a single epitope of a single viral protein (HIV-gp160), and the escape phenomenon caused by viral mutation is still unavoidable, and due to The cost of development, production and use of broad-spectrum neutralizing antibodies is much higher than that of small-molecule anti-HIV drugs, and there is no advantage in combination therapy. Therefore, a large number of anti-AIDS broad-spectrum neutralizing antibody drugs under research are difficult to be used in clinical practice.

Method used

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  • Nucleic acid construct for AIDS gene therapy
  • Nucleic acid construct for AIDS gene therapy
  • Nucleic acid construct for AIDS gene therapy

Examples

Experimental program
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Effect test

Embodiment 1

[0072] Example 1: Structural Design of Expressing HIV Neutralizing Antibodies and Amphotropic Anti-HIV Neutralizing Antibody Molecules

[0073] The structure of the anti-HIV neutralizing antibody and amphotropic anti-HIV neutralizing antibody molecule used in the present invention is:

[0074] 1. Amphotropic anti-HIV neutralizing antibody molecules: from the N-terminal to the C-terminal of the protein molecule are: signal peptide-anti-HIV-gp160 broad-spectrum neutralizing antibody single-chain variable region fragment (VH-ggggsnlinker-VL) — ggggsnlinker — single chain variable region fragment of anti-human CD4 antibody (VH-ggggsnlinker-VL) — ggggsnlinker — human IgG1 CH2 — human IgG1 CH3. The indicated sequence is translated in the cell as a dimer and secreted out of the cell. The expected mature molecular structure is as Figure 2A It was shown that the gene structure such as Figure 3A show.

[0075] 2. Anti-HIV broad-spectrum neutralizing antibody: from the N-terminal t...

Embodiment 2

[0076] Example 2: Gene Design for Expressing HIV Neutralizing Antibodies

[0077] A total of 4 anti-HIV neutralizing antibody gene expression frameworks were constructed, namely:

[0078] 1. Anti-HIV broad-spectrum neutralizing antibody 10E8V4-5R+100cF (see SEQ ID No: 1 for the protein sequence) (see SEQ ID No: 2 for the DNA sequence)

[0079] 2. Anti-HIV broad-spectrum neutralizing antibody PGT128 (see SEQ ID No: 3 for the protein sequence) (see SEQ ID No: 4 for the DNA sequence)

[0080] 3. Amphophilic anti-HIV neutralizing antibody molecule KL-BsHIV01 comprising anti-HIV neutralizing antibody 10E8V4-5R+100cF-scfv (see SEQ ID No:5 for the protein sequence) (see SEQ ID No:6 for the DNA sequence)

[0081] 4. Amphotropic anti-HIV neutralizing antibody molecule KL-BsHIV02 comprising anti-HIV neutralizing antibody PGT128-scfv (see SEQ ID No: 7 for the protein sequence) (see SEQ ID No: 8 for the DNA sequence)

[0082]1 and 2 include heavy chain signal peptide, anti-HIV-1-gp41-MP...

Embodiment 3

[0084] Example 3: Gene Therapy Vector Constructs for HIV Neutralizing Antibodies

[0085] like Figure 4B , 4C As shown, the above monoclonal antibody molecular gene expression framework was cloned into the latest generation of lentiviral vector pKL-kan-lenti-EF1α-WPRE ( Figure 1A ) (see SEQ ID No: 9 for the DNA sequence). The lentiviral vector includes: 5' LTR, wherein the promoter region of LTR is replaced with CMV promoter; ψ packaging signal; retroviral export element RRE; cPPT; promoter CBH; Polynucleotides; post-transcriptional regulatory elements are wPRE; PPT; ΔU3 3'LTR; and poly A signal. The neutralizing antibody gene expression framework designed in Example 2 was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; it was cloned into the lentiviral vector backbone pKL-kan-lenti-EF1α-WPRE by homologous recombination methods well known in the art Between the multiple cloning sites EcoRI / EcoRV on the website, the sequence information was confirmed by sequencin...

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Abstract

The invention relates to the technical field of biological medicines, in particular to a nucleic acid construct for AIDS gene therapy, which comprises a plurality of polynucleotides for coding single-chain variable fragments of an anti-AIDS neutralizing antibody and polynucleotides for coding an immune globulin Fc fragment. The single-chain variable region fragment of the anti-AIDS neutralizing antibody comprises a single-chain variable region fragment capable of being specifically combined with HIV and a single-chain variable region fragment capable of being specifically combined with a CD4 receptor. The construction body can be used for gene therapy of AIDS caused by HIV virus infection, can be used for expressing an amphiphilic / polyphilic neutralizing antibody with broad-spectrum and high-efficiency neutralizing activity in vivo and in vitro, and can be used for clinical research and new drug research and development of AIDS gene therapy drugs delivered by recombinant viruses or non-viral vectors.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a nucleic acid construct for AIDS gene therapy. Background technique [0002] AIDS (acquired immune deficiency syndrome, AIDS) caused by human immunodeficiency virus (human immunodeficiency virus, HIV) infection is one of the most harmful infectious diseases in the world today. With the development of anti-AIDS drugs, the expected survival period of AIDS patients who have been actively treated has been greatly extended, but the side effects and limitations of cocktail therapy, as well as the gradual emergence of drug-resistant strains still pose a serious threat to the majority of AIDS patients. The long-term economic and social burden, the obvious decline in the quality of life and the obvious pain. [0003] The broadly neutralizing antibodies (bNAb) with HIV-1 neutralizing activity produced in a small number of elite infected persons over several years can efficiently and ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/864C12N15/62C12N7/01C12N5/10C07K16/46A61K39/42A61P31/18
CPCC12N15/86C12N7/00C07K16/1063C07K16/2812A61P31/18C12N2740/15021C12N2740/15043C12N2750/14121C12N2750/14143C07K2319/02C07K2319/30C07K2317/31C07K2317/622C07K2317/76A61K2039/505C12N2710/00Y02A50/30C12N2740/16043C07K16/1045C07K2317/33
Inventor 吴昊泉孙保贞党颖
Owner KANGLIN BIOTECHNOLOGY (HANGZHOU) CO LTD
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