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Novel mechanism to control RNA virus replication and gene expression

A kind of RNA virus and virus technology, applied in the field of new mechanisms to control RNA virus replication and gene expression, can solve problems such as limited neurotoxicity and side effects

Pending Publication Date: 2022-02-01
BOEHRINGER INGELHEIM INT GMBH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical development of VSV is limited by potential neurotoxic side effects shown in laboratory animals

Method used

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  • Novel mechanism to control RNA virus replication and gene expression
  • Novel mechanism to control RNA virus replication and gene expression
  • Novel mechanism to control RNA virus replication and gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0235] Example 1: Generation of a protease-regulated ON switch, VSV-P-prot

[0236] To generate tunable switches to control the activity of RNA viruses, we developed a system that incorporates autocatalytically active protease sequences into genes essential for VSV gene expression and replication ( figure 1 a). In our first ON-switch construct, we introduced the HIV protease dimer into the cofactor of VSV polymerase, the P-protein (SEQ ID NO: 1), resulting in VSV-P-prot. It was previously shown that intramolecular insertion sites do not affect P protein function (Das et al., J Virol., 2006, 80(13):6368-6377). Dimerization is required for HIV protease function. To facilitate immediate post-translational proteolytic activity, gene insertion constructs were designed to contain two copies of the HIV protease (PR) linked by a flexible linker ( figure 1 b. 2) (Krausslich, PNAS, 1991, 88(8): 3213-3217). Flexible linkers (SEQ ID NO: 8 and 9) were also applied upstream and downstre...

Embodiment 2

[0239] Example 2: VSV-P-prot can be regulated in a dose-dependent manner and by various HIV protease inhibitors

[0240] To test whether the amprenavir-dependent activity of VSV-P-prot is generally applicable to other members of the HIV protease inhibitor class, BHK cells were treated with the second-generation compounds saquinavir (10 μM) and indinavir (10 μM ) were incubated together, and then infected with VSV-P-prot at an MOI of 0.01. Consistent with the amprenavir effect, both inhibitors promoted viral gene expression (GFP signaling) and viral replication (plaque formation) ( Figure 7 ), demonstrating the universal targeting of the HIV protease-based VSV ON switch system. Lopinavir (10 μM) and other HIV protease inhibitors were also shown to modulate VSV-P-prot (data not shown).

[0241] Amprenavir doses for virus production and initial studies were selected based on previously described APV plasma concentrations in APV orally treated patients (Sadler et al., Antimicro...

Embodiment 3

[0242] Example 3: VSV-Pprot lacks neurotoxicity and intracranial spread

[0243] It is well known that once VSV enters the CNS space, it can cause significant neurotoxicity in experimental animals. VSV glycoproteins display a strong affinity for neurons, and anterograde and retrograde axonal spread have been described. To determine to what extent VSV-P-prot abolished neurotoxicity compared to normal VSV, we employed direct stereotaxic injection into the striatum of mice. Intracranial infusion of wild-type VSV-dsRed (2x105TCID 50 , 2 μl) resulted in profound neurotoxic signs ( Figure 9 A), manifested by hindlimb paralysis, lack of coordination, hunched position, and severe weight loss within 2 days of injection ( Figure 9 C). For humane reasons, all mice must be euthanized within 4 days ( Figure 9B). In stark contrast, when VSV-P-prot was injected into the brain at the same dose, there were no signs of neurotoxicity. When treated simultaneously with amprenavir and rit...

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Abstract

The present invention relates to a novel mechanism to control RNA virus replication and gene expression using a conditional protease approach using a specific protease inhibitor for regulation. More specifically it relates to a single-stranded RNA virus, preferably of the order Mononegavirales, comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease and a cleavage site for said protease. The protease can be inhibited using a protease inhibitor and hence the protease and the cleavage site for said protease form a regulatable switch. By changing the insertion site of the regulatable switch from an INTRA- to an INTER-molecular location in the at least one protein essential for viral transcription and / or replication, the effect of the protease inhibitor can be altered from an ON switch to an OFF switch. RNA virus may further encode a heterologous protein, the expression of which is then regulated by regulating viral activity. The ON switch may also be used in an RNA virus to directly regulate heterologous protein expression. Further provided are in vivo and in vitro uses of said virus with conditional viral activity or heterologous protein expression.

Description

technical field [0001] The present invention relates to novel mechanisms for the control of RNA virus replication and gene expression using a conditional protease approach regulated using protease-specific inhibitors. More particularly, it relates to single-stranded RNA viruses, preferably of the order Single-stranded Antiviridae, comprising a multinuclear nucleus encoding at least one protein necessary for viral transcription and / or replication, a protease and a cleavage site for said protease. nucleotide sequence. A protease can be inhibited using a protease inhibitor, whereby the protease and the cleavage site of said protease form an adjustable switch. By changing the insertion site of the tunable switch from an intramolecular position in at least one protein necessary for viral transcription and / or replication to an intermolecular position, the effect of a protease inhibitor can be changed from an ON switch to an OFF switch. RNA viruses can also encode heterologous prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/57C07K14/145C12N15/86A61K39/205A61P35/00C12R1/93
CPCC12N7/00C07K14/005C12N15/86C12Y304/23016A61K39/12A61P35/00C12N2760/20221C12N2760/20222C12N2760/20243C12N2760/20232C12N2760/00032C07K14/81C12N2760/20211
Inventor 迈克·多萝特·霍尔姆冯拉尔伊曼纽尔·海尔曼亚尼内·坎佩尔吉多·沃尔曼丽莎·埃格雷尔贝内迪克特·霍弗
Owner BOEHRINGER INGELHEIM INT GMBH
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