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1. WO2020229893 - LIVE IMAGING SYSTEM TO VISUALIZE THE RETRO-TRANSCRIBED VIRAL DNA GENOME

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Publication Number WO/2020/229893
Publication Date 19.11.2020
International Application No. PCT/IB2020/000456
International Filing Date 11.05.2020
IPC
C12N 15/86 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
79Vectors or expression systems specially adapted for eukaryotic hosts
85for animal cells
86Viral vectors
C07K 14/21 2006.1
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
14Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
195from bacteria
21from Pseudomonadaceae (F)
C12Q 1/00 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
CPC
C07K 14/195
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
14Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
195from bacteria
C07K 2319/80
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
2319Fusion polypeptide
80containing a DNA binding domain, e.g. Lacl or Tet-repressor
C12N 15/86
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
79Vectors or expression systems specially adapted for eukaryotic hosts
85for animal cells
86Viral vectors
C12N 2740/16043
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
2740Reverse Transcribing RNA Viruses
00011Reverse Transcribing RNA Viruses
10011Retroviridae
16011Human Immunodeficiency Virus, HIV
16041Use of virus, viral particle or viral elements as a vector
16043viral genome or elements thereof as genetic vector
C12Q 1/18
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
02involving viable microorganisms
18Testing for antimicrobial activity of a material
G01N 2333/16
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
2333Assays involving biological materials from specific organisms or of a specific nature
005from viruses
08RNA viruses
15Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
155Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
16HIV-1, HIV-2
Applicants
  • INSTITUT PASTEUR [FR]/[FR]
  • NEOVIRTECH S.A.S. [FR]/[FR]
Inventors
  • DI NUNZIO, Francesca
Agents
  • GUTMANN, Ernest
Priority Data
62/84620110.05.2019US
Publication Language English (en)
Filing Language English (EN)
Designated States
Title
(EN) LIVE IMAGING SYSTEM TO VISUALIZE THE RETRO-TRANSCRIBED VIRAL DNA GENOME
(FR) SYSTÈME D'IMAGERIE EN DIRECT POUR VISUALISER LE GÉNOME D'ADN VIRAL RÉTRO-TRANSCRIT
Abstract
(EN) A recombinant lentiviral vector comprising a coding sequence for an OR protein fused to a coding sequence for a fluorescent protein or a subunit of a fluorescent protein, and a promoter active in human cells operatively linked to the coding sequences. A recombinant lentivirus comprising a recombinant genome comprising an RNA that generates an ANCH sequence upon retrotranscription. A recombinant eukaryotic cell comprising a genomically integrated DNA copy of the recombinant lentiviral vector. A method of observing lentiviral DNA in a eukaryotic cell, comprising: providing a recombinant eukaryotic cell that produces a fusion protein comprising an OR protein, fused to a fluorescent protein or a subunit of a fluorescent protein; infecting the recombinant eukaryotic cell with a recombinant lentivirus comprising a recombinant genome comprising an RNA that generates an ANCH sequence upon retrotranscription, under conditions sufficient for reverse transcription of the recombinant lentiviral genome comprising an ANCH sequence; allowing the OR protein to bind to the ANCH sequence; and detecting the fluorescent protein or subunit of the fluorescent protein to thereby observe the lentiviral DNA in the eukaryotic cell. This tool can be suitable also for in vivo applications (e.g. humanized mice) as well as for screening of new antiretroviral compounds. The HIV-1 ANCHOR system can be extended to the study of other viruses or for the screening of antiviral compounds, e.g. against SARS-CoV2.
(FR) L'invention concerne un vecteur lentiviral de recombinaison comprenant une séquence codante pour une protéine OR fusionnée à une séquence codante pour une protéine fluorescente ou une sous-unité d'une protéine fluorescente, et un promoteur actif dans des cellules humaines fonctionnellement liées aux séquences codantes. L'invention concerne également un lentivirus de recombinaison comprenant un génome de recombinaison comprenant un ARN qui génère une séquence ANCH lors de la rétrotranscription. Une cellule eucaryote de recombinaison comprend une copie d'ADN génomiquement intégrée du vecteur lentiviral de recombinaison. L'invention concerne également un procédé d'observation d'ADN lentiviral dans une cellule eucaryote, consistant à : utiliser une cellule eucaryote de recombinaison qui produit une protéine de fusion comprenant une protéine OR, fusionnée à une protéine fluorescente ou à une sous-unité d'une protéine fluorescente; infecter la cellule eucaryote de recombinaison avec un lentivirus de recombinaison comprenant un génome de recombinaison comprenant un ARN qui génère une séquence ANCH lors de la rétrotranscription, dans des conditions suffisantes pour la transcription inverse du génome lentiviral de recombinaison comprenant une séquence ANCH; permettre à la protéine OR de se lier à la séquence ANCH; et détecter la protéine fluorescente ou la sous-unité de la protéine fluorescente pour ainsi observer l'ADN lentiviral dans la cellule eucaryote. Cet outil peut également être approprié pour des applications in vivo (par exemple des souris humanisées) ainsi que pour le criblage de nouveaux composés antirétroviraux. Le système ANCHOR VIH-1 peut être étendu à l'étude d'autres virus ou pour le criblage de composés antiviraux, par exemple contre le SARS-CoV2.
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