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1. WO2000032815 - MOLECULAR CONJUGATE TO PROMOTE HOMOLOGOUS GENETIC RECOMBINATION

Publication Number WO/2000/032815
Publication Date 08.06.2000
International Application No. PCT/US1998/025309
International Filing Date 27.11.1998
Chapter 2 Demand Filed 24.06.2000
IPC
A61K 47/48 2006.01
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
47Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
48the non-active ingredient being chemically bound to the active ingredient, e.g. polymer drug conjugates
A61K 48/00 2006.01
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
48Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
C12N 15/90 2006.01
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
87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
90Stable introduction of foreign DNA into chromosome
CPC
A61K 47/645
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
47Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
50the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
51the non-active ingredient being a modifying agent
62the modifying agent being a protein, peptide or polyamino acid
64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
645Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
A61K 48/00
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
48Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
C12N 15/90
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
87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
90Stable introduction of foreign DNA into chromosome
Applicants
  • RATNER, Paul, L. [US]/[US]
Inventors
  • RATNER, Paul, L.
Agents
  • KELLY, Patrick, D.
Priority Data
Publication Language English (EN)
Filing Language English (EN)
Designated States
Title
(EN) MOLECULAR CONJUGATE TO PROMOTE HOMOLOGOUS GENETIC RECOMBINATION
(FR) CONJUGUE MOLECULAIRE PROMOTEUR DE RECOMBINAISON GENETIQUE HOMOLOGUE
Abstract
(EN)
A method and composition are disclosed for transfecting eukaryotic cells using a nucleic acid segment coupled to a site-specific chromosome-binding poylpeptide. The polypeptide-nucleic acid molecular conjugate preferably includes a recombinant DNA (rDNA) sequence, and is referred to herein as a polypeptide-linked-rDNA (PLR molecule). One example of a PLR molecule comprises an rDNA segment containing (a) a nucleotide sequence from a normally functioning structural gene, and (b) a selectable marker gene coding for antibiotic resitance, coupled by means of a covalent crosslinking reagent to a site-specific chromosome-binding polypetide (such as a transcription regulating polypeptide that binds to a specfic site in chromosomal DNA). After the PLR molecule enters a cell, by means such as electroporation, biolistics, etc., the chromosome-binding polypeptide enables transport of the PLR molecule through the cytosol and into the nucleus, using a nuclear localization sequence (NLS) domain of the polypeptide, or into a mitochondrion, using a mitochondrial leader peptide sequence. Inside the nucleus or mitochondrion, the polypeptide scans the chromosomes until it binds to a specific chomosomal binding site. This places the rDNA segment of the PLR molecule near a target site (such as an abnormal gene) in the chromosome. The desired rDNA segment contained in the PLR molecule has nucleotide sequence homology with the target gene, to enable cellular recombination enzymes to replace the targeted gene sequence with the desired gene sequence.
(FR)
L'invention concerne un procédé et une composition permettant de transfecter des cellules eucaryotes à l'aide d'un segment d'acide nucléique couplé à un polypeptide qui se lie à un site spécifique des chromosomes. Ce conjugué moléculaire polypeptide-acide nucléique contient de préférence une séquence d'ADN recombinant (ADNr), et est appelé ici « molécule PLR » (polypeptide-linkedDNA). Une molécule PLR comprend par exemple un segment d'ADNr contenant (a) une séquence nucléotidique d'un gène structurel à fonctionnement normal, et (b) un gène marqueur à choix, codant pour la résistance aux antibiotiques, couplé au moyen d'un agent de couplage covalent à un polypeptide formant une liaison avec un site spécifique du chromosome (par exemple un polypeptide régulateur de la transcription qui se lie à un site spécifique de l'ADN chromosomique). Après que la molécule PLR pénètre dans une cellule par des moyens tels que l'électroporation ou la biolistique, etc. le polypeptide liant le chromosome assure le transport de la molécule PLR à travers le cytosol et à l'intérieur du noyau, à l'aide d'un domaine du polypeptide comprenant une séquence de localisation nucléaire (NLS), ou à l'intérieur d'une mitochondrie, à l'aide d'une séquence peptidique guide mitochondrial. A l'intérieur du noyau ou de la mitochondrie, le polypeptide explore le chromosome jusqu'à ce qu'il puisse se lier sur un site de liaison chromosomique spécifique. Ce processus permet de placer le segment d'ADNr de la molécule PLR à proximité du site cible (p. ex. un gène anormal) dans le chromosome. Le segment désiré d'ADNr contenu dans la molécule PLR présente une homologie de séquence nucléotidique avec le gène cible de manière à permettre aux enzymes de recombinaison cellulaire de remplacer la séquence génétique cible par la séquence génétique désirée.
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