EP1068225A2 - Nouveaux composes destines a la transfection d'adn - Google Patents

Nouveaux composes destines a la transfection d'adn

Info

Publication number
EP1068225A2
EP1068225A2 EP99919208A EP99919208A EP1068225A2 EP 1068225 A2 EP1068225 A2 EP 1068225A2 EP 99919208 A EP99919208 A EP 99919208A EP 99919208 A EP99919208 A EP 99919208A EP 1068225 A2 EP1068225 A2 EP 1068225A2
Authority
EP
European Patent Office
Prior art keywords
leu
cell
vitro
vivo
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP99919208A
Other languages
German (de)
English (en)
Inventor
Eric Argirios Kitas
Ernst-Jürgen SCHLAEGER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics GmbH filed Critical Roche Diagnostics GmbH
Priority to EP99919208A priority Critical patent/EP1068225A2/fr
Publication of EP1068225A2 publication Critical patent/EP1068225A2/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • Proteins and other macromolecules are transfected into cells for therapeutic and screening purposes. For example, immunization is enhanced by introducing an immunogenic protein into a cell, so that it is more efficiently processed and presented on the surface of the cells, thereby enhancing the immunogenic response.
  • Negatively charged macromolecules which act inside a cell are transported past the cell membrane into the cytoplasm where they exert their effect.
  • Factors which enhance or hinder transcription of DNA can be used in a screening test to verify the transcription of a gene of interest. These transcription assays are very well known for use in screening compounds to determine their effect against a particular macro- molecule, for example a cell receptor.
  • EP-A-784 984 and Legendre et al. describe conjugates of a lipid and a basic, membrane disturbing peptide that bind polynucleotides and anionic macromolecules can be used for transfection of cells.
  • the peptide portion of the conjugate consists of natural amino acids linked by a natural amide binding.
  • the present invention is directed to novel compounds which avoid disadvantages associated with known transfection agents.
  • reversed amide backbone refers to retro-peptides which characteristically have the same composition as its parent peptide, but the sequence is reversed, i.e., n, ... 3, 2, 1 instead of 1, 2, 3, ... n when reading both in the N to C direction. Both have normal peptide bonds.
  • n n, ... 3, 2, 1 instead of 1, 2, 3, ... n when reading both in the N to C direction. Both have normal peptide bonds.
  • R 3 is a basic, membrane disturbing peptide with a reversed amide backbone.
  • R 3 is Gln-Gln-Arg-Lys-Arg-Lys-Ile-T -Ser-Ile-Leu-Ala-Pro-Leu-Gly-Thr-Thr-Leu-Val-Lys- Leu-Val-Ala-Gly-Ile-NH-CH[CONH 2 ]-(CH 2 )-.
  • the peptide has a reversed amide backbone.
  • R 3 comprises at least 50%, more preferably 65%, and even more preferably 80% of D-amino acids or derivatives thereof. In the most preferred embodiment all amino acids are D-amino acids.
  • D-amino acid refers to naturally as well as non-naturally D- ⁇ - amino carbonic acids or derivatives thereof.
  • R 3 also comprises membrane-disturbing peptide sequences such as magainin, cecropin, defensins, etc., or chimers of such e.g., cecropin-melittin (Hancock & Lehrer TIBTECH, 1998, vol. 16, 82-88) synthesized as the retro-inverso peptides and derivatized to allow conjugation to lipids, analogous to the methods as described below, especially analogous in fashion to Example 1.
  • membrane-disturbing peptide sequences such as magainin, cecropin, defensins, etc., or chimers of such e.g., cecropin-melittin (Hancock & Lehrer TIBTECH, 1998, vol. 16, 82-88) synthesized as the retro-inverso peptides and derivatized to allow conjugation
  • the most preferred compound is
  • a further embodiment of the present invention refers to the peptide portion of R 3 , especially to the intermediate peptide Gln-Gln-Arg-Lys-Arg-Lys-Ile-Trp-Ser-Ile-Leu-Ala-Pro-Leu-Gly- Thr-Thr-Leu-Val-Lys-Leu-Val-Ala-Gly-Ile-Cys-NH 2 or derivatives and/or salts and/or sol- vates thereof having a reversed amide backbone and consisting of at least 50%, more preferably 65%, and even more preferably 80% of D-amino acids or derivatives thereof. In the most preferred embodiment all amino acids are D-amino acids.
  • D-amino acid refers to naturally as well as non-naturally D- ⁇ -amino carbonic acids or derivatives thereof.
  • the reaction of a peptide of the formula R 3 SH with a compound of formula R-SZ can be carried out in an appropriate solvent or solvent mixture which solubilizes both reactants.
  • the compound of formula R-SZ can be dissolved in an organic solvent, e.g., in chloroform.
  • the peptide R 3 SH is suitably dissolved in aqueous buffer solution, such as phosphate buffer, that contains an appropriate amount of an water-miscible organic solvent such as acetonitrile to accomplish the formation of a single phase reaction system.
  • DNA for use in the present invention are plasmids and genes, especially those for which gene therapy protocols have been launched such as cystic fibrosis transmembrane regulator (CFTR), adenosine deaminase (ADA), thymidine kinase (tk) and HLA B7; as well as reporter genes such as beta-galactosidase, luciferase, green fluorescence protein (gfp), chloramphenicol transferase and alpha- 1 antitrypsin.
  • Other examples of DNA are oligodeoxynucleotides and their analogues used as antisense, aptamer or 'triple-helix' agents.
  • RNA are ribozymes or oligoribonucleotide antisense molecules.
  • number of negative charges means the number of negatively charged phosphates in the backbone.
  • the optimal ratio within these ranges depends on the cell to be transfected and is readily ascertained by one of skill in the art to which this invention pertains.
  • transfection competent molecules include cationic lipids as described by J.B. Behr in Bioconjugate Chem., 1994, 5,382089 and X. Gao and L. Huang in Gene Ther., 1995, 2, 710-722; polycations as described by A.V. Kabanov and V.A.: Kabanov in Bioconjugate Chem., 1995, 6, 7-20; peptides and polymers and other non-viral gene delivery systems as described by F.D. Ledley in Human Gene Therapy, 1995, 6, 1129-1144.
  • Fmoc-amino acids (2.5 equiv.) were activated with an equivalent amount of TATU (L. A. Carpino J. Am. Chem. Soc. 1993, 115, 4397-4398) and DIPEA. Fmoc depro- tection was achieved with 20% piperidine in DMF.

Abstract

L'invention concerne des conjugués de lipides et de peptides basiques modifiés perturbant la membrane, lesdits conjugués étant caractérisés en ce que les peptides comprennent un squelette amidé inversé. Elle se rapporte en particulier aux composés correspondant à la formule (I) dans laquelle R1 et R2 représentent un groupe fonctionnel hydrocarbyle d'un acide carboxylique saturé ou insaturé à chaîne droite ou ramifiée ou d'un groupe fonctionnel phospholipidique, R3 représentant un peptide basique modifié perturbant la membrane qui possède un squelette amidé inversé, Y représentant alkylène C¿2-10? et X représentant C(O)-NH- ou -S-S-. L'invention se rapporte également aux sels de ces composés.
EP99919208A 1998-04-07 1999-04-07 Nouveaux composes destines a la transfection d'adn Ceased EP1068225A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP99919208A EP1068225A2 (fr) 1998-04-07 1999-04-07 Nouveaux composes destines a la transfection d'adn

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP98106302 1998-04-07
EP98106302 1998-04-07
EP98124837 1998-12-30
EP98124837 1998-12-30
EP99919208A EP1068225A2 (fr) 1998-04-07 1999-04-07 Nouveaux composes destines a la transfection d'adn
PCT/EP1999/002361 WO1999051629A2 (fr) 1998-04-07 1999-04-07 Nouveaux composes destines a la transfection d'adn

Publications (1)

Publication Number Publication Date
EP1068225A2 true EP1068225A2 (fr) 2001-01-17

Family

ID=26149151

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99919208A Ceased EP1068225A2 (fr) 1998-04-07 1999-04-07 Nouveaux composes destines a la transfection d'adn

Country Status (5)

Country Link
EP (1) EP1068225A2 (fr)
JP (1) JP2002510706A (fr)
AU (1) AU3706599A (fr)
CA (1) CA2327367A1 (fr)
WO (1) WO1999051629A2 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19925143A1 (de) * 1999-06-02 2000-12-07 Aventis Pharma Gmbh Neue liposomale Vektorkomplexe und deren Verwendung für die Gentherapie
GB9915074D0 (en) * 1999-06-28 1999-08-25 Cortecs Plc Ligand-binding composition
US7737108B1 (en) * 2000-01-07 2010-06-15 University Of Washington Enhanced transport using membrane disruptive agents
CN1313327A (zh) * 2000-03-15 2001-09-19 上海博德基因开发有限公司 一种新的多肽——人接头粘连分子12和编码这种多肽的多核苷酸
CA2352645A1 (fr) * 2000-07-28 2002-01-28 F. Hoffmann-La Roche Ag Compositions pour la transfection avec de l'adn
GB0024428D0 (en) * 2000-10-05 2000-11-22 King S College Absorption enhancers

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0784984B1 (fr) * 1996-01-17 2003-07-02 F. Hoffmann-La Roche Ag Molécules compétentes à la transfection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9951629A2 *

Also Published As

Publication number Publication date
JP2002510706A (ja) 2002-04-09
AU3706599A (en) 1999-10-25
WO1999051629A2 (fr) 1999-10-14
CA2327367A1 (fr) 1999-10-14
WO1999051629A3 (fr) 1999-12-23

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