Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Mixing of two or more solid polymers; mixing of solid...
Reexamination Certificate
2000-01-21
2002-06-18
Fredman, Jeffrey (Department: 1655)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Mixing of two or more solid polymers; mixing of solid...
C424S078010, C424S078080, C424S230100, C435S173300, C260S66500B
Reexamination Certificate
active
06407178
ABSTRACT:
The present invention relates to novel cationic polymers which can be used for forming complexes of cationic polymers and of therapeutically active substances comprising at least one negative charge and the corresponding complexes, useful in particular for the transfer of a therapeutically active substance, in particular a nucleic acid, into a target cell.
Genetic diseases can be explained in particular by a dysfunction in the expression of specific genes or by the expression of mutated polypeptides which are nonfunctional in at least one cell type. The therapeutic solution which appears to be most appropriate for this type of condition is to transfer into specific target cells extracted and then reintroduced into the human body, or directly into the affected organs, the genetic information capable of correcting the defect observed. This may be for example the gene encoding the CFTR protein in the case of cystic fibrosis or the gene encoding dystrophin in the case of Duchenne's myopathy. In the context of this approach, also called gene therapy, the genetic information is introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body (ex vivo method), or directly in vivo into the appropriate tissue. Many publications also describe the use of a gene therapy protocol in order to obtain in the target cells the expression of a protein of therapeutic value by introducing the corresponding genetic information. The therapeutic value may for example lie in the possibility of eliminating a tumor, or failing this to slow down its progression, by transferring into the target cancer cells immunostimulatory genes (immunotherapy) which are capable of inducing or of activating a cell-mediated immune response toward the tumor, or the administration of genes encoding cytokines, of cytotoxic genes conferring toxicity on the cells expressing them, for example the tk gene of the Herpes Simplex virus type 1 (HSV-1), or of antioncogenes, such as for example the gene associated with retinoblastoma or p53, or of polynucleotides capable of inhibiting the activity of an oncogene, such as for example the antisense molecules or the ribozymes capable of degrading the messenger RNAs specific for the oncogenes.
During the past 30 years, several studies have described techniques relating to the transfer of this genetic information into cells, in particular mammalian cells. These different techniques may be divided into two categories. The first category relates to physical techniques such as microinjection, electroporation or particle bombardment which, although effective, are largely limited to applications in vitro and whose use is cumbersome and delicate. The second category involves techniques relating to molecular and cell biology for which the genetic material to be transferred is combined with a vector of a biological or synthetic nature which promotes the introduction of said material.
Currently, the most efficient vectors are viral, in particular adenoviral or retroviral, vectors. The techniques developed are based on the natural properties which these viruses possess for crossing the cell membranes, for escaping degradation of their genetic material and for causing their genome to penetrate into the cell nucleus. These viruses have already been the subject of many studies and some of them are already used experimentally as vectors for genes in humans for the purpose, for example, of a vaccination, an immunotherapy or a therapy intended to make up for a genetic deficiency. However, this viral approach has some limitations, in particular linked to the risks of dissemination in the host organism and in the environment of the infectious viral particles produced, to the risk of artefactual mutagenesis by insertion into the host cell in the case of retroviral vectors, and to the induction of immune and inflammatory responses in vivo during the therapeutic treatment. Accordingly, alternative, nonviral systems for transferring polynucleotides have also been developed.
There may be mentioned for example coprecipitation with calcium phosphate, the use of cationic lipids such as DOTMA: N-[1-(2,3-dioleyl-oxyl)propyl]-N,N,N-trimethylammonium (Felgner et al., 1987, PNAS, 84, 7413-7417), DOGS: dioctadecylamido-glycylspermine (Behr et al., 1989, PNAS, 86, 6982-6986 or Transfectam™), DMRIE: 1,2-dimiristyloxypropyl-3-dimethylhydroxyethylammonium and DORIE: 1,2-diooleyl-oxypropyl-3-dimethylhydroxyethylammonium (Felgner et al., 1993, Methods 5, 67-75), DC-CHOL: 3&bgr;-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (Gao and Huang, 1991, BBRC, 179, 280-285), DOTAP™ (McLachlan et al., 1995, Gene Therapy, 2, 674-622) or Lipofectamine™; or the use of polymers coupled to ligands recognized by a membrane receptor (for a review see Cotten and Wagner, 1993, Current Opinion in Biotechnology, 4, 705-710).
However, one of the major problems encountered when it is desired to transfer genes into target cells lies in the difficulty of causing the penetration of the nucleic acids because in particular of their polyanionic nature which prevents their passage across the cell membranes. The use of cationic polymers which can combine with the nucleic acids by electrostatic bonds makes it possible to solve this problem, at least partially. Thus, the document WO 95/24221 describes the use of dendritic polymers, document WO 96/02655 the use of polyethyleneimine, or of polypropyleneimine and the documents U.S. Pat. No. 5,595,897 and FR 2,719,316 the use of conjugates of polylysine.
The applicant company has now defined novel cationic polymers possessing particularly advantageous properties for the transfer into cells of therapeutically active substances comprising negative charges, in particular nucleic acids. Furthermore, these polymers have the advantage of being easily accessible, in particular by chemical synthesis, and inexpensive. They have a very low toxicity to cells, which constitutes a considerable advantage in the field of gene therapy.
The present invention relates first of all to a cationic polymer of formula I:
in which n is a whole number varying from 0 to 5 and p is a whole number varying from 2 to 20,000, more particularly p varies from 10 to 18,000 and advantageously from 200 to 1000,
characterized in that:
at least 10%, advantageously from 30 to 80%, preferentially 70%, of the free NH
2
functions are substituted with identical or different hydrophilic R groups;
said cationic polymer may in addition comprise at least one targeting element combined covalently or not with the free NH
2
functions and/or with said hydrophilic R groups provided that said cationic polymer contains at least 20%, preferably at least 30%, of free NH
2
functions.
The invention relates more particularly to a cationic polymer defined by the following formula II:
Advantageously, said cationic polymer is defined by the formula III:
The polymers of formula II and III exhibit the characteristics as defined above for the polymer of more general formula I.
According to the present invention, “hydrophilic group” is understood to mean a group comprising at least one hydrophilic function. It may be for example a hydrophilic function chosen from the amine, hydroxyl, amide and ester functions.
These hydrophilic functions may be directly combined with the free NH
2
functions of the polymer through an N—C bond, or indirectly via an arm. In the latter case, the invention relates, for example, to a cationic polymer for which R is chosen from the groups:
R′—C═O, and;
—(CH
2
)n′—R′
where R′ designates a group containing at least one hydrophilic function and n′ is a whole number varying from 1 to 5.
According to an advantageous embodiment, the hydrophilic group R or R′ consists of a polymer exhibiting hydrophilic properties, such as for example polyethylene glycol (PEG) or its derivatives, for example a methoxy-PEG, polyvinylpyrrolidone, poly-methyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polylactic
Boussif Otmane
Delair Thierry
Kolbe Hanno V. J.
Veron Laurent
Burns Doane Swecker & Mathis L.L.P.
Chakrabarti Arum Kr.
Fredman Jeffrey
Transgene S.A.
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