Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-11-29
2001-07-10
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S093200
Reexamination Certificate
active
06258791
ABSTRACT:
The present invention relates to a product, which combines a nucleic acid and a substance which leads to disorganization of the extracellular matrix of a cell, in particular hyaluronidase, for simultaneous or consecutive use, or use which is staggered over time, with said nucleic acid being transported by an infectious viral particle or in the form of a synthetic vector. The invention also relates to the use of such a product for the purpose of promoting the transfer of the nucleic acid in question into a cell or into a host organism. The invention is of particular use in the field of gene transfer or gene therapy.
The extracellular matrix consists of protein and polysaccharide molecules which are assembled in a dense, organized network in the extracellular space of most tissues. It plays an important physiological role in maintaining the tissue architecture, and acting as a reservoir for trophic factors, chemoattractants and cell-binding factors. Hyaluronan (or hyaluronic acid) is a ubiquitous constituent of the vertebrate extracellular matrix. This linear polysaccharide, which is based on glucuronic acid and glucosamine [D-glucuronic acid 1-&bgr;-3)N-acetyl-D-glucosamine(1-b-4)], is able to exert an influence on the physicochemical characteristics of the matrices by means of its property of forming very viscous solutions. Hyaluronic acid also interacts with various receptors and binding proteins which are located on the surface of the cells. It is involved in a large number of biological processes such as fertilization, embryonic development, cell migration and differentiation, wound-healing, inflammation, tumor growth and the formation of metastases.
Hyaluronic acid is hydrolyzed by hyaluronidase. Its hydrolysis leads to disorganization of the extracellular matrix. Hyaluronidase is present in a large number of the body's biological liquids. It is produced by the acrosome of the spermatozoa and, in this situation, ensures their penetration into the ovum during fertilization. During embryogenesis, it promotes migration of the embryonic cells toward the territories which they are to colonize.
Besides its action in plasticity and tissue differentiation, the hyaluronic acid/hyaluronidase couple is also thought to be involved in certain pathological processes. Thus, this enzyme is exploited by cancer cells for extending tumors and for the angiogenesis which ensures that these tumors receive nutritional support. Hyaluronidase is cosecreted in a large number of venoms (snakes, lizards, fish, scorpions, bees, spiders, etc.) and, in this situation, is thought to increase the diffusion of these venoms in the body of the prey. Hyaluronidase is also involved in the fusogenic action of viruses such as MLV (Moloney leukemia virus) or CAEV (caprine arthritis encephalitis virus). It may also act as an agent for diffusing viruses during a bacterial contamination, as in the case of infections with herpes (Romano and Moisseiev, Metab. Pediatr. Syst. Ophtalmol. 1982, 6: 361-365).
Furthermore, hyaluronidase has been used for many years for a variety of applications in human clinical medicine: for example as an antiedema agent (Lasonil, Thiomucase), as an agent for diffusing medicines which have been injected by the intramuscular or subcutaneous route (Hyaluronidase Choay), as an anti-cancer agent, in formulating local anesthetics (Lewis-Smith, Br. J. Plast. Surg. 1986, 39: 554-558), or else as an agent for reducing myocardial lesions following an infarct. The possibility of using hyaluronidase in the field of DNA transfection has already been mentioned by Dubensky et al. (Proc. Natl. Acad. Sci. USA, 1984, 81: 7529-7533). This document demonstrates that a more uniform transfection is obtained in vivo by coinjecting plasmid DNA and a mixture of collagenase and hyaluronidase. However, the beneficial effect is not obtained with a plasmid DNA which has not previously been precipitated with calcium phosphate. Similarly, WO 95/26718 relates to a method for transferring exclusively naked DNA into cells, which method consists in using an agent which facilitates penetration of the said naked DNA into the interior of the cells. Thus, it is difficult to conceive of exploiting this technology in gene therapy using unprecipitated vectors or viral particles.
The present invention is directed towards extending the therapeutic potential of hyaluronidase to the field of gene transfer, in particular in gene therapy. It is important to have available tools which promote the distribution of gene vectors or the expression of genes within the host organism in order to improve the efficacy of this novel technology. The present invention provides an advantageous solution to this problem. The ability of hyaluronidase to promote the transfer of a nucleic acid into, or its expression in, a cell or host organism has now been demonstrated. As the examples which follow show, the intramuscular administration of a solution of hyaluronidase a few hours before injecting recombinant adenovirus into the same muscle appreciably increases expression of the recombinant gene. This combined use improves the therapeutic effect and makes it possible to employ reduced vector doses.
For this reason, the present invention relates to a combination product, which comprises at least one substance which leads to disorganization of an extracellular matrix of a host, and at least one nucleic acid of interest, for simultaneous or consecutive administration, or administration which is staggered over time, with the said nucleic acid being transported by an infectious viral particle or in the form of a synthetic vector.
The term “extracellular matrix”, which is well known in the field of the art, is elaborated upon in the introductory section. Within the meaning of the present invention, “substance which leads to disorganization of the extracellular matrix” denotes any substance which acts on the integrity of the matrix, in particular exerting a total or partial degrading or destabilizing action on at least one of the constituents of the said matrix or on the bonds which unite these various constituents. While it is possible, within the context of the present invention, to have recourse to a known substance, it is also possible, if the substance is protein in nature, to have recourse to a mutant which contains one or more mutations, produced by addition, deletion and/or substitution, of one or more amino acids of the native protein, a functional fragment or else a chimeric protein which is derived from fusing sequences of varied origin. For the purposes of the present invention, the said substance can also be modified by the chemical, enzymic, etc. route with the aim of increasing its activity, its stability or else its tropism with regard to one particular cell type. The choice of a substance for use in the present invention is wide. As an indication, it can be selected from the substances which possess collagenase activity, dispase activity, trypsin activity or pronase activity.
According to one advantageous embodiment, preference is given to using a substance which is able to hydrolyze the polysaccharides which are generally present in extracellular matrices, very particularly hyaluronic acid. In this regard, a substance possessing hyaluronidase activity is very particularly suitable for implementing the invention. The hyaluronidases are described in Kreil (Protein Sci., 1995, 4: 1666-1669). The hyaluronidase can be a hyaluronidase which is derived from a mammalian, reptilian or hymenopteran hyaluronate glycanohydrolase, from a hyaluronate glycanohydrolase from the salivary gland of the leech, or from a bacterial, in particular streptococcal, pneumococcal and clostridial hyaluronate lyase. Of these, preference is very particularly given to bovine testicular hyaluronidase. Preference will be given to using a substance which exhibits a degree of homology of at least 70%, advantageously of at least 90% and, preferably, of at least 95%, with the sequence of an hyaluronidase or a functional fragment of this hyaluronidase, with the import
Brunovskis Peter
Burns Doane Swecker & Mathis L.L.P.
Priebe Scott D.
Transgene S.A.
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