Gene therapy of diseases associated with the immune system, using a cell-specific active compound which is regulated by the cell cycle

Abstract

A DNA sequence is described for the gene therapy of diseases associated with the immune system. In its essential elements, the DNA sequence is composed of an activator sequence, a promoter module and a gene for the active substance. The activator sequence is activated in a cell-specific or virus-specific manner and this activation is regulated by the promoter module in a cell cycle-specific manner. The choice of activator sequence and active substance depends on the indication area. The DNA sequence is inserted into a viral or non-viral vector which is supplemented by a ligand having affinity for the target cell. Depending on the choice of activator sequence and active substance, the following can be treated by administering the DNA sequence: defective formation of blood cells autoimmune diseases and allergies and, in addition, rejection reactions against transplanted organs chronic arthritis viral and parasitic infections and, in addition, prophylaxis of viral, bacterial and parasitic infections, and leukaemias.

Description

[0001] A DNA sequence is described for the gene therapy of diseases associated with the immune system.[0002] In its essential elements, the DNA sequence is composed of an activator sequence, a promoter module and a gene for the active substance.[0003] The activator sequence is activated in a cell-specific or virus-specific manner and this activation is regulated by the promoter module in a cell cycle-specific manner. The choice of activator sequence and active substance depends on the indication area. The DNA sequence is inserted into a viral or non-viral vector, which vector is supplemented with a ligand having affinity for the target cell.[0004] Depending on the choice of activator sequence and active substance, the following can be treated by administering the DNA sequence:[0005] defective formation of blood cells[0006] autoimmune diseases and allergies and, in addition, rejection reactions against transplanted organs[0007] chronic arthritis[0008] viral and parasitic infections a...

AI Technical Summary

Benefits of technology

0079] The promoter module CDE-CHR-Inr was discovered in the context of a detailed investigation of the G2-specific expression of the human cdc25C promoter. The starting point was finding a repressor element (cell cycle dependent element; CDE) which is responsible for switching off the promoter in the G1 phase of the cell cycle (Lucibello et al., EMBO U. 14, 132 (1995)). Using genomic dimethyl sulfate (DMS) footprinting and functional analyses (FIGS. 1 and 2), it was possible to demonstrate that the CDE binds a repressor (CDE-binding factor; CDF) in a G1-specific manner and in this way gives rise to conscription inhibition in non-proliferating (G0) cells. The CDE, which is located in the region of the basal promoter, depends, in its repressing function, on an upstream activating sequence (UAS). This led to the conclusion that the CDE-binding factor inhibits the transcription-activating effect of 5'-bound activator proteins in a cell cycle-dependent manner, i.e. in both non-proliferating cells and in the G1 phase of the cell cycle (FIG. 3).

Problems solved by technology

rejection of transplanted organs due to the immune system not being adequately inhibited

poor vaccination results and chronic infections, for example by viruses, as a consequence of immune deficiency

As is well known, the current therapeutic possibilities for diseases of this nature are inadequate.

A common feature of therapy with all cytokines is the disadvantage that they usually have to be administered parenterally every day over a relatively long period of time and, furthermore, that, for their greatest possible efficacy, several cytokines either have to be injected one after the other in the necessary hierarchical sequence or corresponding cytokines have to be present in adequate concentration in the body.

The increased dose which is consequently required constitutes, due to the high level of expenditure involved in producing cytokines, a substantial cost factor which considerably restricts the use of cytokines.

Over and above this, some cytokines give rise, in the therapeutic dose range, to substantial side effects.

Despite improved antiinflammatory and immunosuppressive medicaments, chronic arthritis is a disease for which only inadequate therapeutic measures are available and which substantially reduces the quality of life and can even shorten life expectancy (Pincus et al., Bull. Rheum. Dis. 41, 1 (1992)).

10% of the population of the western world suffers from arthritis) arthritis constitutes a substantial cost factor for national economies.

In view of these medicinal and economic problems, chronic arthritis represents a challenge for pharmaceutical research.

However, it can already be predicted today that, irrespective of their nature, medicaments which are administered orally or parenterally will have difficulty in reaching the region of joint inflammation in adequate concentration since they have to diffuse through the synovial capillaries and then passively through the synovial membrane into the joint cavity and from there into the cells lining the joint (Evans et al., Gene Therapeutics, J. Wolff, Editor, page 320, Birkhuser, Boston 1994).

This diffusion is additionally made more difficult by the fact that the vascularization of the synovial membrane is significantly reduced in rheumatoid arthritis, for example (Stevens et al., Arthritis Rheum.

While intraarticular injection of medicaments circumvents the problem of diffusion, the dwell time of the medicament in the joint is so short, owing to the high reabsorption rate, that repeated intraarticular injections over a relatively long period of time are required.

These injections are in turn associated with the considerable risk of a joint infection.

In addition, they can give rise to substantial side effects on account of the high local concentration of the medicament which is required.

In principle, however, these methods for gene therapy which have hitherto been proposed suffer from considerable disadvantages:

This in itself puts a strain on the patient and carries the risk of a joint infection.

In the second place, synovial cells can only be isolated with great difficulty and in small numbers.

Consequently, the injection of synovial cells which are transduced in vitro suffers from substantial problems and will usually not be technically possible to achieve or only possible to achieve with considerable effort.

Favored by the local inhibition of the immune reaction, and brought about by the causal factors of chronic arthritis, there would be the risk of an intensified pathological immune and inflammatory reaction once the activity of the antiarthritic substances had subsided, but no extensive alleviation or curing of the arthritis.

However, the efficacy of both these approaches to therapy is only limited (Sloane et al., Histophathol.

However, it has so far not been possible to use these molecular biological changes for clinical therapeutic methods.

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