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Sylvia Higgins Memorial Essay - Medical Category Winner, 2006

Sarah Liskowich, a first-year medical student at the University of Saskatchewan, Saskatoon, has provided LESS with an intriguing theory in her winning essay. This theory could, in the future, result in an effective treatment for lupus without the side-effects of current treatments. Sarah's background includes two undergraduate degrees in Biochemistry and Education.

Using Antisense Oligonucleotides in the Treatment of Systemic Lupus Erythematosus

Abstract

The flow of genetic information in the cell moves from DNA to RNA via transcription and from RNA to protein via translation. The expression of proteins such as the antinuclear antibodies produced in systemic lupus erythematosus can be inhibited, thereby blocking the autoimmune response that leads to this disease’s debilitating symptoms. Blocking the expression of proteins can occur during translation of mRNA to protein by several different mechanisms. One method used to inhibit protein synthesis is to inhibit the translation of mRNA in a regulatory region using antisense oligonucleotides. Antisense oligonucleotides (oligos) are short chains of DNA or RNA nucleotides which have complementary sequences to regulatory regions in mRNA molecules. These oligos anneal to mRNA molecules, and block their translation into protein. By preventing the production of cytokine proteins such as the interleukins which are responsible for stimulating T cells in SLE, the immune system cascade of events which causes B cells to release autoantibodies is effectively blocked. By preventing the immune reaction, the symptoms of this disease would cease.

INTRODUCTION

The flow of genetic information in the cell moves from DNA to RNA via transcription and from RNA to protein via translation. One can prevent the expression of the proteins such as the antinuclear antibodies produced in (SLE) that are responsible for creating the autoimmune response leading to its debilitating symptoms. Blocking the expression of proteins can occur at the level of transcription, which prevents the production of the mRNA that gives rise to proteins. One can also block the translation of mRNA to protein by several different mechanisms. One method used to inhibit protein synthesis is to inhibit the translation of mRNA in a regulatory region using antisense oligonucleotides.

WHAT ARE ANTISENSE OLIGONUCLEOTIDES?

Antisense oligonucleotides (oligos) are short chains of DNA or RNA nucleotides which are complementary to regulatory regions in mRNA molecules. These oligos anneal to mRNA molecules, and thus block translation. If the sequence of the bases in the mRNA regulatory region is AUGCAACGAGGA, the corresponding antisense oligonucleotide of RNA would be UACGUUGCUCCU. The complementary antisense oligodeoxynucleotide of DNA would be TACGTTGCTCCT.

Antisense oligonucleotides of RNA can be produced in vivo by incorporating an expression vector in the host genome via gene therapy. One can use a plasmid vector containing a complementary sequence of the oligo to produce it in vivo. This antisense oligonucleotide will anneal specifically to the mRNA molecule forming an RNA-RNA complex. This reaction is reversible and its chemical kinetics is dependant upon the Kd (dissociation constant) value. If the Kd value is high, the RNA oligo will dissociate from the mRNA molecule readily. Therefore, translation inhibition will be lost because the mRNA molecule will become available to attach to the translation machinery in the ribosome of a cell and produce the corresponding protein.

Antisense oligodeoxynucleotides of DNA anneal to mRNA specifically to form a DNA-RNA hybrid, which can be digested by a ribonuclease, such as RNAseH. This degradative enzyme will cleave DNA-RNA linkages, therefore destroying the mRNA molecule. This will prevent the mRNA from being translated into a functional protein, ultimately preventing translation of proteins which cause disease symptoms. This is a non-reversible reaction which permanently inhibits protein synthesis. One disadvantage of antisense oligodeoxynucleotides is that they must be chemically synthesized, and extracellularly added to the system by injection into the bloodstream. These oligo must also be added continuously to maintain translation inhibition in newly synthesized mRNA molecules produced by the targeted cell as the oligo are not recycled in vivo.

Antisense oligodeoxynucleotides (DNA) are preferred over antisense oligonucleotides (RNA) in the treatment of diseases such as SLE, because the action of the ribonuclease permanently destroys protein translation. Therefore, experiments using bacterial retrons to produce DNA oligos in vivo are being attempted. Retrons are 1.3 to 3 Kb genetic units found in the bacterial species Myxococcus xanthus. Retrons can be transferred into Escherichia coli bacterial host genomes to express antisense oligodeoxynucleotides. The retron produces an RNA molecule with reverse transcriptase activity. This molecule acts as a primer to produce multiple copies of single stranded DNA which is attached to a short RNA molecule at an internal G residue by a 2’, 5’ – phosphodiester linkage.

One can replace the upper stem portion of the transcript with a complementary sequence to the desired antisense oligodeoxynucleotide. Consequently, the DNA produced will be a short oligo which is complementary to the mRNA, and can anneal to SLE causing mRNA molecules specifically to inhibit their translation. This has been shown using antisense oligodeoxynucleotides which inhibit the production of the major outer lipoprotein in Escherichia coli by inhibiting its mRNA. RNAse H can then be used to cleave the DNA-RNA hybrid formed between the mRNA and DNA oligo, permanently inhibiting its translation.

MODIFICATIONS TO ANTISENSE OLIGODEOXYNUCLEOTIDES

To increase the efficacy of the action of antisense oligodeoxynucleotides, they can be chemically modified. These modifications increase the stability of these oligos by making them less susceptible to degradation by enzymes in vivo. This gives these oligos a higher probability of reacting with target mRNA before they are destroyed by endogenous enzymes or excreted from the cell via exocytosis.

Presently, the most effective modification to antisense oligodeoxynucleotides is the conversion of their phosphodiester bonds to phosphorothiate bonds. This chemical modification is accomplished by replacing one of the oxygen atoms on the phosphate group with a sulphur atom. This allows experimenters to use shorter oligos, because they are more stable. Chemically modified oligos have longer half lives in vivo, because they are not degraded by endogenous enzymes. Fakler et al (1994) exhibited phosphorothiate (PS) modified oligos effectively inhibited translation 88 to 95% of the time, as compared to 8 to 13% by unmodified oligos in Xenopus oocyte α-amino, 3-hydroxy, 5-methyl, 4-isoxazole propionate receptors.

APPLICATION OF ANTISENSE OLIGONUCLEOTIDES IN HUMAN DISEASE

For an antisense oligonucleotide of DNA or RNA to be effective in the treatment of human disease, it must meet six criteria. First, the oligo must be synthesized easily and in bulk. Unfortunately, currently they are unable to do this, but large-scale oligo synthesis techniques are being commercially pursued. Secondly, the oligo must be stable in vivo so that it can act on its target before it is degraded. Modification of the phosphodiester bonds between nucleotides to phophorothiate bonds allow these oligos to have greater stability as previously discussed.

The third criterion is that the oligo must be able to enter the target cell. The longer the oligo is, the more difficult it is for it to enter the cell. This is due to the steric hindrance of larger molecules trying to fit into the small intermembrane spaces to get across the plasma membrane. Antisense oligodeoxynucleotides are also polyanionic (negatively charged) in nature, therefore they are repulsed by the plasma membrane which contains negatively charged polar head groups.

The PS-modified oligos are taken up by the cell membrane by three main mechanisms. It was shown using fibroblasts that a 80 KDa transport protein could transport antisense oligonucleotides across a membrane channel in a calcium dependent manner. These oligos must compete with other polyanionic molecules such as dextran sulfate, to bind to the transport protein. However, these molecules will be transported across the membrane if they are present in high enough concentration. PS-modified antisense oligodeoxynucleotides can also be transported across the membrane via endocytosis and pinocytosis. The oligos will approach the cell membrane, causing an invagination to encircle these molecules. Once the oligos are inside the cell, they are surrounded by the phopholipid bilayer of the cell membrane, forming a vesicle. The mechanism used to exit these vesicles has not been determined, but researchers know that the vesicles do not bind to lysosomes to release the oligos.

The fourth criterion for antisense oligodeoxyoligonucleotides is that they must be retained by the target cell. Oligos must interact with the mRNA to inhibit translation before they are removed from the cell via exocytosis. One technique to prevent exocytosis from occurring is to modify the 5’ terminus of the oligo by adding a cholesteryl (chol)-moiety. This increases the retention of the oligo in the cell, likely due to steric hindrance of the cholesterol group interacting with the cell membrane.

The fifth criterion is that the oligo must interact with cellular targets to be effective in inhibiting translation. Both DNA and RNA molecules interact with protein in vivo, therefore many regions of these molecules are bound up by the proteins, making them unavailable to interact with antisense oligodeoxynucleotides. Stem loop regulatory regions in the 5’-untranslated region of the mRNA molecules are often exposed to interact with translation machinery; therefore these regions provide a good location to target mRNA molecules.

The final criterion is that the oligo should not act in a non-sequence specific manner with other macromolecules. The greatest advantage of the antisense oligodeoxynucleotides is their specificity. This allows these molecules to be targeted to a specific region in target mRNA molecules or regulatory proteins, because they could alter the metabolism of the cell in a detrimental manner.

USING ANTISENSE OLIGODEOXYNUCLEOTIDES TO TREAT SYSTEMIC LUPUS ERYTHEMATOSUS

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by a loss of tolerance to self antigens. The stimulated immune system mistakenly attacks a person’s own body cells. It is characterized by the production of antinuclear antibodies specifically targeted against double stranded DNA (ds DNA), the production of immune complexes and the activation of the complement system. The immune response sets off a reaction leading to inflammation and tissue damage (vascular lesions).

The disease process of SLE is not well understood, but it is believed that the Helper T lymphoid cells stimulate B cells to produce antinuclear autoantibodies that signal an immune response against the body’s own cells. The helper T cells also secrete and stimulate cytokines, which aid in healing, but can lead to damage when inappropriately excreted since they cause B cells to produce autoantibodies. Studies indicate that the three critical cytokines which are overproduced in SLE patients are IL-1, IL-6, and IL-10. Therefore, the role of these three cytokines is critical in finding an appropriate treatment for SLE.

Interleukin 1 (IL-1) is a cytokine that is produced by T-lymphocytes (as well as other immune cells) and is responsible for the co-stimulation of other T-lymphocytes and activation of the inflammatory response. Once IL-1 is released in SLE patients, it causes a cascade of stimulation of other T-cells that act to stimulate B-cells to produce autoantibodies. Interleukin 6 (IL-6) is a cytokine that is produced by B-cells and T-cells and is responsible for stimulating the final differentiation of antigen activated B-cells. This allows the B-cells to produce multiple copies of specific autoantibodies which further exacerbate the immune response cascade and leads to inflammation and vascular lesions in SLE patients. Interleukin 10 (IL-10) is a cytokine that is produced by T-cells and is also responsible for stimulating T-cells and B-cells, and therefore further contributes to this immune reaction cascade. By selectively inhibiting IL-1, IL-6, and IL-10 expression, one can limit the symptoms created by the autoimmune response by interrupting this cascade in SLE patients.

There is the potential to utilize the PS-modified antisense oligodeoxynucleotides (oligos) in the treatment of SLE, as it is currently being researched to treat HIV-1. Generalized immune suppression using corticosteroids is currently being used to treat SLE, but the use of oligos to specifically block the translation of cytokines allows a more specific interference with the components of the immune response that are causing SLE. This would minimize the side effects and problems associated with suppressing the entire immune system.

These oligos can be taken up by lymphoid cells (B and T cells) where SLE localizes. Antisense oligonucleotides also hybridize with mRNA molecules at 37°C, indicating that they could be effective at human body temperature to treat SLE. Although SLE has genetic variability between the self antigens produced, the production of the cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and interleukin-10 (IL-10) in its autoimmune response have been consistently shown in mice and humans suffering from SLE. Therefore, antisense oligodeoxynucleotides could be produced against the mRNA molecule produced by the genes for IL-1, IL-6 and IL-10 to prevent their translation into protein, thereby suppressing the erroneous immune response that causes damage in SLE patients.

CONCLUSION

Antisense oligodeoxynucleotides can be targeted to the 5’ regulatory region of the mRNA of IL-1, IL-6, and IL-10 specifically to prevent their translation, and therefore shut down the immune response in SLE. By specifically targeting the 5’ regulatory region of the mRNA of these cytokines, translation could be prevented without affecting other parts of the immune system. By preventing the translation of the IL-6 mRNA molecules by targeting the 5’ regulatory region, an autoimmune response that includes a cascade of T-cell and B-cell production and the proliferation of autoantibodies against double stranded DNA will be prevented. Once oligos can be synthesized in bulk, controlling the disease through translation inhibition in SLE patients shows great potential.