Method for inducing a tolerogenic immune response
Inventors
Assignees
Publication Number
US-10813987-B2
Publication Date
2020-10-27
Expiration Date
2032-09-21
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Abstract
The subject technology relates generally to compositions and methods for producing plasmid DNA of a desired quality. In addition, it relates to the discovery of Escherichia coli (E. coli) bacteria with a constitutive methylase gene stably incorporated into the chromosomal DNA and uses thereof.
Core Innovation
The invention provides compositions and methods for producing plasmid DNA of a desired quality and methylation level, specifically through the use of Escherichia coli (E. coli) bacteria engineered to contain a constitutively expressed methylase gene, such as SssI methylase, stably incorporated into their chromosomal DNA. This enables the cost-effective and controlled production of plasmid DNA with adjustable methylation of CpG dinucleotides, which is crucial for applications in gene therapy and DNA vaccination.
The problem addressed is the immunogenicity and short duration of gene expression associated with unmethylated CpG dinucleotides in plasmid DNA produced by standard bacterial strains, which can lead to undesirable immune responses and reduced therapeutic efficacy. Existing methods, such as in vitro methylation or use of a methylase-encoding plasmid, pose significant challenges for reproducible, large-scale, pharmaceutical-grade DNA production due to contamination and inconsistent methylation.
The patent also describes a method for overcoming the toxicity associated with expressing certain methylase genes in bacteria by first amplifying the gene-containing plasmid in vitro, followed by direct chromosome integration, thus avoiding lethal expression levels. The resulting engineered bacteria allow for the precise and reproducible modulation of plasmid DNA methylation, tailored via promoter strength, facilitating the generation of DNA vaccines or gene therapy vectors that promote tolerogenic immune responses and prolonged gene expression.
Claims Coverage
The claims present inventive features relating to the use of partially methylated polynucleotides encoding autoantigens and pro-apoptotic proteins for inducing tolerogenic immune responses to treat autoimmune diseases.
Inducing tolerogenic immune response to an autoantigen using specifically methylated polynucleotides
A method for inducing a tolerogenic immune response to an autoantigen in a mammalian subject with an autoimmune disease, involving the intradermal administration of: - A first polynucleotide encoding the autoantigen (or fragment), with at least about 50% CpG dinucleotide methylation. - A second polynucleotide encoding a pro-apoptotic protein (or functional fragment), with about 15% or less CpG dinucleotide methylation, where the pro-apoptotic protein is selected from BAK, BAX, BIM, Death Receptor 3 (DR3), Death Receptor 4 (DR4), Death Receptor 5 (DR5), and a FAS receptor. - The polynucleotides are administered at a ratio ranging from about 2:1 to about 7:1 (μg:μg), resulting in a combined CpG methylation level of about 40% to about 45%. - The method is applicable to a range of autoimmune diseases, including but not limited to type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and psoriasis.
In summary, the inventive features center on the administration of autoantigen-encoding and pro-apoptotic protein-encoding polynucleotides with tailored CpG methylation levels and specific dosage ratios to induce tolerance as a therapeutic approach for autoimmune disorders.
Stated Advantages
The technology enables cost-effective, reproducible, and scalable production of plasmid DNA with controlled and adjustable CpG methylation levels suitable for pharmaceutical and gene therapy applications.
It allows precise modulation of tolerogenic immune responses by adjusting the methylation status of DNA vaccines, enhancing their potency in treating immune-mediated inflammatory disorders.
The approach overcomes toxicity issues associated with methylase gene expression in bacteria by integrating the gene into the chromosome, leading to stable and non-lethal expression levels.
The method permits the production of plasmid DNA without contamination from methylase-encoding plasmids and ensures consistent methylation, which is essential for therapeutic applications.
Mixing hypomethylated and hypermethylated DNAs achieves higher efficacy for certain conditions, such as type 1 diabetes, compared to fully methylated or unmethylated DNA alone.
Documented Applications
Production of plasmid DNA for use as DNA vaccines or gene therapy agents with controlled CpG methylation to induce tolerogenic responses in the treatment of autoimmune diseases.
Treatment of autoimmune diseases, including but not limited to type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis, celiac disease, and organ transplant rejection.
Treatment of transplant recipients, such as in skin transplant scenarios, by administering methylated polynucleotides encoding donor antigens and pro-apoptotic proteins.
Production of modified plasmids encoding allergens, autoantigens, cancer antigens, donor antigens, or pro-apoptotic proteins for immune modulation and therapeutic applications.
Methods for increasing regulatory T cell (Treg) expression in mammals via administration of methylated plasmid DNA vaccines generated using the described E. coli strains.
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