Method for inducing a tolerogenic immune response
Inventors
Assignees
Publication Number
US-12133886-B2
Publication Date
2024-11-05
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 with desired CpG methylation levels by using engineered strains of Escherichia coli (E. coli) bacteria. Specifically, these bacteria have a constitutively expressed methylase gene, such as SssI, stably incorporated into their chromosomal DNA and under the control of a constitutive promoter. This stable, chromosomal integration enables the production of plasmid DNA with adjustable and reproducible levels of CpG methylation to meet specific quality standards for pharmaceutical applications.
The problem addressed by this technology is the inability of conventional bacterial systems to efficiently and consistently produce CpG-methylated plasmid DNA for use in gene therapy and DNA vaccines. Traditional approaches, such as in vitro methylation or using plasmids encoding methylases, suffer from issues such as inconsistent methylation due to variations in plasmid copy number, contamination with methylase-encoding plasmids, the risk of high methylase expression being toxic to bacteria, and challenges in scaling for industrial production.
By engineering E. coli strains to harbor a chromosomal copy of a methylase gene under a constitutive promoter, the invention circumvents these limitations, allowing the cost-effective and reproducible production of plasmid DNA with controlled intermediate methylation levels (such as 10% to 50% CpG methylation). These methylation levels can be tuned by selecting promoters of varying strength. The technology also includes innovative methods for incorporating toxic genes into bacterial chromosomes by in vitro amplification and direct transformation, overcoming issues related to gene toxicity during plasmid replication.
Claims Coverage
The independent claims define two main inventive features centered on specific compositions containing methylated polynucleotides or methylated plasmid DNAs at defined methylation levels for the induction of a tolerogenic immune response.
Composition of first and second methylated polynucleotides with defined CpG methylation levels
A composition comprising: - A first methylated polynucleotide encoding a pro-apoptotic protein having a CpG methylation level of about 15% or less. - A second methylated polynucleotide encoding an antigen and having a CpG methylation level of about 25%. This composition is intended for use in the induction of a tolerogenic immune response against the antigen. The claim covers any antigen and pro-apoptotic protein fitting these requirements, as well as any ratio that ensures the specified methylation levels.
Composition of first and second methylated plasmid DNAs with defined CpG methylation levels
A composition comprising: - A first methylated plasmid DNA encoding a pro-apoptotic protein and having a CpG methylation level of about 15% or less. - A second methylated plasmid DNA encoding an antigen and having a CpG methylation level of about 25%. This composition is also for use in inducing a tolerogenic immune response against the antigen. The claim specifies plasmid DNAs as the polynucleotide form and requires the defined methylation statuses for both components.
The independent claims focus on the inventive combination of methylated nucleic acid constructs, with precise CpG methylation levels, for inducing a tolerogenic immune response. They cover both general polynucleotide and specific plasmid DNA forms, defining the necessary features for desired immune outcomes.
Stated Advantages
The invention allows production of plasmid DNA with adjustable and reproducible CpG methylation levels, enhancing consistency and scalability for pharmaceutical use.
By inserting a methylase gene into the bacterial chromosome under a constitutive promoter, the method avoids contamination with methylase-encoding plasmids and toxicity issues seen with plasmid-based expression.
Controlled methylation of plasmid DNA can enhance gene therapy efficacy, prolong therapeutic gene expression, and reduce immune-based adverse reactions.
The ability to fine-tune CpG methylation enables optimization for specific therapeutic applications and individualized treatment approaches.
Documented Applications
Production of plasmid DNA for gene therapy applications, providing desired levels of methylation to minimize immune response and prolong gene expression.
Preparation of DNA vaccines for inducing tolerogenic or immunoregulatory responses, such as in the treatment of autoimmune diseases, allergy, asthma, organ transplant rejection, and cancer.
Treatment of autoimmune diseases, including but not limited to type 1 diabetes, by administering compositions containing specific ratios of hypo- and hypermethylated DNA constructs encoding autoantigens and pro-apoptotic proteins.
Treating transplant recipients to promote tolerogenic responses and reduce graft rejection rates.
Applications in allergy treatment, including peanut, pollen, and cat allergies, via DNA vaccination using optimally methylated constructs.
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