A method for delivering an isolated polynucleotide such as DNA or RNA, to the interior of a cell in a mammal comprising the injection of an isolated polynucleotide into a muscle of the mammal where the polynucleotide is taken up by the cells of the muscle and exerts a therapeutic effect on the mammal. The method can be used to deliver a therapeutic polypeptide to the cells of the mammal, to provide an immune response upon in vivo translation of the polynucleotide, to deliver antisense polynucleotides, to deliver receptors to the cells of the mammal or to provide transitory gene therapy.

Induction of a protective immune response in a mammal by injecting a DNA sequence

Circulating nucleic acids (CNAs) and cancer--a survey.

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Sata) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.

/pdf/analysis-of-repetitive-element-dna-methylation-by-methylight-tr3c5njakq.pdf

Analysis of repetitive element DNA methylation by MethyLight

Background Targeted therapies have markedly changed the treatment of cancer over the past 10 years However, almost all tumors acquire resistance to systemic treatment as a result of tumor heterogeneity, clonal evolution, and selection Although genotyping is the most currently used method for categorizing tumors for clinical decisions, tumor tissues provide only a snapshot, or are often difficult to obtain To overcome these issues, methods are needed for a rapid, cost-effective, and noninvasive identification of biomarkers at various time points during the course of disease Because cell-free circulating tumor DNA (ctDNA) is a potential surrogate for the entire tumor genome, the use of ctDNA as a liquid biopsy may help to obtain the genetic follow-up data that are urgently needed Content This review includes recent studies exploring the diagnostic, prognostic, and predictive potential of ctDNA as a liquid biopsy in cancer In addition, it covers biological and technical aspects, including recent advances in the analytical sensitivity and accuracy of DNA analysis as well as hurdles that have to be overcome before implementation into clinical routine Summary Although the analysis of ctDNA is a promising area, and despite all efforts to develop suitable tools for a comprehensive analysis of tumor genomes from plasma DNA, the liquid biopsy is not yet routinely used as a clinical application Harmonization of preanalytical and analytical procedures is needed to provide clinical standards to validate the liquid biopsy as a clinical biomarker in well-designed and sufficiently powered multicenter studies

/pdf/circulating-tumor-dna-as-a-liquid-biopsy-for-cancer-430362ojbn.pdf

Circulating Tumor DNA as a Liquid Biopsy for Cancer

While various clinical applications especially in oncology are now in progress such as diagnosis, prognosis, therapy monitoring, or patient follow-up, the determination of structural characteristics of cell-free circulating DNA (cirDNA) are still being researched. Nevertheless, some specific structures have been identified and cirDNA has been shown to be composed of many “kinds.” This structural description goes hand-in-hand with the mechanisms of its origins such as apoptosis, necrosis, active release, phagocytosis, and exocytose. There are multiple structural forms of cirDNA depending upon the mechanism of release: particulate structures (exosomes, microparticles, apoptotic bodies) or macromolecular structures (nucleosomes, virtosomes/proteolipidonucleic acid complexes, DNA traps, links with serum proteins or to the cell-free membrane parts). In addition, cirDNA concerns both nuclear and/or mitochondrial DNA with both species exhibiting different structural characteristics that potentially reveal different forms of biological stability or diagnostic significance. This review focuses on the origins, structures and functional aspects that are paradoxically less well described in the literature while numerous reviews are directed to the clinical application of cirDNA. Differentiation of the various structures and better knowledge of the fate of cirDNA would considerably expand the diagnostic power of cirDNA analysis especially with regard to the patient follow-up enlarging the scope of personalized medicine. A better understanding of the subsequent fate of cirDNA would also help in deciphering its functional aspects such as their capacity for either genometastasis or their pro-inflammatory and immunological effects.

/pdf/origins-structures-and-functions-of-circulating-dna-in-2da7sm8ntv.pdf

Origins, structures, and functions of circulating DNA in oncology

Small amounts of DNA circulate freely in plasma or serum, but the mechanism of release is not known. To determine if DNA is actively excreted from viable cells, we utilized real-time PCR to measure the proportion of Alu repeat sequences compared to the beta-globin gene in serum and lymphocyte DNA in 27 cancer patients and 22 healthy controls. The proportion of Alu compared to beta-globin was significantly greater in serum DNA than in lymphocyte DNA both in control subjects (p = 0.003) and in cancer patients (p < 0.001). Overall, the proportion was similar in cancer and control patients (p = 0.79). Further experiments showed that the beta-globin gene was not more vulnerable to degradation by nuclease action than Alu sequences. Our results lead us to conclude that active DNA release is likely to play a significant role in the origin of circulating DNA.

Alu repeat sequences are present in increased proportions compared to a unique gene in plasma/serum DNA: evidence for a preferential release from viable cells?

Summary

Human lymphocytes obtained from donors exhibiting different allotypes were separated into B- and T-enriched subpopulations and cultured in the presence or absence of Herpes simplex virus inactivated by U.V. Isolated B or T cell suspensions did not produce any antiherpetic activity. The B lymphocytes cultruredl in the presence of the supernatant collected from virus-exposed T cells or in the presence of DNA extracted from this supernatant, synthesized an antiherpetic antibody carrying allotypic marker of the T cell donor.

The role of extracellular DNA in the transfer of information from T to B human lymphocytes in the course of an immune response.

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Alu repeat sequences are present in increased proportions compared to a unique gene in plasma/serum DNA: evidence for a preferential release from viable cells?

The role of extracellular DNA in the transfer of information from T to B human lymphocytes in the course of an immune response.