Specifications of qPCR based epigenetic immune cell quantification.

OBJECTIVES: Immune monitoring is an important aspect in diagnostics and clinical trials for patients with compromised immune systems. Flow cytometry is the standard method for immune cell counting but faces limitations. Best practice guidelines are available, but lack of standardization complicates compliance with e.g., in vitro diagnostic regulations. Limited sample availability forces immune monitoring to predominantly use population-based reference intervals. Epigenetic qPCR has evolved as alternative with broad applicability and low logistical demands. Analytical performance specifications (APS) have been defined for qPCR in several regulated fields including testing of genetically modified organisms or vector-shedding. METHODS: APS were characterized using five epigenetic qPCR-based assays quantifying CD3+, CD4+, CD8+ T, B and NK cells in light of regulatory requirements. RESULTS: Epigenetic qPCR meets all specifications including bias, variability, linearity, ruggedness and sample stability as suggested by pertinent guidelines and regulations. The assays were subsequently applied to capillary blood from 25 normal donors over a 28-day period. Index of individuality (IoI) and reference change values were determined to evaluate potential diagnostic gains of individual reference intervals. Analysis of the IoI suggests benefits for individual over population-based references. Reference change values (RCVs) show that changes of approx. Fifty percent from prior measurement are suggestive for clinically relevant changes in any of the 5 cell types. CONCLUSIONS: The demonstrated precision, long-term stability and obtained RCVs render epigenetic cell counting a promising tool for immune monitoring in clinical trials and diagnosis.

The Ratio of Regulatory (FOXP3+) to Total (CD3+) T Cells Determined by Epigenetic Cell Counting and Cardiovascular Disease Risk: A Prospective Case-cohort Study in Non-diabetics.

BACKGROUND: Experimental and clinical evidence indicate that inflammatory processes in atherogenesis and the development of cardiovascular complications are promoted by a loss of regulatory T cell (Treg)-mediated immunological tolerance to plaque antigens. Yet, the association between alterations of systemic Treg frequency and cardiovascular disease incidence remains uncertain. METHODS: A nested case-cohort study was conducted within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg, comprising a random subcohort (n=778) and primary cases of myocardial infarction (MI, n=276) and ischemic stroke (n=151). Pre-diagnostic FOXP3+ Treg and total CD3+ T-lymphocyte (tTL) frequencies in blood were measured by epigenetic-based, quantitative real-time PCR-assisted cell counting. RESULTS: Multivariate, Prentice-weighted Cox regression analyses revealed that lower Treg/tTL ratios were not associated with the risk of either MI (lowest vs. highest sex-specific quartile; hazard ratio: 0.72, 95% confidence interval: 0.46 to 1.13; Ptrend=0.51) or stroke (HR: 0.90, 95% CI: 0.51 to 1.60; Ptrend=0.78). There were no correlations of Treg/tTL ratios with C-reactive protein, HbA1c, and various lipid parameters. CONCLUSIONS: Among middle-aged adults from the general population, imbalances in the relative frequency of Tregs within the total T cell compartment do not confer an increased risk of MI or stroke.

Impaired NK cells and increased T regulatory cell numbers during cytotoxic maintenance therapy in AML.

Cyclic cytotoxic maintenance therapy can be applied to patients with AML in post-remission. We studied the immune status of AML patients in complete remission and the effect of maintenance therapy on different immune cell populations. Patients in complete remission had reduced NK, TH and Treg counts and a reduced NK activation capacity. In the course of cytotoxic maintenance therapy, NK counts further declined, while TH and Treg cells increased, with lower proliferative potential of TH cells. We conclude that immunotherapeutic approaches in post-remission have to consider reduced NK cell function and further impairment of cellular immune responses during cytotoxic therapy.

The cellular ratio of immune tolerance (immunoCRIT) is a definite marker for aggressiveness of solid tumors and may explain tumor dissemination patterns.

The adaptive immune system is involved in tumor establishment and aggressiveness. Tumors of the ovaries, an immune-privileged organ, spread via transceolomic routes and rarely to distant organs. This is contrary to tumors of non-immune privileged organs, which often disseminate hematogenously to distant organs. Epigenetics-based immune cell quantification allows direct comparison of the immune status in benign and malignant tissues and in blood. Here, we introduce the "cellular ratio of immune tolerance" (immunoCRIT) as defined by the ratio of regulatory T cells to total T lymphocytes. The immunoCRIT was analyzed on 273 benign tissue samples of colorectal, bronchial, renal and ovarian origin as well as in 808 samples from primary colorectal, bronchial, mammary and ovarian cancers. ImmunoCRIT is strongly increased in all cancerous tissues and gradually augmented strictly dependent on tumor aggressiveness. In peripheral blood of ovarian cancer patients, immunoCRIT incrementally increases from primary diagnosis to disease recurrence, at which distant metastases frequently occur. We postulate that non-pathological immunoCRIT values observed in peripheral blood of immune privileged ovarian tumor patients are sufficient to prevent hematogenous spread at primary diagnosis. Contrarily, non-immune privileged tumors establish high immunoCRIT in an immunological environment equivalent to the bloodstream and thus spread hematogenously to distant organs. In summary, our data suggest that the immunoCRIT is a powerful marker for tumor aggressiveness and disease dissemination.

The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting.

Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord-derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3'-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3'-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications.

Versatile and efficient genome editing in human cells by combining zinc-finger nucleases with adeno-associated viral vectors.

Zinc-finger nucleases (ZFNs) have become a valuable tool for targeted genome engineering. Based on the enzyme's ability to create a site-specific DNA double-strand break, ZFNs promote genome editing by activating the cellular DNA damage response, including homology-directed repair (HDR) and nonhomologous end-joining. The goal of this study was (i) to demonstrate the versatility of combining the ZFN technology with a vector platform based on adeno-associated virus (AAV), and (ii) to assess the toxicity evoked by this platform. To this end, human cell lines that harbor enhanced green fluorescence protein (EGFP) reporters were generated to easily quantify the frequencies of gene deletion, gene disruption, and gene correction. We demonstrated that ZFN-encoding AAV expression vectors can be employed to induce large chromosomal deletions or to disrupt genes in up to 32% of transduced cells. In combination with AAV vectors that served as HDR donors, the AAV-ZFN platform was utilized to correct a mutation in EGFP in up to 6% of cells. Genome editing on the DNA level was confirmed by genotyping. Although cell cycle profiling revealed a modest G2/M arrest at high AAV-ZFN vector doses, platform-induced apoptosis could not be detected. In conclusion, the combined AAV-ZFN vector technology is a useful tool to edit the human genome with high efficiency. Because AAV vectors can transduce many cell types relevant for gene therapy, the ex vivo and in vivo delivery of ZFNs via AAV vectors will be of great interest for the treatment of inherited disorders.

Fate of recombinant adeno-associated viral vector genomes during DNA double-strand break-induced gene targeting in human cells.

Recombinant vectors based on adeno-associated virus (rAAV) are promising tools to specifically alter complex genomes through homologous recombination (HR)-based gene targeting. In a therapeutic setting, an AAV donor vector will recombine with a mutant target locus in order to correct the mutation directly in the genome. The low frequency of HR in mammalian cells can be significantly improved by insertion of a DNA double-strand break (DSB) into the target locus through expression of a site-specific endonuclease. Here, we have scrutinized the fate of rAAV vector genomes during DSB-induced gene targeting and assessed the targeting frequency and the targeting ratio as a risk-benefit indicator. In various human cell lines carrying a mutated enhanced green fluorescent protein locus with a recognition site for the homing endonuclease I-SceI, rAAV-transduced cells were assayed by flow cytometry and by quantitative allele-specific polymerase chain reaction to assess HR and unspecific integration events. Under optimal conditions gene-targeting frequencies of 65% and targeting ratios of 2:1 were achieved, that is, more gene correction than unspecific integrations. The gene-targeting frequency was highly dependent on rAAV vector design, the cell line, and on the presence of a DSB in the target locus. Although expression of I-SceI led to a significant increase in gene targeting, it did not augment unspecific integration. In conclusion, our results reveal the side effects associated with rAAV-mediated gene targeting, but also its great potential for precise genome engineering in a therapeutic context.

Targeted chromosomal gene modification in human cells by single-stranded oligodeoxynucleotides in the presence of a DNA double-strand break.

A DNA double-strand break (DSB) cannot be tolerated by a cell and is dealt with by several pathways. Here, it was hypothesized that DSB induction close to a targeted mutation in the genome of a mammalian cell might attract oligodeoxynucleotide (ODN)-directed gene repair. A HEK-293-derived cell line had been engineered harboring a single target locus with open reading frames encoding the living-cell reporter proteins LacZ and EGFP, the latter translationally decoupled by a DNA spacer with a unique I-SceI recognition site for defined DSB induction. To enable expression of a fluorescent LacZ-EGFP fusion protein, single-stranded (ss) ODNs (80 or 96 nucleotides long) spanning the DSB were designed to fuse both reading frames by altering a few base-pair positions, deleting 59 bp or introducing a 10-bp fragment. The ssODNs alone generated few EGFP-positive cells. With I-SceI transiently expressed, more than 0.3% of cells revealed EGFP expression 7 days after transfection, with up to 96% of the loci faithfully corrected, depending on the ssODN used. During these correction events, the ssODN did not become physically incorporated into the chromosome, but served only as information template. Unwanted insertional mutagenesis also occurred. Both observations have important implications for gene therapy.