Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting.

OBJECTIVE: We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. STUDY DESIGN: Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. RESULTS: In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89-100%) and 99.7% specificity (95% confidence interval, 98.5-99.9%). CONCLUSION: Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.

Quantification of fetal DNA by use of methylation-based DNA discrimination.

BACKGROUND: Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in noninvasive prenatal diagnostics. Most applications rely on the qualitative detection of fetal nucleic acids to determine the genetic makeup of the fetus. This method leads to an analytic dilemma, because test results from samples that do not contain fetal DNA or are contaminated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to maternal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma. METHODS: The assay used methylation-sensitive restriction enzymes to eliminate the maternal (unmethylated) fraction of the DNA sample. The undigested fetal DNA fraction was then coamplified in the presence of a synthetic oligonucleotide to permit competitive PCR. The amplification products were quantified by single-base extension and MALDI-TOF MS analysis. RESULTS: Using 2 independent markers, (sex determining region Y)-box 14 (SOX14) and T-box 3 (TBX3), we measured a mean of 151 copies of fetal DNA/mL plasma and a mean fetal fraction of 0.13 in samples obtained from pregnant women. We investigated 242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensitivity and specificity for the assay of 99% and 100%, respectively. CONCLUSIONS: By investigating several regions in parallel, we reduced the measurement variance and enabled quantification of circulating cell-free DNA. Our results indicate that this multiplex methylation-based reaction detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma.

Autologous transplantation of endothelial progenitor cells genetically modified by adeno-associated viral vector delivering insulin-like growth factor-1 gene after myocardial infarction.

The regenerative potential of bone marrow-derived endothelial progenitor cells (EPCs) has been adapted for the treatment of myocardial and limb ischemia via ex vivo expansion. We sought to enhance EPC function by the efficient genetic modification of EPCs in a rat model of myocardial infarction. Peripheral blood EPCs were expanded and transduced, using adeno-associated virus (AAV). AAV-mediated EPC transduction efficacy was 23 ± 1.2%, which was improved by 4.0- to 7.2-fold after pretreatment with the tyrosine kinase inhibitor genistein. Adult rats (n = 7 in each group) underwent myocardial infarction by left anterior descending coronary artery occlusion, and received autologous EPCs transduced by AAV-IGF-1 or AAV-lacZ into the periinfarct area. Echocardiography demonstrated that cardiac function in the IGF-1-EPC group was significantly improved compared with the lacZ-EPC control group 12 weeks after myocardial infarction. In addition, IGF-1-expressing EPCs led to reduced cardiac apoptosis, increased cardiomyocyte proliferation, and increased numbers of capillaries in the periinfarct area. AAV expression was limited to the targeted heart region only. Pretreatment with genistein markedly improved AAV transduction of EPCs. IGF-1-expressing EPCs exhibit favorable cell-protective effects with tissue-limited expression in rat heart postinfarction.

Analysis of angiogenesis induced by local IGF-1 expression after myocardial infarction using microSPECT-CT imaging.

Insulin-like growth factor-1 (IGF-1) has been found to exert favorable effects on angiogenesis in prior animal studies. This study explored the long-term effect of IGF-1 on angiogenesis using microSPECT-CT in infarcted rat hearts after delivering human IGF-1 gene by adeno-associated virus (AAV). Myocardial infarction (MI) was induced in Sprague-Dawley rats by ligation of the proximal anterior coronary artery and a total of 10(11) AAV-CMV-lacZ (control) or IGF-1 vectors were injected around the peri-infarct area. IGF-1 expression by AAV stably transduced heart muscle for up to 16 weeks post-MI and immunohistochemistry revealed a remarkable increase in capillary density. A (99m)Tc-labeled RGD peptide (NC100692, GE Healthcare) was used to assess temporal and regional alpha(v) integrin activation. Rats were injected with NC100692 followed by (201)Tl chloride and in vivo microSPECT-CT imaging was performed. After imaging, hearts were excised and cut for quantitative gamma-well counting (GWC). NC100692 retention was significantly increased in hypoperfused regions of both lacZ and IGF-1 rats at 4 and 16 weeks post-MI. Significantly higher activation of alpha(v) integrin was observed in IGF-1 rats at 4 weeks after treatment compared with control group, although the activation was lower in the IGF-1 group at 16 weeks. Local IGF-1 gene delivery by AAV can render a sustained transduction and improve cardiac function post-MI. IGF-1 expression contributes to enhanced alpha(v) integrin activation which is linked to angiogenesis. MicroSPECT-CT imaging with (99m)Tc-NC100692 and quantitative GWC successfully assessed differences in alpha(v) integrin activation between IGF-1-treated and control animals post-MI.

Role of cyclic AMP-dependent kinase response element-binding protein in recombinant adeno-associated virus-mediated transduction of heart muscle cells.

Recombinant adeno-associated virus (rAAV) vectors represent a promising approach to gene delivery for clinical use. Published data indicate that rAAV vector genomes persist in vivo as episomal chromatin in the skeletal muscle of nonhuman primates. In this study, we assessed the interconnection between the transcription factor cyclic AMP response element-binding protein (CREB) and recombinant AAV serotype 2 vector genomes after transduction in vitro and in vivo. rAAV-mediated myocyte transduction was potently blocked in the hearts of mice expressing CREB-S133A, which is a CREB-S133A dominant-negative mutant. Isoproterenol, a strong CREB activator, prominently increased rAAV transduction and the increase was abrogated by silencing the CREB gene with small interfering RNA. In addition, rAAV infection of muscle cells mildly but significantly induced CREB protein phosphorylation at serine-133, and was capable of stimulating CREB-dependent transcription from a reporter plasmid. Using chromatin immunoprecipitation and immunoblotting assays, both CREB and p300 were found to physically associate with two different rAAV genomes. Accordingly, CREB/p300 appears to have a role in rAAV transduction to establish active vector transcription in heart muscle cells.

Utility of vascular endothelial specific peptides for enhancement of adeno-associated virus-mediated gene transfer.

Vascular endothelial cells (EC) have been targeted for the treatment of pathological conditions such as atherosclerosis, hypercholesterolemia, post-angioplasty restenosis and hypertension. Non-pathogenic adeno-associated virus (AAV) has been shown as a good gene delivery tool in a variety of cell lines as well as in animal models. However, AAV has been reported to induce less endothelial cell transduction. AAV vector alone transduced HUVEC much lower than other cell lines including Hela, PAC1, and C2C12. Preincubation of AAV vector with EC membrane specific peptides markedly increased AAV transduction of HUVEC. On the contrary, those peptides did not affect AAV expression in other cell types. These EC-specific peptides may be a strategy for enhancement of AAV mediated-gene expression.

A novel variant of ileal bile acid binding protein is up-regulated through nuclear factor-kappaB activation in colorectal adenocarcinoma.

Ileal bile acid binding protein (IBABP) is the only cytosolic protein known to bind and transport bile acids. Because IBABP is reportedly up-regulated in colorectal cancer, it has been suggested as a link between bile acids and the risk of colorectal cancer. However, in this study, we show that IBABP is not up-regulated. Rather, a novel transcript of the IBABP gene, which encodes an additional 49 NH(2)-terminal amino acid residues, is up-regulated in colorectal cancer (P < 0.001). The novel transcript, called IBABP-L, is also distinct from IBABP because its transcription is controlled by nuclear factor-kappaB (NF-kappaB) rather than by the farnesoid X receptor. Most significantly, IBABP-L is necessary for the survival of HCT116 colon cancer cells in the presence of physiologic levels of the secondary bile acid deoxycholate. Collectively, the studies point toward a unique bile acid response pathway involving NF-kappaB and IBABP-L that could be useful for diagnosis and could potentially be targeted for therapeutic benefit.

Long-term stable expression of human growth hormone by rAAV promotes myocardial protection post-myocardial infarction.

Recombinant adeno-associated virus (rAAV)-based gene therapy represents a promising approach for the treatment of heart diseases. It has been shown that growth hormone (GH) exerts a favorable effect on cardiovascular function in clinical and animal studies. This study explores a chronic stage after myocardial infarction and the potential therapeutic effects of delivering a human GH gene by rAAV following coronary artery ligation in Sprague-Dawley rats. rAAV vectors stably transduced heart muscle for up to 22 weeks after myocardial infarction (MI). Overexpression of GH via rAAV vectors significantly improved not only cardiac function but also LV pathologic remodeling was attenuated post-MI compared to the control rAAV-lacZ injected group. rAAV-mediated expression of GH also resulted in a significant induction of several angiogenic genes such as eNOS, VEGF and bFGF in rat hearts. Immunohistochemistry revealed an increase in capillary density and proliferation of cells and a decrease in the number of TUNEL-positive cardiomyocytes in the rAAV-GH group. Based on these data, we conclude that rAAV-mediated GH delivery can render a long-term transduction in the infarcted heart and improve cardiac function through promoting angiogenesis and proliferation of cells and protecting cardiomyocytes from ischemia-induced apoptosis.

Use of recombinant adeno-associated viral vectors as a tool for labeling bone marrow cells.

We have tested the feasibility of using recombinant adeno-associated virus (rAAV) vectors as a tool for labeling bone marrow (BM) cells in vivo. We infected BM cells of donor FVB mice with rAAV vectors containing the lacZ gene for 2 h. We then injected the rAAV-infected cells to lethally irradiated-recipient FVB mice. Peripheral blood (PB), BM and spleen harvested at 4 weeks after BM transplant (BMT) demonstrated stable engraftment in beta-galactosidase (beta-gal) expression. In contrast, Dil-labeling displayed only a faint signal 4 weeks after BMT. To analyze the kinetics of BM cells, we injected vascular endothelial growth factor (VEGF), which promotes mobilization of BM cells. Administration of VEGF protein significantly increased the rAAV-mediated beta-gal expression in PB and BM of recipient mice. Moreover, when myocardial infarction was induced in BMT mice, the ischemic area exhibited significant beta-gal staining in rAAV-labeled BMT group. rAAV vectors programmed stable transduction in BM cells in vivo through rapid infection. rAAV appears to represent a useful vector for labeling BM cells ex vivo prior to BMT for analysis of cardiovascular therapeutic purposes.