Oligonucleotide delivery by nucleofection does not rescue the reduced proliferation phenotype of gene-edited cells.

Gene editing using single-stranded oligonucleotides (ODNs) can be used to reverse or create a single base mutation in mammalian cells. This approach could be used to treat genetic diseases caused, at least in part, by a nucleotide substitution. The technique could also be used as a tool to establish single base polymorphisms at multiple sites and thus aid in creating cell lines that can be used to define the basis for drug resistance in human cells. A troubling outcome of the gene-editing reaction is the effect on normal growth of cells that have undergone nucleotide exchange. In this work, we attempt to overcome this reduced proliferation phenotype by changing the method by which the ODN is introduced into the target cell. Using a series of assays that measure gene editing, DNA damage response, and cell viability, we report that chemically modified ODNs, the most active form of ODN for gene editing, can be used successfully if introduced into the cell by the method of nucleofection. Unlike electroporation, which has been used as the standard mode of ODN delivery, one new result is that nucleofection does not induce a dramatic loss of viability within the first 24 hours after the start of gene editing. In addition, and importantly, ODNs introduced to the cell by nucleofection do not activate the DNA damage response pathway as dramatically as ODNs introduced by electroporation. These 2 novel findings are encouraging for the application of gene editing in other systems. However, reduced proliferation phenotype is still observed when the population of corrected cells is monitored out to 8 days, and thus, delivery by nucleofection does not solve the proliferation problem encountered by cells bearing an edited gene.

DNA damage response pathway and replication fork stress during oligonucleotide directed gene editing.

Single-stranded DNA oligonucleotides (ODNs) can be used to direct the exchange of nucleotides in the genome of mammalian cells in a process known as gene editing. Once refined, gene editing should become a viable option for gene therapy and molecular medicine. Gene editing is regulated by a number of DNA recombination and repair pathways whose natural activities often lead to single- and double-stranded DNA breaks. It has been previously shown that introduction of a phosphorotioated ODN, designed to direct a gene-editing event, into cells results in the activation of γH2AX, a well-recognized protein biomarker for double-stranded DNA breakage. Using a single copy, integrated mutant enhanced green fluorescent protein (eGFP) gene as our target, we now demonstrate that several types of ODNs, capable of directing gene editing, also activate the DNA damage response and the post-translational modification of proliferating cell nuclear antigen (PCNA), a signature modification of replication stress. We find that the gene editing reaction itself leads to transient DNA breakage, perhaps through replication fork collapse. Unmodified specific ODNs elicit a lesser degree of replication stress than their chemically modified counterparts, but are also less active in gene editing. Modified phosphothioate oligonucleotides (PTOs) are detrimental irrespective of the DNA sequence. Such collateral damage may prove problematic for proliferation of human cells genetically modified by gene editing.

Fetal mortality: timing of racial disparities.

Whether or not racial disparities exist in fetal mortality rate (FMR) statistics depends upon the methodology used to calculate the rates. While there appears to be consensus that there is a black-white disparity in late gestation (> or = 28 weeks), the issue is unclear for early gestation (20-27 weeks). To clarify this issue, we assessed disparities in FMR for singleton fetal deaths and live births between non-Hispanic blacks and non-Hispanic whites in three counties of Missouri using gestational age- and weight-specific analyses. These analyses demonstrated statistically significant disparities for non-Hispanic whites when fetal deaths occurred < 28 weeks gestation and also at weights < 1,000 g. Statistically significant disparities for non-Hispanic blacks were not evident until gestation was > or = 32 weeks or weights were > or = 2,500 g. The results of these analyses were consistent with each other and suggest that the non-Hispanic black disparity in FMR is a late gestational issue. The lack of disparity for non-Hispanic blacks and the disparity for non-Hispanic whites during earlier gestation or with low weights were associated with the disparate rates for very preterm live birth.

Perinatal periods of risk analysis of infant mortality in Jackson County, Missouri.

The perinatal periods of risk (PPOR) methodology provides an easy-to-use analytical approach to infant mortality that helps focus community initiatives for improving maternal and infant health. Because few analyses have been published, many public health practitioners may be unfamiliar with PPOR. This article demonstrates the application of PPOR analysis using infant mortality in Jackson County, Missouri. While the PPOR consists of two phases, this analysis was restricted to the initial phase of the overall process. The second phase builds on the initial findings and prioritizes the contributing factors of fetal/infant mortality so that targeted interventions can be developed. For Jackson County, the PPOR analysis found that racial and geographic disparities existed and, for very low-birth-weight infants, different interventions strategies may be needed on the basis of race. In addition, a mother who experienced a fetal or infant death was more likely to have had a medical risk factor, to have smoked cigarettes, to have started prenatal care after the first trimester or received no prenatal care, and to have been nulliparous.