Incidence of Abnormal Findings During Pelvic Examinations in Women Aged 21-35 Years.

To describe the incidence of abnormal gynecologic examination findings in asymptomatic compared with symptomatic patients during preventive visits, we conducted a retrospective study of 1,121 visits for patients between the ages of 21 and 35 years from January 2017 to March 2017. Only 1.2% (95% CI, 0.5%,1.9%) of asymptomatic patients had abnormal findings on pelvic examination, compared with 32.4% (95% CI, 27.0%, 37.8%) of those with symptoms ( P ≤.001). In symptomatic patients, the most common symptoms were vaginal discharge (25.1%), pelvic pain (16.4%), and vaginal bleeding (15.7%). In asymptomatic patients, the most common findings were bacterial vaginosis and Candida infection. Asymptomatic patients presenting for a routine preventive visit have low rates of abnormalities detected on examination, and routine pelvic examinations should be re-considered.

RNA Trans-Splicing Targeting Endogenous ß-Globin Pre-Messenger RNA in Human Erythroid Cells.

Sickle cell disease results from a point mutation in exon 1 of the ß-globin gene (total 3 exons). Replacing sickle ß-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting ß-globin pre-messenger RNA among human erythroid cells. Binding domains from random ß-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous ß-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous ß-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.

Total body irradiation must be delivered at high dose for efficient engraftment and tolerance in a rhesus stem cell gene therapy model.

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) gene therapy applications. However, low gene marking was previously observed in gene therapy trials, suggesting that RIC might be insufficient for (i) opening niches for efficient engraftment and/or (ii) inducing immunological tolerance for transgene-encoded proteins. Therefore, we evaluated both engraftment and tolerance for gene-modified cells using our rhesus HSC gene therapy model following RIC. We investigated a dose de-escalation of total body irradiation (TBI) from our standard dose of 10Gy (10, 8, 6, and 4Gy), in which rhesus CD34(+) cells were transduced with a VSVG-pseudotyped chimeric HIV-1 vector encoding enhanced green fluorescent protein (GFP) (or enhanced yellow fluorescent protein (YFP)). At ~6 months after transplantation, higher-dose TBI resulted in higher gene marking with logarithmic regression in peripheral blood cells. We then evaluated immunological tolerance for gene-modified cells, and found that lower-dose TBI allowed vigorous anti-GFP antibody production with logarithmic regression, while no significant anti-VSVG antibody formation was observed among all TBI groups. These data suggest that higher-dose TBI improves both engraftment and immunological tolerance for gene-modified cells. Additional immunosuppression might be required in RIC to induce tolerance for transgene products. Our findings should be valuable for developing conditioning regimens for HSC gene therapy applications.