Nucleated Red Blood Cells in Children With Sickle Cell Disease Hospitalized for Pain.

Acute chest syndrome (ACS) and transfusion requirements are common and difficult to predict during hospitalizations for acute vaso-occlusive episodes (VOE) among individuals with sickle cell disease (SCD). This study examined the relationship between nucleated red blood cell (NRBC) counts during hospitalization for VOE and development of ACS or transfusion requirement among children with SCD. Retrospective chart review was performed for 264 encounters of patients with SCD hospitalized for uncomplicated VOE who had NRBC count data at admission during a 5-year period. Multivariable logistic regression analysis was conducted to determine the relationship of admission and change in NRBC ([INCREMENT]NRBC) to ACS/transfusion requirement. Overall, 44 of 264 (16.7%) encounters resulted in ACS, transfusion, or both. Admission NRBC was not associated with development of ACS/transfusion requirement. Among 125 of 264 (47.3%) encounters in which a subsequent CBC was obtained, greater increases in NRBCs and greater decrease in hemoglobin were significantly associated with ACS/transfusion requirement (OR, 2.72; 95% CI, 1.16, 6.35; P=0.02 and OR, 2.52; 95% CI, 1.08, 5.89; P=0.03, respectively). Our finding that an increase in NRBC counts was associated with development of ACS/transfusion requirement suggests that [INCREMENT]NRBCs may represent a useful biomarker for predicting complications in children with SCD hospitalized for VOE.

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.

Kinetics of lentiviral vector transduction in human CD34(+) cells.

Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34(+) cells. We transduced HeLa and CD34(+) cells with self-inactivating HIV-1 vector at low and tenfold higher multiplicity of infection (MOI) and evaluated vector amounts at various time points based on the rationale that if a given step was not limiting, tenfold greater vector amounts would be obtained at the tenfold higher MOI. We observed slower internalization (>60 min), a peak in reverse transcription at 24 hours, and completion of integration at 3 days in CD34(+) cells. In HeLa cells, there were approximately tenfold greater amounts at high MOI at all time points. When compared with HeLa cells, CD34(+) cells exhibited larger differences in vector amounts between high and low MOIs at 2-6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34(+) cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts did not decrease once detected among CD34(+) cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34(+) cells in vitro and in xenografted mice. These data suggest that it is the initiation of reverse transcription that limits lentiviral transduction of human CD34(+) cells. Our findings provide an avenue for optimizing human CD34(+) cell transduction.