Utility of assessing CD3+ cell chimerism within the first months after allogeneic hematopoietic stem-cell transplantation for acute myeloid leukemia.

After allogeneic hematopoietic stem-cell transplantation (alloHSCT), the chimerism assay is used to monitor cell engraftment and quantify the respective proportions of donor/recipient cells in blood or bone-marrow samples. Here, we aimed to better assess the utility of determining CD3+ cell chimerism within the first 6 months post alloHSCT. One hundred and thirty five patients diagnosed with acute myeloid leukemia were enrolled in this study. We observed significantly lower overall survival and relapse free survival for patients without full donor chimerism (<95%, <98%, <99%) in whole blood at Day 30, as well as at Day 90 after alloHSCT, than for patients with full donor chimerism. This outcome was not observed when assessing selected CD3+ cells. However, at Day 90, patients with discordant whole blood versus selected CD3+ cell chimerism showed both significantly lower overall survival and relapse free survival, giving an interest to assess selected cells chimerism.

Strength and muscle specificity of a compact promoter derived from the slow troponin I gene in the context of episomal (gutless adenovirus) and integrating (lentiviral) vectors.

BACKGROUND: Gutless adenovirus (helper-dependent adenoviral vector; HDAd) and lentiviral vectors (LV) are attractive vectors for the gene therapy of muscle diseases. Because the organization of their DNA (episomal versus integrated) differs, we investigated whether the strength and specificity of ΔUSEx3, a novel muscle-specific promoter previously tested with plasmid, were maintained in the context of these vectors. METHODS: Two HDAds expressing ß-galactosidase regulated by ΔUSEx3 or CAG [cytomegalovirus (CMV) enhancer/ß-actin promoter], and three LV expressing green fluorescent protein regulated by ΔUSEx3, CMV or a modified skeletal α-actin promoter (SPcΔ5-12), were constructed. Gene expression was compared in cell culture and after intravenous (HDAd only) and intramuscular injection of mice. RESULTS: Irrespective of the vector used, ΔUSEx3 remained poorly active in nonmuscle cells and tissues. In myotubes, ΔUSEx3 was as strong as CMV and SPcΔ5-12, although it was ten-fold weaker than CAG, a proven powerful promoter in muscle. In cell culture, ΔUSEx3 activity in the context of LV was more stable than CMV, indicating it is less prone to silencing. In the context of HDAd, the behavior of ΔUSEx3 in skeletal muscle mirrored that of cell culture (10% of the CAG activity and half the number of transduced fibers). Surprisingly, in muscles treated with LV, ΔUSEx3 activity was five-fold lower than SPcΔ5-12. CONCLUSIONS: The data obtained in the present study confirm that ΔUSEx3 is a strong and robust muscle-specific promoter in the context of HDAd (cell culture and in vivo) and LV (cell culture). However, it was less efficient in vivo in the context of LV.

Strong muscle-specific regulatory cassettes based on multiple copies of the human slow troponin I gene upstream enhancer.

High-level tissue-specific expression of recombinant proteins in muscle is an important issue for several therapeutic applications. To achieve this goal, we generated several constructs containing one to five copies of the upstream enhancer (USE) of 160-bp of the human slow troponin I gene, linked to that gene's minimal promoter. We also tested constructs made with one to four copies of a 100-bp deletion of USE (DeltaUSE) reported to drive pan-muscle-specific expression in transgenic mice. These constructs were evaluated by measuring the activity of the reporter gene beta-galactosidase (beta-gal). In cell culture, these multimerized enhancers retained tissue specificity and their transcriptional strength increased with the number of enhancer copies. In myotube cultures (which still contain nondifferentiated cells), constructs containing four and five USE copies were stronger than the cytomegalovirus (CMV) early enhancer/promoter and slightly weaker than the hybrid CMV enhancer/beta-actin (CB) promoter. Those containing three USE, or four DeltaUSE copies were similar in strength to CMV. After electrotransfer of plasmid DNA into the mouse tibialis anterior muscle, the strengths of the two constructs (USEx3 and DeltaUSEx3) were tested; as measured by beta-gal activity in the total muscle lysate and by the number of transduced fibers, they were similar to CMV and CB. Muscle fiber typing, after electrotransfer of the soleus muscle, showed that DeltaUSEx3 and USEx3 were active in slow and fast fibers. The tissue specificity of these two constructs was also evaluated by hydrodynamic plasmid injection through the tail vein. Although significant beta-gal expression was measured in the liver when CMV was tested, no expression above background level was detected with USEx3 and DeltaUSEx3. The strength, muscle specificity, and small size of these transcriptional elements render them very attractive for gene therapy applications.