Laser-Supported CD133+ Cell Therapy in Patients with Ischemic Cardiomyopathy: Initial Results from a Prospective Phase I Multicenter Trial

Objectives: This study evaluates the safety, principal feasibility and restoration potential of laser-supported CD133+ intramyocardial cell transplantation in patients with ischemic cardiomyopathy. Methods: Forty-two patients with severe ischemic cardiomyopathy (left ventricular ejection fraction (LVEF) >15% and 3 myocardial segments) determined in the preoperative MRI was inversely correlated with a LVEF increase after laser-supported cell therapy (p = 0.024). Conclusions: This multicenter trial demonstrates that laser-supported CD133+ cell transplantation is safe and feasible in patients with ischemic cardiomyopathy undergoing CABG, and in most cases, it appears to significantly improve the myocardial function. Importantly, our data show that the beneficial effect was significantly related to the extent of transmural delayed enhancement, suggesting that MRI-guided selection of patients is mandatory to ensure the effectiveness of the therapy. Trial Registration: EudraCT 2005-004051-35) Controlled-Trials.com ISRCTN4999863

Ranking noncanonical 5 ' splice site usage by genome-wide RNA-seq analysis and splicing reporter assays

Most human pathogenic mutations in 5' splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in similar to 1% of all human 5'ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5'ss are used. In this work, we systematically examined noncanonical 5' splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2.4 billion gapped reads covering 269,375 exon junctions. From both approaches, we consistently derived a noncanonical 5'ss usage ranking GC > TT > AT > GA > GG > CT. In our competition splicing reporter assay, noncanonical splicing was strictly dependent on the presence of upstream or downstream splicing regulatory elements (SREs), and changes in SREs could be compensated by variation of Ul snRNA complementarity in the competing 5'ss. In particular, we could confirm splicing at different positions (i.e., -1, +1, +5) of a splice site for all noncanonical dinucleotides weaker than GC. In our comprehensive RNA-seq data set analysis, noncanonical 5'ss were preferentially detected in weakly used exon junctions of highly expressed genes. Among high-confidence splice sites, they were 10-fold overrepresented in clusters with a neighboring, more frequently used 5'ss. Conversely, these more frequently used neighbors contained only the dinucleotides GT, GC, and TT, in accordance with the above ranking

Altered HIV-1 mRNA Splicing Due to Drug-Resistance-Associated Mutations in Exon 2/2b

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient

Study inclusion and exclusion criteria.

<p>Study inclusion and exclusion criteria.</p

Preoperative indicators for the improvement of the LVEF.

<p>While the preoperative global LVEF values were not correlated with the postoperative increase of the LVEF (A), we found an inverse correlation of the preoperative amount of transmural delayed enhancement (TDE) with the postoperative increase of the LVEF (B). The association of a preoperatively larger percentaged area of hibernating myocardium with a higher increase of the LVEF was not statistically significant (C).</p

Quality of life after cell therapy.

<p>At all postoperative time points, the physical limitation score of the Seattle Angina Questionnaire (SAQ) was significantly improved (40.2±1.4 after 3 months, 40.9±1.5 after 6 months, 43.7±1.0 after 12 months, versus 28.4±1.5 preoperatively). Pre, preoperatively. Separated values, outliers included in the statistical analysis. Asterisk, p<0.0001 versus pre.</p

Transmural delayed enhancement (TDE) and the postoperative increase of the LVEF.

<p>TDE in more than three myocardial segments (at screening MRI) resulted in significantly less increase of the LVEF after 6 months, as compared to patients with three or less affected segments (1.0±1.4 versus 10.0±4.3, p = 0.042).</p

Blood serum level analyses.

<p>Blood serum level analyses.</p

Preoperative patients' characteristics.

<p>Preoperative patients' characteristics.</p

Endpoint events.

<p>Endpoint events.</p