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    • We revealed the PGE enhanced transduction of human HSPCs

    2018-11-09

    We revealed the PGE2-enhanced transduction of human HSPCs by VSV-G-pseudotyped LV, possibly by acting on the endocytosis-dependent entry pathway. Applying PGE2 makes shorter ex vivo manipulation protocols that feature a single transduction cycle clinically viable, mitigating the negative effect of prolonged culture on progenitor cell engraftment and allowing to double the number of patients that can be treated with a given vector batch with respect to current clinical standards. Importantly, the enhancing effect of PGE2 on LV transduction has recently been reported by an independent group (Heffner et al., 2016). In contrast to other pharmacological interventions that enhance transduction but may also have broader effects on HSC biology and/or cause toxicity (Wang et al., 2014; Petrillo et al., 2015), PGE2 was non-toxic, did not change the biological properties of the cells, and has already been safely employed in an ex vivo setting to increase CB engraftment in patients (Cutler et al., 2013). We did not note a significant increase in engraftment levels in the PGE2 groups, as could be expected from published data (Goessling et al., 2011). Further studies on diverse cell sources employing ultra-short ex vivo protocols are warranted to understand whether the promoting effects of PGE2 on LV transduction and engraftment capacity are functionally linked to a common mechanism and whether both effects can be exploited contemporaneously. The shortened PGE2 protocol can be readily applied to current ex vivo gene therapy trials and should translate clinically into accelerated hematologic recovery. We provide evidence here that CD34+CD38− isradipine transduce at higher levels than total CD34+ cells, consistent with a recent report (Baldwin et al., 2015). In vitro, the effect was most robust (1) when cell populations were sorted before transduction (Figure S4C), (2) when saturating vector doses were used (Figure S4B), and (3) when PGE2 was employed (Figure 3B). We hypothesize that the lack of cell division of CD34+CD38− cells during the first days of culture (Figure 4F) may give more time for infecting LV to complete integration before DNA replication, thus transmitting a copy of each provirus to both daughter cells, whereas the rapidly dividing CD38+ progenitors may randomly sort the LV pre-integration complexes between the two daughter cells. Considering that PGE2 relieves an early transduction block, applying it to purified CD34+CD38− cells may provide added benefits in terms of gene transfer efficiency. Robust gene transfer into HSCs may allow reducing the intensity of the conditioning regimen and implementing non-genotoxic and stem cell-specific conditioning strategies (Palchaudhuri et al., 2016). Regarding safety, avoiding the infusion of CD38+ progenitors carrying semi-random LV integrations would reduce the integration load 10-fold, thereby decreasing the chances of gain-of-function events due to insertional mutagenesis, which may originate from immortalized progenitor cells (Cavazzana-Calvo et al., 2010; Stein et al., 2010; Braun et al., 2014). We argue that gene therapy based on transduced, purified CD34+CD38− cells is clinically feasible and offers advantages over alternative strategies that have previously been employed in clinical trials to obtain HSC-enriched cell populations, including positive selection for CD133+ cells (Gordon et al., 2003) or CD34+CD90+ cells (Michallet et al., 2000; Negrin et al., 2000). Combining a negative (CD38) with a positive (CD34) selection marker allows using a sequential, bead-based selection procedure that can be made GMP compliant and implemented in centers that already possess standard cell separation equipment. The 10-fold gain in HSC purity with respect to CD34+ cells is higher than what can be achieved with the CD133+ (2×) or the CD34+CD90+ (up to 5×) selection strategies. Furthermore, we show that almost all long-term repopulation activity is captured within the CD34+CD38− fraction, while, in agreement with CB data (Notta et al., 2011), substantial numbers of HSCs are contained within the CD34+CD90– fraction of mPB, which would be discarded in the case of a CD34+CD90+ selection. While more stringent gating on CD90high cells may increase HSC purity, it would at the same time aggravate the loss of CD90–/low HSCs.