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Regulation of F8 promoter activity
in HA mice receiving FVIII under the control of the ubiq- uitous 3-phosphoglycerate kinase promoter, reinforcing the importance of a targeted expression of FVIII to avoid a specific immune response.
Discussion
One of the major challenges in the design of gene trans- fer vectors over the years, has involved the selection of cell type- or tissue-specific promoters suitable for restoring transgene expression in a defined disease setting. In gene therapy, transcriptional targeting represents a way to restrict mRNA and protein expression to a specific cell type, even if transduction of viral vectors is likely to occur in multiple cell types.36 In the context of F8 gene transfer, several attempts have been made to achieve a selective and stable gene expression in various cell types. This has been challenging due to the complexity of FVIII synthesis, its size and immunogenic behavior, therefore highlighting the need for novel approaches. Using previous in silico analyses performed on the native pF8,23 we investigated in the present study the profile of TF potentially involved in promoting the expression of FVIII in EC. We identified and demonstrated in vitro that the Ets family of TF plays a fundamental role in the modulation of pF8 activity, offer- ing regulatory elements that can be explored to promote FVIII expression in a targeted manner. Further, we defined a minimal pF8 required to efficiently drive FVIII expres- sion in vivo, potentially overcoming a number of the cur- rent obstacles associated with F8 gene delivery.37
With LSEC now accepted as the main FVIII producing cells,15–18 understanding the key EC FVIII-specific regulato- ry elements is essential in promoting FVIII expression. The Ets-family of TF met our restricted criteria, with Ets-1 and -2 having the greatest number of BS across the pF8. More importantly, they are known to have essential roles in embryonic endothelial cell survival and in adult angiogen- esis.28 In adults the expression of Ets-1 is maintained at very low levels in the resting endothelium and is induced after specific stimuli. In particular, Ets-1 is reported to be up-regulated in response to pro-angiogenic and pro- inflammatory stimuli such as TNF-α, hepatocyte growth factor and platelet-derived growth factor.38 In addition, hypoxia has been demonstrated to regulate Ets-1 expres- sion through the hypoxia inducible factor-1.29 Further, LSEC are known to be physiologically exposed to decreas- ing oxygen pressure along the liver lobule.39
In our case, by overexpressing Ets-1 and Ets-2, both independently and collectively, we clearly demonstrate the involvement of Ets-1 in the regulation of pF8 activity. Interestingly, the transactivation efficiency of Ets-1 appeared to be potentiated by the presence of Ets-2, likely via protein-protein interactions. Co-operation between Ets-1 and -2 has been shown to be involved in the reacti- vation of the TERT gene in cancer.40 In this context, the co- operative interaction of Ets-1 and Ets-2 promotes the recruitment of p52 to a mutant form of the TERT promot- er enhancing TERT expression when the non-canonical NF-κB signaling is activated. In our case, we observed this phenomenon in ECV-304 but not in HEK293T cells, sug- gesting the existence of cell-specific regulation with respect to F8 expression. Both Ets-1 and Ets-2 are tightly regulated transcription factors,41 and it might be that in specific cells, like HEK293T, the forced expression of one
is able to bypass the establishment of a co-operative inter- action. A similar regulation is not surprising for F8 due to the necessity of a tight control, since it has been demon- strated that high expression of FVIII can cause cellular stress and increased immunogenicity.42
In order to clarify the relevance of the Ets-BS for the reg- ulation of pF8, we opted to generate several reduced sized pF8 progressively removing single or multiple Ets sites. This approach demonstrated that the -342 bp region, in which three Ets-1 and one Ets-2 sites were maintained, represented a minimal functional promoter in vitro. Further reduction of the pF8 to 246 bp, which excluded an addi- tional Ets-BS, caused a strong suppression of basal pF8 activity and preserved a minimal response to Ets-1 and Ets-2 overexpression. Of note, we identified that the -223 to -217 Ets-1 (E2) site is the most relevant regulatory ele- ment needed to achieve the maximal up-regulation of pF8 activity. In recent years, several mutations targeting the nucleotide positions ranging from -218 to -221, encom- passing the E2 site, have been described to be responsible for different degrees of HA.43 It has been clearly shown that these nucleotides are part of a highly conserved region where any change compromises the functionality of the pF8. Our in vitro studies highlight the importance of the -218 to -221 sequence, identifying it as the most essen- tial Ets-1 responsive element on pF8. The use of a CRISPRa system to transactivate pF8, reinforces this find- ing, highlighting the E2 site as being essential for pF8 reg- ulation. While only a modest up-regulation of pF8 was detectable in ECV-304, it is likely that the physiological expression of Ets-1 hampers the efficient binding of the sgRNA to the E2 site. The use of CRISPRa also supports the concept of the binding of Ets to the E2 site.
The in vivo delivery of LV expressing the GFP or FVIII transgene under the control of each described shortened promoter, reinforces the results obtained in vitro. All LV- treated mice displayed GFP expression in liver Lyve-1+ cells, most likely LSEC,44 resembling the pattern detectable using pF8-1175. Nevertheless, a few F4/80+ macrophages expressed GFP, especially with respects to the two longer pF8. This is not surprising because we previously described that the complete absence of macrophage tar- geting using an endothelial promoter, was only obtained by inserting a multiple miRNA target (mirT) sequence rec- ognized by the hematopoietic-specific miRNA 142-3p (mirT-142-3p).45
F8 gene transfer in HA mice confirmed the power of each tested promoter to drive long-term and stable FVIII expression without the appearance of inhibitors. Importantly, therapeutic levels of FVIII activity were detectable in all treated mice at similar levels to those observed in mice delivered with the control pF8-1,175 promoter. Such results are encouraging with respect to the current obstacles associated with the achievement of an efficient F8 gene delivery.
There are some limitations in our study. While Ets TF seem to be involved in pF8 regulation, the direct binding of Ets-1 and Ets-2 to pF8, and their specific role in LSEC remains to be confirmed. Further, the difficulty to isolate and maintain primary LSEC for extended periods in cul- ture,46 limits the use of luciferase assays.
Taken together, our in vitro and in vivo results have iden- tified the region from 0 to -342 as a minimal pF8 which preserves its activity and Ets-response and is comparable to the full-length pF8. Evidence for a role of Ets-1 and the
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