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Regulation of F8 promoter activity
When considering gene therapy approaches aimed at restoring and sustaining FVIII activity, the liver is consid- ered the primary target organ, as it is the principal site of FVIII synthesis and possesses the necessary tolerogenic properties.8 The identity of liver cells capable of synthe- sizing and releasing FVIII has generated an extensive debate over the years.9 This has significantly influenced the understanding of the regulatory elements involved in promoting the preferential expression of FVIII in liver cells. The F8 promoter (pF8), reported to be a 1.2 kb region upstream of the F8 translation start site, was first described in 1984.10 With hepatocytes originally consid- ered the major source of FVIII,11,12 the first in vitro studies aimed at elucidating the transcription factors (TF) respon- sible for pF8 activation, were performed using hepato- cyte-derived cell lines. In hepatocytes, Figueiredo and McGlynn described the region from -279 and -64 to be responsible for maximal promoter activity.13,14 They iden- tified and confirmed the binding of several hepatic TF, such as CCAAT/enhancer-binding proteins (C/EBPα and C/EBPb), and hepatocyte nuclear factor 1 (HNF-1) and 4 (HNF-4). Other TF binding sites (TFBS) on pF8 were also identified in this study, however, to date their involve- ment has never been thoroughly investigated.
While heavily debated, it has recently been demonstrat- ed that liver FVIII production predominantly occurs in the liver sinusoidal endothelial cells (LSEC),15–18 which repre- sent a principal but not exclusive source.19–22 In fact, detec- tion of FVIII mRNA in many tissues, suggests that a highly complex and likely tissue-specific transcriptional regula- tion exists. Recently, our group described pF8’s ability to direct a specific and long-term FVIII expression in LSEC after a lentiviral vector (LV)-delivery in HA mice.23 Importantly, this targeted restoration of FVIII did not trig- ger an immune response, one of the major obstacles for the successful treatment of HA patients. In the present study, we used data from an in silico analysis of the pF8 region,23 to extrapolate and assess the role of the most rep- resented endothelial-specific TFBS on F8 transcriptional regulation. Understanding the stimuli and the TF required for maximal promoter activity in endothelial cells (EC), offers an inportant forward step in the development of gene therapeutic approaches for HA. To date, several clin- ical trials using the adeno-associated viral vectors (AAV) to delivery FVIII in HA patients have started. Despite the promising results, some concerns have been raised, like the vector dose, the variability of FVIII activity among the different subjects and the decline of FVIII expression over- time.24 Our optimization of the minimal pF8 size opens up the possibility to explore new perspectives in the field the HA gene therapy by introducing for the first time a pF8 suitable for vectors with a limited expression cassette, like the AAV which, to date, are the only ones successfully used in clinical trials for HA.
Methods
Animal studies
Experiments, described in the Online Supplementary Appendix, were performed according to an approved protocol by the Animal Care and Use Committee of the University of Eastern Piedmont and the Italian Health Ministry, Italy (Project n. DB064.5, date of approval n°492/2016-PR 17/05/2016).
Identification of putative endothelial transcription factors binding sites on the F8 promoter
In silico prediction of TFBS distribution on pF8 was retrieved from a previous analysis performed by Merlin et al.23 using PROMO 3.0.25 In order to identify potential endothelial TFBS, two parameters were considered: the number of consensus sites identified on pF8 using a stringent dissimilarity rate (<3) and the expression and functional role of TF in EC.
Generation of the constructs
The full-length human pF8 (1,175 basepairs [bp]) was excised from a plasmid already available in our laboratory and cloned with XhoI-HindIII into a promoterless pNL1.1 vector at the 5’ of NanoLuc® luciferase reporter gene. Serial deletions of pF8 were generated via polymerase chain reaction using diverse primer sets carrying restriction sites, while E26 transformation-specific (Ets)-core sequence (GGAA) deletions were performed using site-directed mutagenesis, according to the manufacturer’s instructions (Stratagene, San Diego, CA, USA).
Human Ets-1 cDNA was obtained from Origene (#RC227466) while Ets-2 cDNA expressing vector (CMV-Ets-2 in pCDNA 3.1) was available in our laboratory. Ets-1 and Ets-2 DNA binding domains were removed by mutagenesis according to the proto- col described by Follo et al.26 All mutagenesis primers are listed in the Online Supplementary Tables S2 to S4.
The single guide RNA (sgRNAF8.1, sgRNAF8.2 and the con- trol sgRNAF7.5) were designed using ZiFit web tool, as described by Pignani et al.27, scanning for the S. Pyogenes PAM sequence (NGG) both in the sense and antisense strands. dCas9- VPR was a gift from Dr George Church (Addgene plasmid # 63798).
Transient transfection and luciferase assays
For the luciferase reporter assay, ECV-304 and HEK293T cells were seeded in a 24-well plate at a density of 5x104 cells/well 24 hours (h) prior to transfection. Luciferase pF8 reporter plasmids along with Ets-1 and/or Ets-2 expressing constructs, or dCas9- VPR and gRNA, were transfected (1 mg) into cells using the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. Firefly luciferase vector pGL4.54 (TK-Firefly; #E5061, Promega) was used as an internal control. In order to maintain the amount of total DNA, the pUC19 vector was used to transfect cells. After 24 h, the cells were lysed, and luciferase activity assayed per- formed using the Dual-Luciferase Reporter Assay System (Promega). Both firefly and NanoLuc signals were measured at 562 nm using a Victor X microplate reader (PerkinElmer). Results are expressed as mean ± standard deviation (SD) of the fold change, calculated as the average of the ratio of stimulated to non-stimulated promoter.
Statistical analysis
All data were continuous and expressed as an average ± SD. Parametric analysis was used because the groups were balanced with the same number of observations. One-way analysis of variance (ANOVA) was performed to compare changes in pro- moter activity among promoter variants and to separately eval- uate the difference in Ets-response of each promoter tested. Two-way ANOVA was carried out to compare changing in FVIII activity over time and among the mice groups. P-values less than 0.05 were considered statistically significant for the overall test while Bonferroni’s adjustment was used for multiple compar- isons. The statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software).
haematologica | 2021; 106(6)
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