Endothelial-specific transgene expression for gene therapy approaches
Prof. Antonia Follenzi, University of Eastern Piedmont; Prof. Simone Merlin, University of Eastern Piedmont; Prof. Ester Borroni, University of Eastern Piedmont; Dr Chiara Borsotti, University of Eastern Piedmont; Dr Valentina Bruscaggin, University of Eastern Piedmont
University of Eastern Piedmont
Hemophilia A (HA) is a recessive X-linked bleeding disorder that occurs in 1:5000 male new births, it is due to the lack or reduced activity of coagulation Factor VIII (FVIII) and, according to residual FVIII activity, it is classified as severe (<1%), moderate (1-5%) and mild (5-40%). Recently, hepato-directed FVIII-gene therapy clinical studies showed promising data for FVIII production following in vivo gene delivery. Despite the promising data obtained from these clinical studies, the development of anti-FVIII antibodies inhibiting the FVIII activity (inhibitors) remains the major obstacle. In view of these considerations, there is still a huge need to develop a new gene therapy strategy able to produce FVIII efficiently and for long term avoiding the immune responses to the transgene, i.e. inhibitors formation.
The technology is based on sequences derived from Stabilin-2 (Stab-2) promoter to be used as promoter to drive the expression of a therapeutic gene specifically into endothelial cells for medical purposes. The main advantage of these Stab-2-derived sequences is the endothelial-specific expression of a gene of interest. Our preclinical data demonstrated that the use of these sequences restricts the transgene expression in endothelial cells, specifically in sinusoidal endothelial cells of liver, spleen and bone marrow. Additionally, the transgene expression, in particular FVIII, with the use of these sequences is robust and long lasting and it is able to avoid immune responses to the transgene, the main weakness in gene therapy, in several strains of preclinical mouse models.
Our strategy is to exploit the technology of these sequences first in HA. Current clinical trials for haemophilia only involve inhibitor-negative patients. Moreover, the use of non-integrating vectors in pediatric patients could result in transgene loss due to the physiological cell proliferation during natural growth. The long-term FVIII expression avoiding or reverting a pre-existing immunity to FVIII is an hot research topic that could lead to the development of a therapeutic protocol for the treatment of virtually all HA patients, or even only those excluded from current treatments, through collaboration with pharmaceutical companies active in the gene therapy sector. Additionally, these sequences can potentially be exploited for any transgene other than FVIII, thus increasing their therapeutic potential and opening up new treatment options for other genetic diseases.
Currently, the development status is based on in vitro and in vivo data. Using a lentiviral vector platform, we verified the endothelial targeting using a marker gene (GFP), while the therapeutic activity was evaluated using the B domain-deleted FVIII transgene in several strains of preclinical HA mouse model showing stable performances, thus we can estimate that we are at the stage TRL 4.
Italian patent application filed on July 27, 2017. The patent priority has been extended as international application as WO 2019/021238A1. Recently, European and United States patent applications have been filed.
We demonstrated that endothelial-specific transgene expression limits immune response to the transgene when expressed by Lentiviral vectors (PMID 28552407) and with advanced efficiency using the F8 promoter (PMID 30862611). This effect was even more effective using the Stab-2 promoter thus increasing the therapeutic potential of these sequences.