A Novel Platform for Immune Tolerance Induction in Hemophilia A Mice

Abstract

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.

The national blueprint for future basic and translational research to understand factor VIII immunogenicity: NHLBI State of the Science Workshop on factor VIII inhibitors

“…The latter can induce immune tolerance, although recent literature suggests that targeting LSEC, as compared to hepatocytes, may better induce tolerance, perhaps owing to their ability to promote induction of Treg. 5,6,12 Potential reasons for differences in immunogenicity of FVIII produced by varying cell types need to be better addressed.…”

“…Recombinant FVIII is made in various mammalian cell lines (none of which are endothelial cells) and current gene therapies target hepatocytes for FVIII expression. The latter can induce immune tolerance, although recent literature suggests that targeting LSEC, as compared to hepatocytes, may better induce tolerance, perhaps owing to their ability to promote induction of Treg …”

Haemophilia gene therapy: From trailblazer to gamechanger

“…[11][12][13][14][15] The induction of immune tolerance to the coagulation factors after gene therapy is dependent on several variables. [11][12][13][14][15] The induction of immune tolerance to the coagulation factors after gene therapy is dependent on several variables.…”

“…68 If FVIII or FIX is inadvertently expressed in APCs, the risk of inhibitory antibody development is increased, which prevents long-term expression and makes the gene therapy unsuccessful. 15 The immune tolerance that is induced by hepatic FVIII or FIX gene transfer by lentiviral vectors typically requires regulatory T-cell induction. 15 The immune tolerance that is induced by hepatic FVIII or FIX gene transfer by lentiviral vectors typically requires regulatory T-cell induction.…”

“…25,46 The use of hepatocyte-specific promoters can avoid expression in APCs and in this way result in long-term FIX expression. 15,24,35,71 We have shown that introducing a codon-optimized hyperactive FIX-R338L (Padua) gene with either integration-defective or integration-competent lentiviral vectors in haemophilia B mice could significantly augment FIX activity. 15,24,35,71 We have shown that introducing a codon-optimized hyperactive FIX-R338L (Padua) gene with either integration-defective or integration-competent lentiviral vectors in haemophilia B mice could significantly augment FIX activity.…”