Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A

Abstract

Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA.

Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

“…As a first approximation, the in vitro results presented here indicate successful editing among the rare LT-HSC population (CD34 + ,CD90 + ) 49 even with relatively low numbers of CD34 + HSPCs used for editing. For these experiments, the number of nucleofected cells ranged from 2.5x10 5 to 1.0x10 6 for each GSH site being evaluated. In comparison, approved gene therapies against ADA-SCID (Strimvelis TM , Orchard Therapeutics, PLC), MLD (Libmeldy TM , Orchard Therapeutics, PLC) or β-thalassemia (ZYNTEGLO™, betibeglogene autotemcel, Bluebird Bio, Inc.) reportedly require up to 2.0-3.0x10 7 autologous CD34 + HSPCs per kg of body weight, retroviral or lentivirally transduced, as the dose for infusion.…”

“…Thus, identification of GSHs that provide safe and reliable locations for gene addition should be a high priority for gene and cellular therapies. Clinical outcomes for lentivirus-dependent therapies addressing blood disorders (multiple myeloma 1 , β-hemoglobinopathies 2 , coagulopathies 3 , or Fanconi anemia 4 ), would benefit enormously from an available catalog of verified GSH sites that reduce the risks associated with cell engineering. Importantly, the accessibility of primary human CD34+ hematopoietic stem and progenitor cells 5,6 (HSPCs) combined with programmable endonuclease technologies (particularly CRISPR/Cas9 7 ) constitute an exceptional model to assess novel GSH candidates for autologous therapies.…”

“…Clinical outcomes of lentiviral vector-dependent gene therapies addressing blood disorders (e.g. multiple myeloma 4 , β-hemoglobinopathies 5 , coagulopathies 6 , Fanconi anemia 7 , etc.) would improve upon selection of an appropriate GSH from an available catalog of verified GSH sites that reduce the risks associated with cell engineering.…”

Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations

“…This strategy, which involves the deletion of different regions of exon 13, is similar to the one developed for the B domain (exon 14) of factor VIII [ 38 ], reducing its size and then packaging the B-domain deleted molecule into multiple viral vectors without loss of function [ 39 ].…”

Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A

“…LVs were generated using the pEGWI LV system as previously described (Gong et al 2021 ; Chang et al 1999 ). F8BDD construct was created by ligation of the human F8 ( hF8 ) cDNA into the viral vector based on optimized nucleotide sequences between the A2 and A3 domains (Doering et al 2002 ).…”