Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene

Abstract

Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.

Protein-Engineered Coagulation Factors for Hemophilia Gene Therapy

“…It may be informative that several other mammalian FVIII orthologs (Table 1) are superior to hFVIII in this regard. Emerging data also suggest that ectopic FVIII expression induces the unfolded protein response (UPR) and markers for ER-stress 120 both in mammalian cell culture121, 122, 123 and after liver-directed gene transfer using a variety of vector systems in mice 123, 124, 125, 126. FVIII secretion may be inversely related to induction of the UPR and ER-stress because FVIII variants with improved secretion appear to have lower levels of UPR and ER-stress biomarkers compared with controls 121, 123.…”

Modulation of immune responses in lentiviral vector-mediated gene transfer

“…Later, the immuno-regulatory properties of the above-described LV platform have been successfully exploited to control the development of autoimmune type 1 diabetes (T1D) in non-obese diabetic (NOD) mice [81]. The Baculovirus GP64 envelope protein, used as LV pseudotype, has been shown to confer tropism restriction against hematopoietic-lineage cells, thus representing an additional layer of regulation to avoid LV transduction of APCs and improve targeting of expression into hepatocytes to further promote or achieve tolerance to transgene product [82], [83]. The correlation between stable transgene expression targeted to hepatocytes and induction of active Ag-specific tolerance that was highlighted by applications of this LV platform is in line with previous evidences obtained by AAV vectors for liver gene therapy, which also showed requirement for hepato-specific expression to achieve long-term transgene expression and tolerance [65], [75].…”

2021 clinical trials update: Innovations in hemophilia therapy

“…Recently, a phase I trial looking at the feasibility of hematopoietic stem cell gene therapy using platelet FVIII in participants with FVIII inhibitors funded by the National Institutes of Health (USA) opened for enrollment (NCT03818763). Other approaches using lentiviruses are under early investigation 83,84 . Lentiviral vectors provide the capacity for larger gene constructs and, unlike the AAV‐vector approach, the gene of interest integrates into the host's cellular DNA 85 .…”