Integration-deficient Lentiviral Vectors Expressing Codon-optimized R338L Human FIX Restore Normal Hemostasis in Hemophilia B Mice

Abstract

Integration-deficient lentiviral vectors (IDLVs) have been shown to transduce a wide spectrum of target cells and organs in vitro and in vivo and to maintain long-term transgene expression in nondividing cells. However, epigenetic silencing of episomal vector genomes reduces IDLV transgene expression levels and renders these safe vectors less efficient. In this article, we describe for the first time a complete correction of factor IX (FIX) deficiency in hemophilia B mice by IDLVs carrying a novel, highly potent human FIX cDNA. A 50-fold increase in human FIX cDNA potency was achieved by combining two mechanistically independent yet synergistic strategies: (i) optimization of the human FIX cDNA codon usage to increase human FIX protein production per vector genome and (ii) generation of a highly catalytic mutant human FIX protein in which the arginine residue at position 338 was substituted with leucine. The enhanced human FIX activity was not associated with liver damage or with the formation of human FIX-directed inhibitory antibodies and rendered IDLV-treated FIX-knockout mice resistant to a challenging tail-clipping assay. A novel S1 nuclease-based B1-quantitative polymerase chain reaction assay showed low levels of IDLV integration in mouse liver. Overall, this study demonstrates that IDLVs carrying an improved human FIX cDNA safely and efficiently cure hemophilia B in a mouse model.

Gene therapy for hemophilia

“…Alanine substitution mutation resulted in the artificial generation of F.IX-triple, which has ~10-fold greater specific activity than wild-type F.IX (211, 212). Additionally, a naturally occurring F.IX mutation (R338L), termed F.IX Padua, was discovered that has 5-10-fold higher activity (42, 213-215). Although these hyperactive F.IX variants can cause thrombosis at physiological expression levels, the fact that F.IX circulates in plasma in an inactive form makes them apparently safe at the expression levels achieved by gene therapy (~5-10% of normal).…”

Retrovirus-based vectors for transient and permanent cell modification

“…In another report, codon-optimization together with point mutation of the catalytic site (R338L) yielded a >50-fold more potent, naturally occurring hyperactive human coagulation factor IX (FIX). As a strong illustration of the potential of RET for therapeutic application, systemic administration of the improved FIX via LV episomes resulted in complete and long-term cure of hemophilia B in a mouse model [64 ].…”

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

“…27,34,35 IDLVs have garnered significant interest among researchers for precise in vivo analysis of genetic diseases, since they significantly reduce the risk of insertional mutagenesis inherent in integrating delivery platforms. For example, Thrasher et al 36 and Suwanmanee et al 37 have successfully employed IDLVs in mouse models as gene replacement therapies for degenerative retinal disease and hemophilia B, respectively. Furthermore, the efficacy of IDLVs in cancer immunotherapy and as a means of inducing protective immune responses to human pathogens has been characterized in different experimental settings.…”

“…Plates were then washed six times and incubated at 37 C for 2 hr with 100 mL polyclonal rabbit anti-p 24 antibody (catalog # SP451T), diluted 1:500 in RPMI 1640, 10% fetal bovine serum (FBS), 0.25% BSA, and 2% normal mouse serum (NMS; Equitech-Bio). Plates were washed as above and incubated at 37 C for 1 hr with goat anti-rabbit horseradish peroxidase IgG (Santa Cruz Biotechnology), diluted 1:10,000 in RPMI 1640 supplemented with 5% normal goat serum (NGS; Sigma), 2% NMS, 0.25% BSA, and 0.01% Tween 20. Plates were washed as above and incubated with TMB peroxidase substrate (KPL) at room temperature for 10 min.…”