Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Abstract

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

Enhanced Biosynthesis of Coagulation Factor VIII through Diminished Engagement of the Unfolded Protein Response

“…The reverse transcription product was amplified using either hamster XBP1s-specific primers (forward, 5Ј-CTG ATG CCG CAG GTG CA-3Ј and reverse, 5Ј-TGA CAG GGT CCA ACT TGT CCA GAA-3Ј) or primers that amplified both the spliced and unspliced XBP1 isoforms (forward, 5Ј-GCT GGA ACA GCA AGT GGT GGA TTT-3Ј and reverse, 5Ј-TAC TCC ATT TCC CTT GGA CTC CGT-3Ј) as previously described (6). Dilution curves were prepared using dilutions of reverse transcription product to verify the efficiency of the two sets of primers.…”

“…Measurement of fVIII Transcript Levels-FVIII transcript levels were determined using quantitative real-time PCR as previously described (6).…”

“…A standard curve was generated using dilutions of the lenti-BiP expression plasmid. PCR were performed using BiP specific primers (forward, 5Ј-TTG AAT GGC TGG AAA GCC ACC AAG-3Ј and reverse, 5Ј-AGG GCC TGC ACT TCC ATA GAG TTT-3Ј) (IDT) as previously described (6).…”

Replacing bad (F)actors: hemophilia

“…15,16 Furthermore, we identified sequences within p-FVIII that are necessary and sufficient for the greater expression, 11 demonstrated that this differential occurs through reduced engagement of the unfolded protein response pathway, 10 and have used this information to generate humanized, human/porcine (HP) high-expression FVIII constructs that retain this biosynthetic advantage. 17,18 A similar strategy is being pursued in gene therapy applications for hemophilia B through the use of a naturally occurring human FIX variant termed FIX-Padua (R338L) that exhibits greater specific procoagulant activity than wild-type human FIX. Inclusion of this modified FIX has been shown to improve vector and transgene potency in preclinical AAV gene therapy studies.…”

“…This bioengineered FVIII transgene demonstrated equivalent transgene product expression to BDD p-FVIII in vitro and in vivo using both SIV-and HIV-based expression vectors. 11,17,18 An alternative, but also successful, preclinical HSCT gene therapy strategy incorporating nonengineered BDD h-FVIII, and more recently FIX, transgenes involves specific targeting of transgene expression to the megakaryocyte/platelet hematopoietic lineage. This approach was first demonstrated by Poncz et al using transplantation of HSCs from transgenic mice to correct the hemophilia A bleeding phenotype.…”