Preclinical Development of a Hematopoietic Stem and Progenitor Cell Bioengineered Factor VIII Lentiviral Vector Gene Therapy for Hemophilia A

Abstract

Genetically modified, autologous hematopoietic stem and progenitor cells (HSPCs) represent a new class of genetic medicine. Following this therapeutic paradigm, we are developing a product candidate, designated CD68-ET3-LV CD34, for the treatment of the severe bleeding disorder, hemophilia A. The product consists of autologous CD34 cells transduced with a human immunodeficiency virus 1-based, monocyte lineage-restricted, self-inactivating lentiviral vector (LV), termed CD68-ET3-LV, encoding a bioengineered coagulation factor VIII (fVIII) transgene, termed ET3, designed for enhanced expression. This vector was shown capable of high-titer manufacture under clinical scale and Good Manufacturing Practice. Biochemical and immunogenicity testing of recombinant ET3, as well as safety and efficacy testing of CD68-ET3-LV HSPCs, were utilized to demonstrate overall safety and efficacy in murine models. In the first model, administration of CD68-ET3-LV-transduced stem-cell antigen-1 cells to hemophilia A mice resulted in sustained plasma fVIII production and hemostatic correction without signs of toxicity. Patient-derived, autologous mobilized peripheral blood (mPB) CD34 cells are the clinical target cells for ex vivo transduction using CD68-ET3-LV, and the resulting genetically modified cells represent the investigational drug candidate. In the second model, CD68-ET3-LV gene transfer into mPB CD34 cells isolated from normal human donors was utilized to obtain in vitro and in vivo pharmacology, pharmacokinetic, and toxicology assessment. CD68-ET3-LV demonstrated reproducible and efficient gene transfer into mPB CD34 cells, with vector copy numbers in the range of 1 copy per diploid genome equivalent without affecting clonogenic potential. Differentiation of human CD34 cells into monocytes was associated with increased fVIII production, supporting the designed function of the CD68 promoter. To assess in vivo pharmacodynamics, CD68-ET3-LV CD34 cell product was administered to immunodeficient mice. Treated mice displayed sustained plasma fVIII levels and no signs of product related toxicity. Collectively, the findings of the current study support the preclinical safety and efficacy of CD68-ET3-LV CD34.

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 .…”

The Immune Response to the fVIII Gene Therapy in Preclinical Models

“…Several target cell types including adipocytes, mesenchymal stem/progenitor cells, and HSPCs are being pursued for ex vivo gene therapy (37)(38)(39)(40). However, infusion of transduced mesenchymal stem/progenitors and adipocytes has been less successful in preclinical studies than HSPC LV-fVIII gene therapy, and thus HSPC appears to be the leading candidate (24).…”

“…In terms of HSPC LV-fVIII design and preclinical testing, we published a comprehensive set of preclinical studies supporting the clinical testing of an HSPC gene therapy for hemophilia A. The product candidate, referred to as CD68-ET3-LV CD34 + , consists of autologous CD34 + cells transduced with a HIV-1based, monocyte lineage-restricted, self-inactivating LV encoding the high-expression ET3 transgene (Figure 4) (19,24). An Investigational New Drug (IND) application for this product candidate was recently cleared for clinical testing by the United States of America Food and Drug Administration.…”

“…For example, our group discovered that recombinant porcine fVIII is expressed at significantly higher levels than recombinant human fVIII due to apparent avoidance of UPR and more efficient secretion from the cell (10,14,15). Furthermore, this high expression property translates to greater potency in gene therapy applications (16)(17)(18)(19)(20)(21)(22)(23)(24). One can speculate that reduced engagement of UPR also provides a safety benefit to gene therapy applications, wherein liver toxicities (e.g., elevated liver enzyme levels) of unknown origin are being observed in clinical AAV-fVIII gene therapy trials incorporating BDD human fVIII transgenes, extremely high vector doses, and potent, liver-directed promoters.…”

Transcriptional Targeting and MicroRNA Regulation of Lentiviral Vectors

“…Hematopoietic stem cell (HSC) transduction is a typical example of advances made in gene therapy for clinical applications. In a recent study, Doering et al 66 . developed an LV platform containing a bioengineered FVIII with enhanced expression under the control of the CD68 (GP110, macrosialin, or LAMP4) promoter, restricting FVIII expression in myeloid cells for the transduction of autologous HSC CD34 + that upon differentiation in monocytes were able to increase FVIII production and secretion.…”