Curing Hemophilia: Repeated Treatments versus a One-Off Fix
Authors: Ning Li, Radoslaw Kaczmarek
Journal: Molecular Therapy (2020)

Hemophilia A or factor (F)VIII deficiency is an X-linked bleeding disorder that occurs worldwide in approximately 1 of 5,000 male births and is characterized by recurrent debilitating or fatal bleeding episodes. Traditional treatment replaces the missing FVIII protein by administration of concentrate to prevent or stop bleeds. The hemostatic correction is short-lived due to the short (∼12 h) half-life of FVIII, which necessitates frequent intravenous infusions. However, a liver-directed gene therapy using an adeno-associated viral (AAV) vector is now in a phase III clinical trial and has normalized FVIII levels in some individuals. We recently reviewed the ongoing molecular revolution in therapy for hemophilia, which is well on its way to provide patients with an unprecedented range of treatment options that include multiple standard and extended half-life (EHL) factor products, antibody, RNA and gene therapies.1Butterfield J.S.S. Hege K.M. Herzog R.W. Kaczmarek R. A Molecular Revolution in the Treatment of Hemophilia.Mol. Ther. 2020; 28: 997-1015Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar The preclinical pipeline has now been even further expanded, with new reports on gene editing, mRNA delivery, and cell therapy.2Zhang J.P. Cheng X.X. Zhao M. Li G.H. Xu J. Zhang F. Yin M.D. Meng F.Y. Dai X.Y. Fu Y.W. et al.Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse.Genome Biol. 2019; 20: 276Crossref PubMed Scopus (26) Google Scholar, 3Rose M. Gao K. Cortez-Toledo E. Agu E. Hyllen A.A. Conroy K. Pan G. Nolta J.A. Wang A. Zhou P. Endothelial cells derived from patients' induced pluripotent stem cells for sustained factor VIII delivery and the treatment of hemophilia A.Stem Cells Transl Med. 2020; (Published online March 12, 2020)https://doi.org/10.1002/sctm.19-0261Crossref PubMed Scopus (12) Google Scholar, 4Chen C.-Y. Tran D.M. Cavedon A. Cai X. Rajendra R. Lyle M.J. Martini P.G.V. Miao C.H. Treatment of hemophilia A using factor VIII messenger RNA lipid nanoparticles.Mol. Ther. Nucleic Acids. 2020; (Published online April 6, 2020)https://doi.org/10.1016/j.omtn.2020.03.015Abstract Full Text Full Text PDF Scopus (25) Google Scholar Interestingly, lipid nanoparticle (LNP)-based delivery of FVIII-encoding mRNA and gene editing represent molecular approaches that occupy opposite ends of the durability spectrum, the former requiring weekly administrations, with the latter aiming to be a one-off treatment. The weekly intravenous injections of FVIII mRNA-LNPs4Chen C.-Y. Tran D.M. Cavedon A. Cai X. Rajendra R. Lyle M.J. Martini P.G.V. Miao C.H. Treatment of hemophilia A using factor VIII messenger RNA lipid nanoparticles.Mol. Ther. Nucleic Acids. 2020; (Published online April 6, 2020)https://doi.org/10.1016/j.omtn.2020.03.015Abstract Full Text Full Text PDF Scopus (25) Google Scholar would be a marked improvement over the therapy with FVIII concentrates, which must be infused two to three times weekly. The FVIII encoded by the LNP-delivered mRNA is expressed endogenously in hepatocytes and includes an amino acid substitution (F309S) that speeds up secretion and an increase of plasma FVIII levels. The FVIII mRNA-LNPs were formulated in a cell-free in vitro system, which would place that approach a notch above the latest generations of recombinant FVIII concentrates with regards to safety from blood-borne infections. The FVIII-encoding mRNA does not translocate to the nucleus, thus posing no risk of insertional mutagenesis. Also, the approach is not subject to vector packaging limits, which has limited development of AAV vectors for hemophilia A, due to the large size of the F8 cDNA. From a broader perspective, the study illustrates the potential of mRNA delivery for treatment of genetic disease, but it also raises questions about the long-term effects of lifelong repeated infusions of lipid particles. On the downside, the short half-lives of mRNA and FVIII cause a rapid decline of the FVIII level, resulting in seesaw pharmacokinetics, which is also characteristic for prophylaxis with FVIII concentrates. The weekly intervals between infusions were achieved due to the persistence of the FVIII-encoding mRNA for the first 24 h, during which plasma FVIII levels were building up to reach a somewhat supra-physiological peak (∼200%), before dwindling to a trough of ∼1%. Although the half-life of the F309S-FVIII variant was estimated at ∼22 h, it would likely be shorter in humans because of the 15–19 h half-life ceiling imposed by von Willebrand factor (VWF), with which FVIII circulates in plasma. In fact, the FVIII half-life ceiling is the reason why none of the available EHL FVIII products shows more than an ∼1.6-fold extension. A novel fusion protein consisting of FVIII, the D'D3 domain of VWF, immunoglobulin G1 Fc fragment, and XTEN polypeptides is now in phase III trials. The addition of the D'D3 VWF domain uncouples FVIII from endogenous VWF, which extended the FVIII half-life to ∼37.6 h in early trials. A standard prophylaxis dose (25 IU/kg) of that molecule resulted in FVIII levels ∼5% a week after infusion, as reported at the 2019 world congress of the International Society on Thrombosis and Haemostasis.5Lissitchkov T. Rice K. Katragadda S. Willemze A. Benson C. Knobe K. Phase 1 Repeat Dosing with BIVV001: The First Investigational Factor VIII Product to Break through the Von Willebrand Factor-Imposed Half-Life Ceiling.Blood. 2019; 134: 625Crossref Google Scholar This feature would be welcome because the quest for longer-acting therapies is not only driven by the desire to reduce injection frequency, but also to increase trough levels and ultimately achieve steady-state protection from bleeding. The recently approved bispecific antibody emicizumab, which mimics FVIII activity, is infused subcutaneously at weekly, biweekly, or monthly intervals and provides a steady-state hemostatic activity equivalent to ∼15% FVIII, but it differs from FVIII in several important ways,1Butterfield J.S.S. Hege K.M. Herzog R.W. Kaczmarek R. A Molecular Revolution in the Treatment of Hemophilia.Mol. Ther. 2020; 28: 997-1015Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar so the pursuit of longer-lasting or permanent replacement of the missing protein will continue. The new reports on CRISPR/Cas9-mediated gene editing and cell therapy with endothelial cells (ECs) derived from induced pluripotent stem cells (iPSCs) may be taking another step toward the functional cure.2Zhang J.P. Cheng X.X. Zhao M. Li G.H. Xu J. Zhang F. Yin M.D. Meng F.Y. Dai X.Y. Fu Y.W. et al.Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse.Genome Biol. 2019; 20: 276Crossref PubMed Scopus (26) Google Scholar,3Rose M. Gao K. Cortez-Toledo E. Agu E. Hyllen A.A. Conroy K. Pan G. Nolta J.A. Wang A. Zhou P. Endothelial cells derived from patients' induced pluripotent stem cells for sustained factor VIII delivery and the treatment of hemophilia A.Stem Cells Transl Med. 2020; (Published online March 12, 2020)https://doi.org/10.1002/sctm.19-0261Crossref PubMed Scopus (12) Google Scholar Zhang et al.2Zhang J.P. Cheng X.X. Zhao M. Li G.H. Xu J. Zhang F. Yin M.D. Meng F.Y. Dai X.Y. Fu Y.W. et al.Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse.Genome Biol. 2019; 20: 276Crossref PubMed Scopus (26) Google Scholar knocked a F8 transgene encoding B domain-deleted (BDD)FVIII into the albumin locus in a mouse model of hemophilia A. This resulted in stable FVIII levels in the range of 20%–400% for 2 years. The approach delivered constructs encoding Streptococcus pyogenes-derived Cas9, BDDFVIII, and single guide (sg)RNA to hepatocytes using three AAV8 vectors. The transgene insertion proceeded through non-homologous end joining (NHEJ) within intron 11 or 13 of the Alb gene. The albumin locus has been considered a safe harbor for transgene integration and, indeed, the authors did not find any adverse events other than minor decreases in albumin expression (∼5%). The approach promises to overcome the gene dilution problem, which hinders using the AAV-based gene addition in young children, who would benefit the most from early FVIII level normalization by avoiding the onset of joint disease. However, AAV-mediated gene editing spurs safety questions that will need answers before moving to the clinic. The risk of off-target CRISPR/Cas9 effects, even if minimal, will remain a concern because AAV efficiently integrates into Cas9-induced double-strand breaks. Also, persistent expression of Cas9 may lead to genotoxicity or provoke an immune response to gene-edited cells, which may be addressed using self-deleting systems. In an alternative strategy, Rose et al.3Rose M. Gao K. Cortez-Toledo E. Agu E. Hyllen A.A. Conroy K. Pan G. Nolta J.A. Wang A. Zhou P. Endothelial cells derived from patients' induced pluripotent stem cells for sustained factor VIII delivery and the treatment of hemophilia A.Stem Cells Transl Med. 2020; (Published online March 12, 2020)https://doi.org/10.1002/sctm.19-0261Crossref PubMed Scopus (12) Google Scholar corrected hemophilia A in mice by transplanting hemophilic ECs that were lentivirally modified to produce BDDFVIII. The cells originated from uroepithelial cells, which were obtained from hemophilia A patients, reprogrammed into iPSCs and differentiated into ECs.3Rose M. Gao K. Cortez-Toledo E. Agu E. Hyllen A.A. Conroy K. Pan G. Nolta J.A. Wang A. Zhou P. Endothelial cells derived from patients' induced pluripotent stem cells for sustained factor VIII delivery and the treatment of hemophilia A.Stem Cells Transl Med. 2020; (Published online March 12, 2020)https://doi.org/10.1002/sctm.19-0261Crossref PubMed Scopus (12) Google Scholar Upon subcutaneous engraftment, FVIII levels rose over 2 weeks to ∼10%–370%. Of the three approaches discussed, cell therapy comes closest to reproducing the cellular context of FVIII expression, which naturally takes place in liver sinusoidal ECs (LSECs). In the other two strategies, FVIII was expressed ectopically in hepatocytes. Nevertheless, the iPSC-derived ECs differed phenotypically from LSECs, which leaves room for improvement with this method. The engrafted cells did not proliferate extensively, which suggests low risk of tumorigenesis, although longer follow-up will be needed to rule it out. Conversely, low proliferation might necessitate repeat transplantations to prevent cell death-related loss of FVIII expression. None of the three studies shed much light on the immunogenicity of each approach. Immune response to FVIII replacement therapy leads to the development of neutralizing anti-FVIII antibodies and loss of efficacy in approximately 30% of hemophilia patients, while immune responses to AAV gene therapy vectors have long stymied progress of the field and continue to vex clinical trials. The FVIII mRNA-LNP and iPSC-EC studies used immune-deficient mice, while success of the gene-editing attempts relied on temporary but potent immunosuppression. Future studies will need to explore other models and regimens to ensure feasibility. Competition between a myriad of diverse approaches is fierce in current drug development for hemophilia. Only the future will tell which treatments will prevail as the most effective, safe, and beneficial for patients.