Hemophilia A Gene Therapy Research Articles

Gene Therapy in Hemophilia A: Achievements, Challenges, and Perspectives.

Strides in advancements of care of persons with hemophilia include development of long-acting factor replacement therapies, novel substitution and hemostatic rebalancing agents, and most recently approved gene therapy. Several decades of preclinical and clinical trials have led to development of adeno-associated viral (AAV) vector-mediated gene transfer for endogenous production of factor VIII (FVIII) in hemophilia A (HA). Only one gene therapy product for HA (valoctocogene roxaparvovec) has been approved by regulatory authorities. Results of valoctocogene roxaparvovec trial show significant improvement in bleeding rates and use of factor replacement therapy; however, sustainability and duration of response show variability with overall decline in FVIII expression over time. Further challenges include untoward adverse effects involving liver toxicity requiring immunosuppression and development of neutralizing antibodies to AAV vector rendering future doses ineffective. Real-life applicability of gene therapy for HA will require appropriate patient screening, infrastructure setup, long-term monitoring including data collection of patient-reported outcomes and innovative payment schemes. This review article highlights the success and development of HA gene therapy trials, challenges including adverse outcomes and variability of response, and perspectives on approach to gene therapy including shared decision-making and need for future strategies to overcome the several unmet needs.

Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A.

Hemophilia A (HA) is a common X-linked recessive hereditary bleeding disorder. Coagulation factor VIII (FVIII) is insufficient in patients with HA due to the mutations in the F8 gene. The restoration of plasma levels of FVIII via both recombinant B-domain-deleted FVIII (BDD-FVIII) and B-domain-deleted F8 (BDDF8) transgenes was proven to be helpful. FVIII-Padua is a 23.4 kb tandem repeat mutation in the F8 associated with a high F8 gene expression and thrombogenesis. Here we screened a core enhancer element in FVIII-Padua for improving the F8 expression. In detail, we identified a 400 bp efficient enhancer element, C400, in FVIII-Padua for the first time. The core enhancer C400 extensively improved the transcription of BDDF8 driven by human elongation factor-1 alpha in HepG2, HeLa, HEK-293T and induced pluripotent stem cells (iPSCs) with different genetic backgrounds, as well as iPSCs-derived endothelial progenitor cells (iEPCs) and iPSCs-derived mesenchymal stem cells (iMSCs). The expression of FVIII protein was increased by C400, especially in iEPCs. Our research provides a novel molecular target to enhance expression of FVIII protein, which has scientific value and application prospects in both viral and nonviral HA gene therapy strategies.

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

Evolution Directed Gene Engineering (EDGE) Identifies Single Amino Acid Substitutions in FVIII with Six-Fold Increased in Vitro and In Vivo Activity

Background: Current AAV gene therapy for hemophilia A (HA) is limited by low sustained factor VIII (FVIII) levels. In contrast, AAV gene therapy for hemophilia B benefits from the widespread adoption of the single amino acid (AA) substituted high-specific-activity FIX variant R338L. We have previously shown that wild-type (WT) FIX has not evolved for maximal specific activity, but rather has evolutionary conserved AAs that limit procoagulant activity (Samelson-Jones et al. Blood Advances 2021). FVIII may have evolved with similar considerations. Based on this work, we developed a novel screening technique, termed Evolution Directed Gene Engineering (EDGE), to identify new hyperactive single AA substituted FVIII variant transgenes to address the current limitations of HA gene therapy. Aims: To find and characterize single AA substituted FVIII variants that can enhance gene transfer. Methods: EDGE evaluates AA positions fulfilling 2 criteria: 1) highly evolutionary conserved and 2) not associated with the disease phenotype. Here, EDGE compared 33 mammalian FVIII orthologues and then eliminated positions associated with known HA-causing missense mutations. FVIII variants were transiently expressed in Huh7 cells and cultured media was assayed for FVIII activity and antigen. Recombinant FVIII protein of lead candidates were purified to homogeneity and assayed for FVIII activity with one- and two-stage aPTT assays, thrombin generation assay (TGA), and enzyme kinetics studies. The in vivo efficacy of the variants was tested by tail clip assays of HA mice after retro-orbital delivery of the protein or liver-directed AAV gene therapy. Results: Out of 52 AA positions identified by EDGE, we found 12 AA positions in FVIII where single AA substitutions increased specific activity ≥2-fold (Figure 1A). The largest increase in specific activity was 6-fold, which occurred for multiple AA substitutions at position 659. Purified recombinant protein of FVIII-K659M and -K659V both demonstrated 6-fold increased activity in one- and two-stage aPTT assays, but comparable thrombin activation and A2-stability to FVIII-WT. TGA show the FVIII-K659M/V variants have greater than 6-fold improvements in endogenous thrombin potential and peak thrombin generation. Compared to FVIII-WT, FVIII-K659M/V proteins have an increased k cat and decreased appK D which suggests that the mechanism of increased specific activity is due to enhanced interactions with FIXa. This is similar to the mechanism of hyperactivity of FIX-R338L. FVIII-K659M/V also demonstrated enhanced procoagulant activity in vivo. In tail clip injuries of HA mice (n≥4) treated with purified protein, the FVIII-K659M/V proteins restored normal hemostasis at 5-fold lower concentrations than FVIII-WT. When mice were treated with equal doses of 2.5 µg/kg of protein, FVIII-K659M/V restored normal hemostasis while FVIII-WT had no significant change in blood loss (Figure 1B). As expected, 12.5 µg/kg of FVIII-WT protein was required to restore blood loss to WT mouse levels. FVIII-K659 substituted variants also enhanced AAV based gene therapy. HA mice (n=6) treated with AAV8 vectors expressing FVIII-K659C had 6-fold higher plasma FVIII activity than mice (n=6) treated at the same dose with AAV8 vectors expressing FVIII-WT. HA mice that received 4 x 10 12 vg/kg of AAV8 FVIII-K659C had less bleeding than HA mice that received the same dose of AAV8 FVIII-WT, but similar blood loss as WT mice after tail clip injury. Conclusions: The success of EDGE in identifying multiple new high specific activity FVIII variants supports the hypothesis that FVIII-WT is not optimized for specific activity and thus is amenable to bioengineering approaches. The ≥5-fold enhanced in vitro and in vivo hemostatic activity of the FVIII-K659 substituted variants and initial murine gene therapy studies support their translational potential to address current limitations of HA gene therapy. These results also implicate K659 in modulating the FVIIIa/FIXa interaction. EDGE may also be applicable to other gene therapy targets as well.

Second Generation Adeno-Associated Viral Vector Using a Novel Factor VIII Variant with Improved Secretion Achieves Higher Factor VIII Expression in Non-Human Primates

Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in functional factor VIII (FVIII). Adeno-associated virus (AAV)-mediated gene therapy for HA is actively in clinical development, primarily utilizing codon-optimized (CO) wild type (WT) B-domain deleted human FVIII (hFVIII) transgenes. Limitations of these approaches highlight the need to develop novel second generation AAV approaches that improve transgene expression while reducing the vector dose with the fundamental goal of achieving durable levels of FVIII in the normal range while minimizing the potential risk of anti-AAV immune responses and genotoxicity. One of the main challenges of HA gene therapy development is that hFVIII is not expressed efficiently in vitro or in vivo compared to other proteins of similar size. Notably, the FVIII proteins of other species, such as canine and porcine, have superior expression. Previously, we reported that canine FVIII is expressed at higher levels than hFVIII which prompted us to conduct a comparative study between these two proteins (Sabatino 2009). Those studies led us to engineer and characterize a series of FVIII variants around the furin cleavage recognition site (1645-48)(Nguyen 2017) and a secondary cleavage site at 1657-58 in the acidic region 3 (a3). After investigating 15 variants, we identified our best performing variant, hFVIII-Δ3-SP/DE, carrying a deletion of three residues at the furin site (1645-47) (Δ3) (Nguyen 2017) and two amino acid substitutions S1657P and D1658E at the a3 cleavage site. With these minimal modifications, hFVIII-Δ3-SP/DE is secreted significantly more efficiently than WT hFVIII with and without codon-optimization and in conjunction with other improvements in the expression cassette. In the setting of AAV gene therapy, we used AAV8 to deliver WT hFVIII-CO or variant hFVIII-Δ3-SP/DE-CO driven by the modified transthyretin (TTRm) promoter and compared levels of FVIII expression in vivo. Immuno-deficient HA/CD4 KO mice administered AAV8-TTRm-hFVIII-Δ3-SP/DE-CO expressed FVIII levels that were 2-3-fold higher than animals delivered WT FVIII-CO. To investigate this variant in a large animal model, we previously injected AAV8-hFVIII-CO and AAV8-hFVIII-Δ3-SP/DE-CO (2x1012 vg/kg) in an HA dog model tolerized to hFVIII. We observed that the levels of hFVIII expression using the variant were 2 to 4-fold higher than WT hFVIII. Given the success of our variant in HA mice and dogs, we investigated hFVIII-Δ3-SP/DE in non-human primates (NHPs). Animals were negative for neutralizing antibodies to AAV8 prior to vector administration. We delivered AAV8-TTRm-hFVIII-CO or AAV8-TTRm-hFVIII-Δ3-SP/DE-CO to male African green monkeys via intravenous infusion at three vector doses (2x1012, 5x1012, 1x1013 vg/kg)(n=3 per dose per AAV construct). hFVIII antigen levels were measured by hFVIII specific ELISA and are expressed as a percentage of normal levels. Two weeks after AAV administration, NHPs treated with low dose (2x1012 vg/kg) of AAV8-hFVIII-CO expressed a median of 7.4% (range 5.2-8.8%) and AAV8-hFVIII-Δ3-SP/DE-CO expressed 19.2% (range 11.4-72.0%). The mid-dose cohort (5x1012 vg/kg) expressed 17.7% (range 11.8-128.2%) after AAV8-hFVIII-CO and 38.2% (range 12.1-97.0%) after AAV8-hFVIII-Δ3-SP/DE-CO delivery. At 1x1013 vg/kg the AAV8-hFVIII-CO treated NHPs expressed 17.9% (range 12.6-54.3%) while the AAV8-hFVIII-Δ3-SP/DE-CO expressed 114.2% (range 20.2-195.6%). As noted in clinical studies, there is some variability in hFVIII expression in our cohorts. Nevertheless, our data suggests that the delivery of the WT hFVIII transgene did not show a dose response. In contrast, there is a dose-dependent increase in hFVIII-Δ3-SP/DE expression, implying improved secretion correlating with higher levels of FVIII expression. As anticipated, anti-hFVIII antibodies developed to the non-species specific transgene in 16 of 18 animals after 3 weeks post vector administration, leading to a decline in hFVIII expression in most animals. In summary, consistent with our observations in HA mice and dogs, AAV delivery of the Δ3-SP/DE variant in NHPs results in 2 to 5-fold higher dose-dependent levels of FVIII expression than the WT hFVIII. This novel hFVIII variant provides the opportunity to overcome the challenge of inefficient FVIII secretion to improve the efficacy of AAV gene therapy for hemophilia A.

Long-Term Real-World Safety and Efficacy of Liver-Directed AAV Gene Therapy for Severe Hemophilia with and without Inbibitors in Privately Owned Dogs

Background Hemophilia A (HA) and hemophilia B (HB) are inherited bleeding disorders resulting from deficiency of coagulation factors VIII (FVIII) or factor IX (FIX) activity, respectively. HA and HB both occur naturally in dogs. The bleeding phenotype of severe canine HA and HB is analogous to the human patient experience with frequent spontaneous and trauma-induced bleeding. Previous studies of dogs from HA and HB colonies at research institutes have been highly informative about the safety and efficacy of adeno-associated virus (AAV) liver-directed gene therapy for HA and HB. However, these animals are housed in highly controlled environments; in contrast, pet dogs with HA or HB are exposed to real-world challenges that likely affect their bleeding phenotype and the efficacy of novel therapeutics. In this regard, privately owned dogs may better recapitulate the human patient experience. Aims To determine the real-world efficacy of liver-directed AAV gene therapy for HA/HB in privately owned canines with and without inhibitors. Methods Eleven dogs with severe HA and one dog with severe HB were treated with liver-directed AAV gene therapy with canine (c) FVIII or cFIX, as detailed in Table 1. Pre-existing neutralizing antibodies to AAV8 were detected using a luciferase-based transduction inhibition assay. Pre- and post-gene therapy annualized bleeding rates were calculated by cumulative bleeding events divided by the age at gene therapy (pre) or duration of follow-up (post). FVIII activity was determined using a chromogenic assay and FIX activity using a one-stage assay. Results All dogs had AAV8 neutralizing antibody titers ≤ 1:5 at enrollment. The HA dogs (10 males and 1 female) were treated with either dual chain vectors of cFVIII-SQ (n = 2, total dose of 5 x 1013 vg/kg) or single-chain vectors encapsidating high-expression cFVIII variants: cFVIII-ΔF (n = 2, 6x1012 vg/kg) or cFVIII-ΔF/V3 (n = 7, 5.4-9 x 1012 vg/kg). F8 genetic testing revealed intron 22 inversions (n=7), small deletion (n=2), missense (n=1) and complex mutation (n=1) all compatible with severe HA. Of note, one HA dog (PC3) had a pre-existing FVIII inhibitor which was eradicated following gene therapy. In all dogs with available data (n=10), FVIII activity is > 1% with 5 in the moderate hemophilia range and 4 in the mild range with a median FVIII activity of 3.2% (range 1.1-13.8%) after a median duration of 4.1 years (range 2.6-8.9 years). In HA dogs, the ABR post-gene therapy declined by an average of 93%. The sole HB dog carried two missense F9 mutations and was treated with wildtype cFIX at 3 x 1012 vg/kg and has been followed for 4.5 years with FIX activity of 5.7% and has had no bleeding episodes since gene therapy. Three animals have died during follow up. The previously inhibitor positive dog (PC3) died approximately 2 years after gene therapy from an unrelated cause. An HA dog (PC1) treated with dual chain vector with expression in the ~2% normal range died from a bleeding episode at 8.8 years after gene therapy. Notably, many of his bleeds were associated with real-world activities including having his legs being pulled by a toddler. A third HA dog (PC9) was euthanized 3.5 years post-gene therapy after being diagnosed with widely disseminated cancer; the details of the vector integration analysis of his tumor will be reported elsewhere. Conclusion AAV gene therapy was successful at substantially ameliorating the bleeding phenotype in both male and females dogs with > 1 year of follow-up. However, some dogs continued to have bleeding, especially animals with <5% normal cFVIII activity. One animal developed cancer after gene therapy; there were no other safety concerns. As we have described previously, liver-directed AAV gene therapy also induced immune tolerance in an HA dog with a pre-existing inhibitors. We show that AAV liver-directed gene therapy is safe and efficacious in a real-world experience of privately owned dogs with severe HA and HB. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibiting Activated Protein C Cleavage of Factor VIII for Hemophilia a Gene Therapy

Background Despite progress in adeno-associated virus (AAV) mediated factor VIII (FVIII) gene transfer for hemophilia A (HA), durable and sustained FVIII expression sufficient to ameliorate bleeding in all participants is an unrealized goal. Following the success of using the gain of function FIX-Padua variant in hemophilia B gene therapy, we hypothesized that use of an enhanced hemostatic function FVIII would similarly improve HA gene therapy by: 1) permitting use of lower vector doses, thereby overcoming dose-dependent safety and efficacy limitations and 2) achieving efficacy with lower FVIII antigen. We previously published a FVIII variant resistant to activated protein C (APC) cleavage (FVIII-R336Q/R562Q or FVIII-QQ) with normal specific activity and 4-5 fold improved hemostatic function in recombinant protein experiments (Wilhelm et al. Blood 2021). Aims We evaluated the possible clinical translation of FVIII-QQ for HA AAV-mediated gene therapy by determining the hemostatic benefit and potential prothrombotic and immunological risks in mice. Additionally, FVIII-QQ steady-state expression was 2.5 to 5-fold higher than FVIII-WT. We hypothesized this was due to disrupted cleavage by APC or other serine proteases at R336 and/or R562. Results Survival studies in HA/CD4KO and WT/CD4KO mice treated with AAV vectors to express (h)uman FVIII-WT and hFVIII-QQ in the range of mild HA, normal, and supratherapeutic FVIII levels thus far demonstrate no significant differences in survival at 7-11 month follow up or D-dimer levels after 8 months. Additionally, FVIII-QQ mice were generated using CRISPR/Cas9 to introduce R562Q (WT mice are Q336) for endogenous (m)ouse FVIII-QQ expression. Endogenous mFVIII-QQ mice were viable, fertile, normal weight, and D-dimers did not significantly differ from WT mice, while FVIII activity was ~2-fold higher than WT mice. HA immune competent mice treated with either 6 weekly doses of hFVIII-WT or hFVIII-QQ protein (0.2 μg) or AAV vector to express hFVIII-QQ or hFVIII-WT had no significant differences in inhibitor titers. To evaluate hemostasis, HA/CD4KO mice were treated with AAV vector only differing by 2 amino acids (R336, R362) at 3 different doses (n=6-12 mice/cohort) to express hFVIII-QQ or hFVIII-WT and underwent tail clip challenge. Estimated EC50 and EC80 of vector doses required to normalize blood loss to WT mice were 5 and 10-fold lower, respectively, for hFVIII-QQ versus hFVIII-WT supporting a hemostatic benefit to using the hFVIII-QQ transgene. Interestingly, across 3 different vector constructs differing only by F8 codon optimization, we observed hFVIII-QQ antigen values 2.5 to 5-fold higher than in mice expressing hFVIII-WT. Analysis of liver tissue from HA/CD4KO mice treated with the same vector construct and dose to express hFVIII-WT or hFVIII-QQ demonstrated approximately the same mRNA values, but plasma hFVIII-QQ antigen was 4-fold higher than hFVIII-WT and intracellular hFVIII-QQ antigen was the same or lower than hFVIII-WT antigen. Consistent with our in vivo AAV experiments, preliminary studies of intracellular versus secreted hFVIII antigen in BHK stable cell lines expressing hFVIII-WT or hFVIII-QQ demonstrated similar mRNA values with higher relative secreted hFVIII-QQ protein. To further probe the mechanism of higher hFVIII-QQ steady-state expression, we evaluated a panel of serine proteases (APC, FIXa, FXa, plasmin, PACE/furin and thrombin) for cleavage of FVIII-QQ relative to FVIII-WT protein. As expected, hFVIII-QQ was resistant to APC cleavage. Surprisingly, all other serine proteases tested demonstrated cleavage of hFVIII-WT, but not hFVIII-QQ, at R336 and/or R562 suggesting protease attack at these cleavage sites may occur more than previously appreciated, though the physiologic significance remains to be determined. Conclusion Our studies outline the use of hFVIII-QQ transgene expression thus far does not demonstrate safety concerns and confers hemostatic benefit over hFVIII-WT via: 1) improved hemostatic function and 2) higher steady-state expression. Enhanced steady state hFVIII-QQ expression relative to hFVIII-WT was observed across multiple model systems. Preliminary data demonstrate multiple serine proteases can cleave at the described FVIII APC cleavage sites (R336, R562) suggesting hFVIII-QQ may generally be more proteolytically stable thereby permitting improved steady-state expression.

Intravenous Lentiviral Gene Therapy for Hemophilia a: Endothelial-Specific Expression and Increased Glycosylation for Improved FVIII Function

Background: Hemophilia A (HA) is an X-linked monogenic coagulation disorder in the intrinsic coagulation cascade resulting from deficiency of the factor VIII (FVIII, F8) gene. The current treatment of HA is based on protein replacement therapy (PRT) with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment option for HA. Methods: An advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy has been established based on a novel B domain deleted (BDD) F8 gene, which restored all N-glycosylation sites (F8-299) in the B domain with increased coagulation function (DOI:10.1016/j.jbc.2021.101397). LV-F8-299 containing a universal (EF1a) or endothelial-specific (VEC) promoter was investigated in vitro using a human endothelial cell line, EA-hy926, and biologically and immunologically characterized in vivo in F8 knockout (KO) mice and in a non-human primate. Results: We found that LV-VEC-F8-299 (VEC-299) exhibited higher virus packaging (100 folds) and transduction efficiencies than EF1a-299. The F8 protein expression was verified in EA-hy926 by RT-PCR, Western blot and ELISA. We detected F8 activities close to 4 folds above the normal plasma level from the EF1a-299 transduced cells, whereas the VEC-299 expressed only 0.1 fold of the normal plasma F8 level. In vivo studies in F8 KO mice via iv injection of LVs after non-myeloablative radiation conditioning illustrated variable phenotypic corrections as well as anti-F8 immune responses from the two LVs. The iv gene therapy of EF1a-299 and VEC-299 LVs illustrated therapeutic F8 activities over time, in the range of 90% and 40%, respectively, in 60 days. Importantly, VEC-299 steadily reached a very high level (180%) of F8 activities, whereas EF-299 dropped to 3% after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titer showed that the VEC-299 treated mice exhibited the lowest F8 inhibitory immune response over time. We next assessed the distribution of LV F8 gene in a macaque monkey (Macaca fascicularis). The monkey was chemo-conditioned with methylprednisolone (10 mg/kg) and prednisone (2 mg/kg) and iv injected with 1x109 TU/kg LV-VEC-299. The vector copy number (VCN) in body fluids was closely monitored by qPCR. From day 1 to week 14 after injection, the VCN in the blood remained stable (~1%), while in feces and saliva was high on day 1 (~3%), and in urine reached peak (~5%) on day 7, but all of which decreased with time. The VCN in body fluids (blood, urine, faces, saliva and tear) became nearly undetectable after 14 weeks. A second LV injection of 1x109TU/kg LV-VEC-299 was performed using a modified non-myeloablative conditioning with busulfan (2 mg/kg), cyclophosphamide (40 mg/kg) and dexamethasone (3 mg/kg). The VCN in the blood reached 20% from day 1 to day 7, then reduced to 5% and remained stable to week 6 (Figure B). The blood biochemical analyses including Alanine Transaminase (ALT), Aspartate Transaminase (AST), Total Protein (TP) and Albumin (ALB) remained in the normal range before and after treatment. Conclusion: These results demonstrated that the LV-VEC-299 exhibited delivery consistency, high coagulation function and low immunogenicity. The in vivo study supported that iv LV-VEC-299 gene therapy could be a safe, convenient and effective HA gene therapy strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Adeno-Associated Virus Gene Therapy for Hemophilia.

In vivo gene therapy is rapidly emerging as a new therapeutic paradigm for monogenic disorders. For almost three decades, hemophilia A (HA) and hemophilia B (HB) have served as model disorders for the development of gene therapy. This effort is soon to bear fruit with completed pivotal adeno-associated viral (AAV) vector gene addition trials reporting encouraging results and regulatory approval widely anticipated in the near future for the current generation of HA and HB AAV vectors. Here we review the clinical development of AAV gene therapy for HA and HB and examine outstanding questions that have recently emerged from AAV clinical trials for hemophilia and other monogenic disorders.

Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FVIII composed of the human heavy chain and the rat light chain.

Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC + rLC protein had a higher activity than the hHC + hLC protein at comparable expression levels. The specific activity of hHC + rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC + rLC treatment exhibited a visibly higher activity than those treated with hHC + hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC + rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy.

Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A.

Hemophilia A (HA) is caused by mutations in the coagulation factor VIII (FVIII) gene (F8). Gene therapy is a hopeful cure for HA; however, FVIII inhibitors formation hinders its clinical application. Given that platelets promote coagulation via locally releasing α-granule, FVIII ectopically expressed in platelets has been attempted, with promising results for HA treatment. The B-domain-deleted F8 (BDDF8), driven by a truncated ITGA2B promoter, was targeted at the ribosomal DNA (rDNA) locus of HA patient-specific induced pluripotent stem cells (HA-iPSCs). The F8-modified, human induced pluripotent stem cells (2bF8-iPSCs) were differentiated into induced hematopoietic progenitor cells (iHPCs), induced megakaryocytes (iMKs), and mesenchymal stem cells (iMSCs), and the FVIII expression was detected. The ITGA2B promoter-driven BDDF8 was site-specifically integrated into the rDNA locus of HA-iPSCs. The 2bF8-iPSCs were efficiently differentiated into 2bF8-iHPCs, 2bF8-iMKs, and 2bF8-iMSCs. FVIII was 10.31 ng/106 cells in lysates of 2bF8-iHPCs, compared to 1.56 ng/106 cells in HA-iHPCs, and FVIII was 3.64 ng/106 cells in 2bF8-iMSCs lysates, while 1.31 ng/106 cells in iMSCs with CMV-driven BDDF8. Our results demonstrated a high expression of FVIII in iHPCs and iMSCs derived from hiPSCs with site-specific integration of ITGA2B promoter-driven BDDF8, indicating potential clinical prospects of this platelet-targeted strategy for HA gene therapy.

Intravenous Lentiviral Gene Therapy for Hemophilia a: In Vitro and In Vivo Assessment of Universal Versus Tissue-Specific Promoters

Background：Hemophilia A (HA) is an X-linked monogenic coagulation disorder resulting from deficiency of the factor VIII (FVIII, F8) gene in the intrinsic coagulation cascade. The current treatment of HA is based on protein replacement therapy (PRT) through plasma-derived coagulation factors or recombinant proteins with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment for HA.Methods and Materials: We developed an advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy. A selective codon optimized and B-domain deleted human F8 (hF8BDD) gene was synthesized, sequenced and functionally verified. LVs carrying a universal EF1α promoter, or several modified tissue-specific promoters including endothelial- (VEC), endothelial and epithelial- (KDR), and two megakaryocyte-specific (ITGA and Gp) promoters, were biologically and immunologically characterized in vitro using human endothelial and megakaryocytic cell lines, EA-hy926 and DAMI, and in vivo using F8 knockout (KO) mice.Results: We investigated the different LV promoter constructs and found that LV-VEC-F8BDD exhibited the highest virus packaging (1.3x10 9 transduction units/ml) and transduction efficiencies compared with the other LV constructs. Tissue-specific expression of the VEC, KDR, ITGA and Gp promoters was confirmed in EA-hy926 and DAMI cells by RT-PCR, Western blot and ELISA analyses. We detected F8 activities close to 6 folds and 4.5 folds above the normal plasma level from the EF1a LV-F8BDD transduced EA-hy926 cells and DAMI cells, respectively, whereas the VEC LV in EA-hy926 cells and the ITGA-LV in DAMI cells exhibited F8 activities at 1.5 folds and 5 folds above the normal plasma level, respectively. In vivo studies in F8 KO mice via iv injection of LVs after reduced radiation conditioning illustrated preferential vector expression in different cell lineages, with high expression of EF1a vector in CD11b, F4/80 and Ly-6G positive immune cells, and preferential expression of VEC vector in CD31 positive endothelial cells, and ITGA and Gp vectors in CD41 positive megakaryocytes. In addition, we detected variable phenotypic corrections as well as anti-F8 immune responses in the F8 KO mice treated with the different LVs. The iv deliveries of VEC and Gp F8BDD vectors illustrated therapeutic F8 activities over time, around 25% and 8%, respectively, in 60 days, which increased to high levels (80% and 25%, respectively) after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titers (Bethesda assay) of the treated mice showed that the VEC vector exhibited the lowest F8 inhibitory immune response over time (Figure B).Conclusion: Based on the in vitro and in vivo studies, our results suggest that for HA gene therapy, optimal rather than high F8 expression is critical, and tissue-specific expression but not universal expression can reduce adverse inhibitor effect. We demonstrated that the LV-VEC-F8BDD vector displayed high tissue specificity in vivo, and high transgene delivery efficiency, high coagulation function and low immunogenicity. In addition, iv LV gene therapy could be a safe, convenient and effective HA gene therapy strategy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characteristics of the Thrombus Formation in Transgenic Mice with Platelet-Targeted Factor VIII Expression.

Platelet-targeted FVIII gene therapy can efficiently recover bleeding phenotype for hemophilia A (HA), yet characteristics of thrombus formation with this ectopic expression of factor VIII (FVIII) in platelets remain unclear. Here, we generated 2bF8trans mice restrictively expressing human B-domain-deleted FVIII (hBDD FVIII) in platelets on a hemophilic (FVIIInull) mice background. The results showed no statistical difference in clot strength and stability between wild-type (WT) and 2bF8trans mice, but with a prolonged reaction time (R-time), by thromboelastography. Fluid dynamics analysis showed that at the shear rates of 500 to 1,500 s-1, where physiological hemostasis often develops, the thrombi formed in 2bF8trans mice were more stable than those in FVIIInull mice, while at high pathological shear rates (2,500 s-1), mimicking atherosclerosis, thrombus size and fibrin deposition in 2bF8trans mice were less than those in WT mice. Thrombus morphology analysis showed that there was a locally concentrated deposition of fibrin in thrombus at the injured site and fibrin co-localized with activated platelets in 2bF8trans mice. Moreover, a higher ratio of fibrin to platelets was found in thrombus from 2bF8trans mice following laser-induced injury in cremaster arterioles, which might be the underlying mechanism of thrombus stability in 2bF8trans mice at physiological arterial circumstance. These observations suggest that specific morphological features of the thrombi might contribute to the efficacy and safety of platelet-targeted FVIII gene therapy for HA.

Non-genotoxic conditioning facilitates hematopoietic stem cell gene therapy for hemophilia A using bioengineered factor VIII

Hematopoietic stem and progenitor cell (HSPC) lentiviral gene therapy is a promising strategy toward a lifelong cure for hemophilia A (HA). The primary risks associated with this approach center on the requirement for pre-transplantation conditioning necessary to make space for, and provide immune suppression against, stem cells and blood coagulation factor VIII, respectively. Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA alkylation, that increase risk of sterility, infection, and developing secondary malignancies. In the current study, we describe a non-genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies to provide transient immune suppression against the transgene product in a murine HA gene therapy model. This strategy provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor VIII variant, termed ET3. Factor VIII procoagulant activity levels were durably elevated into the normal range and phenotypic correction achieved. Furthermore, no immunological rejection or development of anti-ET3 immunity was observed. These preclinical data support clinical translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for HA.

Gene Therapy for Hemophilia A: Where We Stand.

Hemophilia A (HA) is a hereditary hemorrhagic disease caused by a deficiency of coagulation factor VIII (FVIII) in blood plasma. Patients with HA usually suffer from spontaneous and recurrent bleeding in joints and muscles, or even intracerebral hemorrhage, which might lead to disability or death. Although the disease is currently manageable via delivery of plasma-derived or recombinant FVIII, this approach is costly, and neutralizing antibodies may be generated in a large portion of patients, which render the regimens ineffective and inaccessible. Given the monogenic nature of HA and that a slight increase in FVIII can remarkably alleviate the phenotypes, HA has been considered to be a suitable target disease for gene therapy. Consequently, the introduction of a functional F8 gene copy into the appropriate target cells via viral or nonviral delivery vectors, including gene correction through genome editing approaches, could ultimately provide an effective therapeutic method for HA patients. In this review, we discuss the recent progress of gene therapy for HA with viral and nonviral delivery vectors, including piggyBac, lentiviral and adeno-associated viral vectors, as well as new raising issues involving liver toxicity, pre-existing neutralizing antibodies of viral approach, and the selection of the target cell type for nonviral delivery.

Deciphering the Ets-1/2-mediated transcriptional regulation of F8 gene identifies a minimal F8 promoter for hemophilia A gene therapy.

Amajor challenge in the development of a gene therapy for hemophilia A is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help to optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets- 1/Est-2 combination, while Ets-2 alone was ineffective. Moreover, Ets-1/Ets- 2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of green fluorescent protein (GFP) or FVIII cassettes driven by the shortened promoters, led to GFP expression mainly in endothelial cells in the liver and to longterm FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in hemophilia A gene therapy.

Translational Potential of Immune Tolerance Induction by AAV Liver-Directed Factor VIII Gene Therapy for Hemophilia A.

Hemophilia A (HA) is an X-linked bleeding disorder due to deficiencies in coagulation factor VIII (FVIII). The major complication of current protein-based therapies is the development of neutralizing anti-FVIII antibodies, termed inhibitors, that block the hemostatic effect of therapeutic FVIII. Inhibitors develop in about 20–30% of people with severe HA, but the risk is dependent on the interaction between environmental and genetic factors, including the underlying F8 gene mutation. Recently, multiple clinical trials evaluating adeno-associated viral (AAV) vector liver-directed gene therapy for HA have reported promising results of therapeutically relevant to curative FVIII levels. The inclusion criteria for most trials prevented enrollment of subjects with a history of inhibitors. However, preclinical data from small and large animal models of HA with inhibitors suggests that liver-directed gene therapy can in fact eradicate pre-existing anti-FVIII antibodies, induce immune tolerance, and provide long-term therapeutic FVIII expression to prevent bleeding. Herein, we review the accumulating evidence that continuous uninterrupted expression of FVIII and other transgenes after liver-directed AAV gene therapy can bias the immune system toward immune tolerance induction, discuss the current understanding of the immunological mechanisms of this process, and outline questions that will need to be addressed to translate this strategy to clinical trials.

The Immune Response to the fVIII Gene Therapy in Preclinical Models.

Neutralizing antibodies to factor VIII (fVIII), referred to as “inhibitors,” remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product- and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liver-specific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pre-transplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors.

SsODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs

Given that the cDNA of F8 is too large to be packaged into adeno-associated virus (AAV) capsids, gene transfer of some versions of B-domain-deleted F8 (BDD-F8) for hemophilia A (HA) treatment has been attempted with promising results. Here, we describe an efficient gene correction via single-stranded-oligodeoxynucleotide (ssODN)-mediated in-frame deletion within the B domain of F8 with CRISPR/Cas9 in HA-patient-derived induced pluripotent stem cells (HA-iPSCs). The expression and activity of FVIII was restored in corrected HA-iPSC-derived induced endothelial progenitor cells (C-iEPCs) in vitro and in vivo. The bleeding phenotype was rescued in HA mice after C-iEPC infusion. Our results demonstrate an efficient approach for in situ gene correction via introduction of a tiny deletion using ssODN and CRISPR/Cas9 to reframe the F8 transcript and restore FVIII function in HA-iPSC-derived EPCs with potential clinical impact in HA gene therapy. For the first time, we demonstrated in vitro and in vivo the FVIII function that is encoded by the endogenous F8 gene with a partially deleted B domain. This work also suggests an applicable strategy for genetic correction of other gene frameshift mutations.

Hematopoietic stem cell transplantation gene therapy for hemophilia a using non-genotoxic ANTI-CD117 immunotoxin conditioning

Background & Aim Hematopoietic stem cell transplantation (HSCT) for coagulation factor VIII (fVIII) gene therapy is a potentially curative approach for the treatment of the most common severe bleeding disorder, hemophilia A (HA). Our laboratory has previously established the ability to produce stable therapeutic levels of fVIII in a mouse model of HA following irradiation or chemotherapy and transplantation of Sca-1+ cells, a population containing both HSC and progenitors, genetically modified ex vivo using recombinant lentiviral vector encoding a bioengineered high-expression fVIII variant, termed ET3. Conventional myeloablative regimens for HSCT such as total body irradiation and myeloablative alkylating chemotherapy are associated with adverse side effects, including sterility, hormonal dysregulation and genotoxicity. These risks represent a primary barrier to clinical translation of HSCT gene therapy for HA and many other monogenic diseases. Therefore, the development of targeted, non-genotoxic conditioning agents is critical. Methods, Results & Conclusion We have successfully implemented the use of an immunotoxin bioconjugate comprised of a monoclonal antibody against the stem cell factor receptor c-Kit (anti-CD117) and the ribosome-inactivating protein toxin saporin (sap) for non-genotoxic HSCT conditioning. We achieved consistent depletion of bone marrow LSK (lineage-negative Sca-1+ c-Kit+) and long-term HSC (LSK+ CD150+ CD48−) compartments five days after retro-orbital venous sinus injection of 0.5 mg/kg CD117-sap compared to control (59.2% LSK reduction; 38.9% LT-HSC reduction) in both C57BL/6J wild-type and HA mice. Following CD117-sap conditioning, HA mice were transplanted with 1 × 106 ET3-transduced Sca-1+ cells from GFP+ congenic mice. We observed robust mixed hematopoietic chimerism and myeloid engraftment at two and four weeks post-transplantation in recipients conditioned with CD117-sap compared to control mice (2 wk: 86.4 ± 9.2% vs. 1.2 ± 0.6% GFP+ granulocytes, p = 0.00009; 4 wk: 93.9 ± 5.6% vs. 0.2 ± 0.3% GFP+ granulocytes, p = 0.0018). Moreover, these mice exhibit therapeutic levels of circulating fVIII activity as early as 2 weeks post-transplantation (0.11 IU/ml, p = 0.005). Taken together, these preliminary data are a promising proof-of-concept demonstrating the feasibility of using non-genotoxic immunotoxin conditioning for HA gene therapy.