BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression

Abstract

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608.

Conflict of interest statement

Conflict-of-interest disclosure: B.A.K. has received research funding from Bioverativ, Pfizer, Sangamo, Shire (a Takeda company), and Spark Therapeutics; and has received payment for consulting from BioMarin, Pfizer, Sanofi, and Spark Therapeutics. M.A.E. has received honoraria and consulting fees from Baxalta, a member of the Takeda group of companies, Bayer, CSL Behring, Genentech/Roche, Kedrion, Novo Nordisk, Pfizer, and Sanofi. G.Y. has received honoraria and consulting fees from Bayer, Bioverativ, CSL Behring, Genentech/Roche, Grifols, Kedrion, Novo Nordisk, Pfizer, Shire, a member of the Takeda group of companies, Spark, and UniQure. A.V.D. has received honoraria and consulting fees from Baxalta, Bayer, BioMarin, Bioverativ/Sanofi, CSL Behring, Hema Biologics, Novo Nordisk, Pfizer, Shire, a member of the Takeda group of companies, and uniQure; and is cofounder and member of the board of directors of Hematherix LLC., a biotech company that is developing superFV, a therapy for bleeding complications. S.W.J.M. was an employee of Asklepios and a co-owner of Chatham at the time of the study and may receive remuneration from future development of this program. R.J.S. is the founder and a shareholder at Asklepios BioPharmaceutical and Bamboo Therapeutics, Inc.; holds patents that have been licensed by the University of North Carolina to Asklepios BioPharmaceutical, for which he receives royalties; and has consulted for Baxter Healthcare and has received payment for speaking. I.B. is an employee of Baxalta Innovations GmbH, a member of the Takeda group of companies. M.d.l.R. was an employee of Baxalta Innovations GmbH, a member of the Takeda group of companies, at the time of the study. B.M.R. was an employee of Baxalta Innovations GmbH, a member of the Takeda group of companies, at the time of the study and is a Takeda stock owner. H.R. was an employee of Baxalta Innovations GmbH, a member of the Takeda group of companies, at the time of the study and is a Takeda stock owner. F.S. was an employee of Baxalta Innovations GmbH, a member of the Takeda group of companies, at the time of the study and is a Takeda stock owner. J.C.C. is an employee of Baxalta US Inc., a member of the Takeda group of companies, and is a Takeda stock owner. B.E. is an employee of Baxalta US Inc., a member of the Takeda group of companies. P.E.M. was an employee of the University of North Carolina and was subsequently an employee of Baxalta US Inc., a member of the Takeda group of companies, at the time of the study (with no conflicts to disclose related to this analysis); he holds patents that have been licensed to Asklepios BioPharmaceutical, for which he receives royalties; and he has received research support through the University of North Carolina from Asklepios BioPharmaceutical, Baxter Healthcare, and Novo Nordisk. The remaining authors declare no competing financial interests.

© 2021 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Schematic drawing of the BAX 335 expression cassette. The recombinant vector genome of BAX 335 is designed to express a codon-optimized FIX Padua (R338L) cDNA from the liver-specific murine transthyretin (TTR) promoter/enhancer. The intron fragment from minute virus of mice (MVM) and the bovine growth hormone (BGH) polyadenylation (pA) sequence help improve expression. The expression cassette is flanked by 1 AAV2 wild-type inverted terminal repeat (ITR) sequence (3′ITR) and 1 mutated AAV2 ITR (MUT 5′ITR) to direct preferential replication and packaging of self-complementary (sc) vector DNA. The overall size of the resulting sc genome is 4806 nucleotides (nt). The BAX 335 nucleotide sequence including ITRs is provided in supplemental Figure 1.

Figure 2.

FIX activity, FIX Padua levels, liver enzymes, and PBMC-mediated immune responses to AAV8 post–BAX 335 infusion for participants in dose cohort 2. FIX activity and FIX Padua activity levels post–BAX 335 infusion by participant in relation to bleeding episodes, administration of FIX replacement therapy, and prednisone dosing (participant 8) as well as liver enzyme markers of hepatotoxicity (ALT and aspartate aminotransferase [AST]) are shown. Study week represents nominal collection time (mean values used if ≥1 result in 1 week). The number of exogenous FIX infusions or bleeding episodes within 1 week is indicated. The lower limit of detection (LOD) for FIX Padua–specific activity (<0.020 U) is shown by the gray dashed line. Participant 8 received prophylactic treatment with prednisone. The duration of prednisone treatment is shown in the green bar chart, with the slope indicating dose tapering. The results from IFN-γ ELISpot assays are shown in the lower graph panel for each participant; the reaction of the participant’s PBMCs to AAV8 capsid peptides in 3 separate pools are plotted as the number of spots per million PBMCs. Values were considered positive if they were above the media control (background) by a factor of 2 and were >60 spots per million PBMCs (black dashed line). Maximum results across all pools were presented for each time point.

Figure 3.

FIX activity, FIX Padua levels, liver enzyme levels, and PBMC-mediated immune responses to AAV8 post–BAX 335 infusion for participants in dose cohort 3. FIX and FIX Padua activity post–BAX 335 infusion by participant in relation to bleeding episodes, administration of FIX replacement therapy, and prednisone dosing (participants 6 and 7) as well as liver enzyme markers of hepatotoxicity (ALT and aspartate aminotransferase [AST]) are shown. Study week represents nominal collection time (mean values used if ≥1 result in 1 week). The number of exogenous FIX infusions or bleeding episodes within 1 week is indicated. Participants 6 and 7 received prednisone as a rescue treatment in response to a sudden loss of FIX expression. The duration of prednisone treatment is shown in the green bar charts, with the slope indicating dose tapering. FIX Padua–specific activity values were above the lower limit of detection (LOD) (<0.020 U), which is shown by the gray dashed line. The results from IFN-γ ELISpot assays are shown in the lower graph panel for each participant; the reaction of the participant’s PBMCs to AAV8 capsid peptides in 3 separate pools are plotted as the number of spots per million PBMCs. Values were considered positive if they were above the media control (background) by a factor of 2 and were >60 spots per million PBMCs (black dashed line). Maximum results across all pools were presented for each time point.

Figure 4.

Peak FIX activity by participant, baseline CRM status, and dose cohort. FIX activity was measured in each participant during the 6 months after BAX 335 infusion. Participants, testing either positive or negative for circulating FIX antigen CRM at baseline, received a single infusion of BAX 335 at 1 of 3 doses. FIX measurements were determined by one-stage clotting assay (central laboratory), including measurements within 5 days of an exogenous FIX infusion and measurements from unscheduled study visits.

Figure 5.

Anti-AAV8 NAb responses in mice treated with BAX 335 vs CpG-depleted vector constructs. Anti-AAV8 NAb responses, as a surrogate marker for immune activation via TLR9, indicate lower immunogenicity for CpG-depleted vectors. C57BL/6J mice (8-10 weeks old, n = 6-8 per group) were treated IV with identical doses (4 × 1012 vg/kg) of either one of two AAV8 vectors carrying an FIX Padua coding sequence with different numbers of CpG oligodeoxynucleotides (ODNs): BAX 335 (99 CpG ODNs); CpG-depleted candidate (0 CpG ODNs). Blood was collected 4 weeks later, and the magnitude of the resulting titer of anti-AAV8 NAbs was assayed as a marker of adaptive immune responses to CpG ODNs.