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 (AAV) serotype 8-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetics, effects on FIX activity, and immune responses for dosed participants. Eight adult males (\aged 20-69 years; range FIX activity, 0.5%-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 (SAEs), all considered unrelated to BAX 335. No SAE led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity were reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0%-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 5-11 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. Registered at www.clinicaltrials.gov as NCT01687608.

Novel vectors and approaches for gene therapy in liver diseases

“…Additionally, these immune responses can be mitigated by modifying the CpG sequence content of the recombinant genetic material in order to reduce toll-like receptor 9 (TLR9) pathway activation. 141 Furthermore, very recently, concern has been raised about hepatoxicity associated with systemic administration of rAAV due to the death of three young patients suffering a fatal neuromuscular disorder that received a high vector dose. The three patients were older and heavier than the other treated patients, and more importantly they had evidence of preexisting hepatobiliary disease.…”

Challenges Posed by Immune Responses to AAV Vectors: Addressing Root Causes

“…Since that initial trial, several additional hemophilia B gene therapy trials have been reported, some achieving long-term efficacy, but others recapitulating the loss of transgene expression associated with immune-mediated loss of transduced cells (33). This failed outcome was recently reported in a trial using an AAV8 vector (34), demonstrating that consensus strategies to effectively minimize and manage deleterious capsid CTL response have not yet been fully established. The targeted nature of these efficacy-limiting responses when they are observed is evidenced by liver enzyme elevation concomitant with loss of therapeutic transgene expression.…”

“…If achievable, methylation of CpG motifs to 'humanize' this epigenic attribute is predicted to reduce TLR9 activation. Another approach is the incorporation of TLR9 inhibitory nucleotides sequences into the AAV expression cassette (60).…”

“…Prednisone initiated prior to vector administration and continued for at least 30 days is part of the administration protocol for Zolgensma, the first licensed AAV product approved for systemic administration, and similar protocols are used for many AAV products currently in clinical development. While prophylactic corticosteroids have improved outcomes in AAV clinical trials, they have failed to prevent loss of transgene expression in others ( 34 ), and present risks ( 34 , 43 ). Additional novel immunomodulatory approaches have shown promise.…”

Immunomodulation in Administration of rAAV: Preclinical and Clinical Adjuvant Pharmacotherapies

“…Regulatory T cells (Treg) modulate immune tolerance towards transgene product and capsid that are vital to durable expression of therapeutic protein ( 16 , 17 ). Although the full clinical significance of innate response to rAAV is unclear ( 18 ), unmethylated CpG motifs in rAAV vector genome interact with toll-like receptor (TLR) 9 present in plasmacytoid dendritic cells and Kupffer cells, releasing type I interferons activating cellular and humoral responses in mouse models ( 19 , 20 ), and has been suggested as the cause of loss of expression in a rAAV8 hemophilia B trial ( 21 ). Furthermore, rAAV capsid-targeting TLR2, various DNA sensors, and complement activation may also play a role ( 22 ).…”