According to a new report in the journal Blood, in a clinical trial, treatment of patients with hemophilia B using the gene therapy agent BAX 335 may have been hindered by the presence of CpG-rich areas of coding sequence present in the BAX 335 genetic construct.

BAX 335 is a gene therapy agent for hemophilia B that is based on use of a F9 gene and is constructed with an adeno-associated viral (AAV) vector. It is designed to express factor IX (FIX) Padua, which is a variant of FIX considered to be hyperactive, potentially enhancing FIX expression relative to vector exposure, the study authors explained.

In this first-in-human, open-label phase 1/2 trial ( Identifier: NCT01687608), 8 male patients with hemophilia B were given BAX 335 intravenously at 1 of 3 levels of dosage, which were 2.0 x 1011, 1.0 x 1012, and 3.0 x 1012 vector genomes/kg. Primary study objectives included safety and analyses of laboratory values.

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Out of these patients, 2 received the lowest BAX 335 dosage, 4 received the intermediate dosage, and 2 received the highest dosage. A total of 7 of the patients were evaluable for FIX expression, with BAX 335-based FIX expression and peak FIX activity showing dose dependence. During the first 6 months following treatment, mean levels of peak FIX activity were 2.8% at the lowest dosage, 12.8% at the intermediate dosage, and 45.3% at the highest dosage.

Therapeutic FIX activity was seen in 1 patient for 4 years, but for the remaining patients, peak FIX activity appeared not to be sustained, with most beginning to lose activity after approximately 5 to 11 weeks.

The researchers hypothesized that FIX expression may have been impeded by an immune response related to CpG content in the BAX 335 sequence. They tested this by generating a BAX 335-like construct that included the FIX Padua transgene, but depleted of CpG motifs. When applied to a mouse model, neutralizing antibody titers against the vector were generally higher in mice given BAX 335, in comparison with titers seen in mice treated with the CpG-depleted construct.

In this study, the rate of serious adverse events was 37.5%, reflecting 4 nonlethal events in 3 patients, each of which was deemed unrelated to treatment with BAX 335. Reportedly, clinical thrombosis, inhibitors, and FIX Padua-oriented immune responses were not detected.

“Here, we suggest that innate immune stimulation, potentially driven by CpG enrichment (introduced during codon optimization of the gene expression cassette) could have contributed to the loss of AAV vector–mediated gene expression in a human clinical trial,” the researchers wrote in their report. They indicated further research is required to evaluate this hypothesis.

Disclosures: Some authors have declared affiliations with or received grant support from the pharmaceutical industry. Please refer to the original study for a full list of authors’ disclosures.


Konkle BA, Walsh CE, Escobar MA, et al. BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression. Blood. 2021;137(6):763-774. doi: 10.1182/blood.2019004625