Preclinical evaluation of SPK-8011QQ, an adeno-associated virus gene therapy for people with hemophilia A leveraging the dirloctocogene samoparvovec platform encoding an activated Protein C-resistant B-domain deleted factor VIII Free

Background: Gene therapies for hemophilia A (HA) offer the potential for durable factor (F)VIII expression as a curative treatment option. However, recent clinical programs and approved gene therapies expressing wild-type (WT) B-domain deleted (BDD) FVIII have faced challenges in reaching sufficient and/or durable FVIII levels. SPK-8011QQ is an investigational adeno-associated virus (AAV) gene therapy that leverages the clinically evaluated safety and durability of the SPK-8011 (dirloctocogene samoparvovec) platform to introduce the enhanced-function FVIII-QQ variant (Wilhelm et al. Blood 2021). The optimized payload carries a two-amino acid change (R336Q; R562Q) that has 99.9% identity with the original transgene sequence. As a mode of action, the FVIII-QQ variant confers resistance to cleavage by the anticoagulant activated protein C (APC), thereby enhancing FVIII potency and hemostatic potential. This ongoing study evaluates the preclinical efficacy and safety of a surrogate vector for SPK-8011QQ in vitro, ex vivo and in vivo.

Methods: Surrogate AAV vectors for SPK-8011QQ or SPK-8011 were administered intravenously to FVIII knock-out mice. Seven days post vector administration, plasma samples were collected and analyzed using APC-sensitive chromogenic substrate assay (CSA) and thrombin generation assay (TGA). APC-sensitive assays specifically evaluate FVIII function in the presence of APC, allowing for the differentiation of FVIII-QQ with enhanced potency from WT-BDD-FVIII, which standard assays cannot detect. Differences in FVIII-QQ potency and activity versus WT-BDD-FVIII were evaluated in murine samples using unpaired two-tailed t-test with Welch's correction. In addition, AAV-treated mice were challenged in a 4mm tail-clip bleeding model 7 days post infusion. Concurrently, plasma samples were collected from the same mice to measure FVIII activity (FVIII:C) and antigen levels (FVIII:Ag).

Planned preclinical work to evaluate prothrombotic risks of the FVIII-QQ transgene will be conducted, whereby AAV-treated mice will be challenged with a 3.5% ferric chloride-induced carotid injury model and compared to mice infused with the control AAV (i.e. surrogate SPK-8011 vector). In addition, plasma samples from people with severe HA will be spiked with recombinant FVIII-QQ or WT-BDD-FVIII and evaluated in APC-sensitive CSA and TGA. Results from ongoing preclinical work will be presented once data are available.

Results: Analysis of murine ex vivo samples in APC-sensitive assays resulted in residual FVIII activity increasing by 30% in the CSA (p=0.03), and 24% in the TGA (p=0.12), for the surrogate SPK-8011QQ group compared with the control vector. In the tail-clip model, at comparable FVIII activity levels, mice treated with surrogate SPK-8011QQ exhibited significantly reduced bleeding times and blood loss compared with those treated with surrogate SPK-8011. Analysis of blood loss as a function of FVIII:C demonstrated an approximately 9-fold increase in potency for surrogate SPK-8011QQ (EC₅₀: 12.2% FVIII:C) relative to the control vector (EC₅₀: 112.3% FVIII:C).

The prothrombotic risk assessment in the carotid injury model will be presented once readouts are available. Similarly, findings in severe HA plasma samples spiked with recombinant FVIII-QQ and WT-BDD-FVIII will be discussed.

Conclusions: Surrogate SPK-8011QQ, which leverages the previously clinically evaluated SPK-8011 (dirloctocogene samoparvovec) platform to introduce FVIII-QQ, demonstrates markedly enhanced hemostatic potency under APC-sensitive conditions in ex vivo and in vivo mouse models. Furthering the learnings from the safety and durability of the SPK-8011 platform, our preclinical data collected to date support the ongoing evaluation of SPK-8011QQ, which aims to optimize FVIII potency and hemostatic potential.