Preclinical assessment of an optimized AAV-FVIII vector in mice and non-human primates for the treatment of hemophilia A

Abstract

Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our second-generation expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 × 10¹² vg/kg AAV-Spark100-FVIII or AAV-Spark200-FVIII expressed FVIII antigen levels of 13% ± 2% and 22% ± 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic.

Keywords: AAV; codon optimization; hemophilia A; low AAV dose; optimized vectors; preclinical development.

Graphical abstract

Figure 1

Introduction of furin variants into a first-generation codon-optimized sequence improves hFVIII expression (A) Schematic of the B-domain deleted hFVIII expression cassettes. (B) AAV vectors were delivered at a dose of 4 × 10¹² vg/kg into hemophilia A/CD4 KO mice (n = 4/group). The vectors with and without codon-optimized sequences are labeled as hFVIII-CO and hFVIII, respectively. The Δ3 and Δ4 furin variants were introduced into codon-optimized constructs, generating hFVIII-CO-Δ3 and hFVIII-CO-Δ4, respectively. Human FVIII plasma levels were assayed using ELISA. ∗p < 0.05, Kruskal-Wallis one-way ANOVA vs hFVIII. (C) Hemostatic challenge of AAV-treated animals. The tail clip assay was performed at 6 weeks after vector administration. ∗p < 0.05, Kruskal-Wallis one-way ANOVA; ns, non-significant versus wild-type (WT) PBS. (D) AAV vectors carrying either hFVIII-CO or hFVIII-CO-Δ3 cassettes were administered to hemophilia A/CD4 KO mice at three different doses (2 × 10¹¹, 8 × 10¹¹, and 4 × 10¹² vg/kg). Shown are hFVIII levels measured using ELISA after vector administration. ∗p < 0.05, Kruskal-Wallis one-way ANOVA versus hFVIII-CO Low. Results in panels B-D are represented as mean ± standard error of the mean.

Figure 2

Generation of second-generation codon-optimized FVIII cassettes (A) Wild-type mice (n = 5/group) were hydrodynamically delivered 50 μg of plasmids carrying 1 of the 26 novel codon-optimized sequences, shown as 1–26, and benchmarked against the first-generation codon-optimized cassette (CO). Plasma was collected 24 h after hydrodynamic delivery for FVIII analysis. Average hFVIII levels in the CO group were given a relative value of 1, and all the other groups were normalized to this reference. Red bars indicate the constructs that were selected for further study as AAV vectors. Gray bars indicate the three constructs that were discarded because of low yields. The dotted line indicates 2-fold over CO. ∗p < 0.05 versus CO in groups with FVIII >2-fold difference, one-way ANOVA. (B–E) AAV delivery of second-generation codon-optimized sequences. AAV vectors using the Spark100 capsid and containing the TTRm-hFVIII-CO sequences were administered to hemophilia A/CD4 KO mice at 4 × 10¹² vg/kg. Human FVIII levels were measured by ELISA and followed for 8 or 12 weeks. (B) TTRm-hFVIII-CO compared with codon-optimized sequences 07 and 10. ∗p < 0.05 versus CO, Kruskal-Wallis one-way ANOVA. (C) TTRm-hFVIII-CO compared with codon-optimized sequences 09, 12, and 16. ∗p < 0.05 versus CO, Kruskal-Wallis one-way ANOVA. (D) TTRm-hFVIII-CO compared with codon-optimized sequences 01 and 11. ∗p < 0.05 versus CO, Kruskal-Wallis one-way ANOVA. (E) TTRm-hFVIII-12 compared with a vector containing the TTRm-hFVIII-12 Δ4 furin variant. ∗p < 0.05 versus 12, unpaired t test. Results are represented as mean ± standard error of the mean.

Figure 3

Levels of hFVIII in plasma of cynomolgus macaques after SPK-8005 administration (A–C) Animals received intravenous administration of either 2 × 10¹² (A), 5 × 10¹² (B), or 1 × 10¹³ vg/kg (C) SPK-8005, also known as Spark100-TTRm-hFVIII-07. Lines represent individual animals. Human FVIII plasma levels were assayed using ELISA and represent repeated measurements, obtained by serial bleeding, on the same group of animals during the course of the study (n = 2 or 3 animals per cohort). Human FVIII levels measured in vehicle-treated animals are shown in white squares in all three graphs. ε denotes detection of anti-FVIII antibodies.

Figure 4

Levels of hFVIII in plasma of cynomolgus macaques after SPK-8011 administration (A–C) Animals received intravenous administration of either 2 × 10¹² (A), 6 × 10¹² (B), or 2 × 10¹³ vg/kg (C) SPK-8011, also known as Spark200-TTRm-hFVIII-07. Lines represent individual animals. Human FVIII plasma levels were assayed using ELISA and represent repeated measurements, obtained by serial bleeding, on the same group of animals during the course of the study (n = 3 animals per cohort). ε denotes detection of anti-FVIII antibodies.

Figure 5

Biodistribution of vector genomes in tissues of cynomolgus macaques after SPK-8005 and SPK-8011 administration One microgram of genomic DNA was isolated from the indicated organs 30 days after administration of the vectors and vector genome presence was analyzed by qPCR. (A) Tissue biodistribution following administration of 5 × 10¹² vg/kg of AAV-Spark100 (n = 4 macaques). (B) Tissue biodistribution following administration of 3 × 10¹², 6 × 10¹², or 1.2 X 10¹³ vg/kg AAV-Spark200 (n = 3 macaques per dose). Results are represented as mean vector copy number per haploid genome ± standard error of the mean.