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 × 10 12 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.

Pharmacokinetic analysis identifies a factor VIII immunogenicity threshold after AAV gene therapy in hemophilia A mice

“…However, administration of similar doses to normal NHP (expressing endogenous NHP FVIII) has resulted in rapid and potent inhibitory immune responses, whereas some lower-dose studies appear to avoid such immune responses (supplemental Table 1). 10-14 Data from other preclinical AAV gene therapy studies also suggest a dose and/or FVIII expression–related immunogenicity threshold (supplemental Table 1). 11,15 - 19 It has also been proposed that FVIII dosing intensity and dosing intervals may play a role in the risk of inhibitor development vs. establishing and maintaining FVIII immune tolerance in non–gene therapy settings.…”

“…Furthermore, variants such as BDD porcine FVIII, ET3, X10, N6, V3, and R1645H possess amino acid sequence changes that affect biosynthetic efficiency and/or specific activity. 10,13,18,23 - 28 In addition to transgene design, synthetic promoter/enhancer elements could affect comparative immunogenicity via effects on FVIII expression level or FVIII expressing cell type, which is affected by both vector tropism (eg, AAV serotype) and promoter specificity for a given transduced cell type. 15,19,29 - 34 Finally, natural and recombinant AAV particles are immunogenic in humans and higher AAV capsid loads can induce cellular stress and creation of a proinflammatory microenvironment in the target organ that alters the immunogenicity risk of the transgene product.…”

“…Furthermore, in the context of comparative immunogenicity between different recombinant protein candidates or gene therapy candidates with varying vector designs or dose levels, preclinical models have proven informative (supplemental Table 1). 9 - 19,23,25 - 28,37 - 53 Furthermore, aside from immunogenicity resulting from neoepitopes present in xenoproteins, there may be inherent biochemical properties conserved across FVIII variants that impact overall immunogenicity.…”

First-in-Patient Dose Prediction for Adeno-Associated Virus-Mediated Hemophilia Gene Therapy Using Allometric Scaling

“… e Human FIX or FVIII was expressed in macaques, but immunogenicity data were not reported. f B domain-deleted human FVIII (hFVIII-SQ) transgene was expressed in macaques, and 3 of 7 macaques developed anti-human FVIII antibodies. , g hFVIII-SQ transgene was expressed in macaques, and 3 of 5 macaques developed anti-human FVIII antibodies h Human FVIII was expressed in macaques, and 8 of 9 macaques developed anti-human FVIII antibodies …”

Expression and function of four AAV-based constructs for dystrophin restoration in the mdx mouse model of Duchenne muscular dystrophy

“…Sample sizes of n =5 were used to power the study to determine statistical differences in functional evaluation. Additionally, the study was designed and executed as a proof-of-concept evaluation and based on historical gene-therapy publications with similar sample sizes ( Zhang et al, 2013 ; Elkouby et al, 2021 ). Outlier analyses were run using the ROUT analysis using GraphPad Prism 5 statistical software (Graphpad Software, La Jolla, CA, USA).…”