Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

Abstract

Factor VIII gene transfer with a single intravenous infusion of valoctocogene roxaparvovec (AAV5-hFVIII-SQ) has demonstrated clinical benefits lasting 5 years to date in people with severe hemophilia A. Molecular mechanisms underlying sustained AAV5-hFVIII-SQ-derived FVIII expression have not been studied in humans. In a substudy of the phase 1/2 clinical trial (NCT02576795), liver biopsy samples were collected 2.6–4.1 years after gene transfer from five participants. Primary objectives were to examine effects on liver histopathology, determine the transduction pattern and percentage of hepatocytes transduced with AAV5-hFVIII-SQ genomes, characterize and quantify episomal forms of vector DNA and quantify transgene expression (hFVIII-SQ RNA and hFVIII-SQ protein). Histopathology revealed no dysplasia, architectural distortion, fibrosis or chronic inflammation, and no endoplasmic reticulum stress was detected in hepatocytes expressing hFVIII-SQ protein. Hepatocytes stained positive for vector genomes, showing a trend for more cells transduced with higher doses. Molecular analysis demonstrated the presence of full-length, inverted terminal repeat-fused, circular episomal genomes, which are associated with long-term expression. Interindividual differences in transgene expression were noted despite similar successful transduction, possibly influenced by host-mediated post-transduction mechanisms of vector transcription, hFVIII-SQ protein translation and secretion. Overall, these results demonstrate persistent episomal vector structures following AAV5-hFVIII-SQ administration and begin to elucidate potential mechanisms mediating interindividual variability.

Application of in-vitro-cultured primary hepatocytes to evaluate species translatability and AAV transduction mechanisms of action

“…Human liver biopsy samples were obtained per protocol in the phase 1/2 trial; all participants provided written informed consent, and trial procedures aligned with the Declaration of Helsinki and local regulations. 15 , 27 …”

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

“…This was recently highlighted in trials evaluating rAAV-FVIII for the treatment of hemophilia A where a high degree of variation in FVIII activity levels was observed among participants. 2, 41 One could envision using an RNAi-based approach to titrate transgene expression to within the therapeutic window and thereby mitigate potential negative consequences associated with over-production of the therapeutic protein, such as increased thrombotic risk in the case of high FVIII levels. In addition to initial dose titration after rAAV dosing, RNAi-based approaches could enable transgene expression to be dynamically modified as the disease state evolves.…”

Gene Therapy and Hemophilia: Where Do We Go from Here?

“…A small number of liver biopsy tissue evaluations did not find evidence of stress to the endoplasmic reticulum. 34 Eighty-six percent of patients developed increased transaminases and 79% were treated with reactive transient immunosuppression, with resolution in 96% of the cases. 29 This vector product is currently under review by regulatory authorities in Europe and the United States (US).…”

“… 40 However, other causes could include details of the vector construct, manufacturing methods, vector titering methods, transduction efficiency including AAV binding and internalization at cell surface, escape from endosomes, entry into the nucleus, DNA annealing or second-strand synthesis, episome and concatemer formation, mRNA transcription, and translation and post-translational processing – much of this is not well understood. 34 , 41 …”

“… 52 A small number of patients after AAV administration, with diagnoses of hemophilia, acute intermittent porphyria and lipoprotein lipase deficiency, had liver biopsies revealing a low frequency of integrations, but no concern for integration at oncogenic sites and no evidence of abnormal pathology. 34 , 53 , 54 To date, no AAV vector-related malignancy has been identified across all trials in hemophilia. A single case of hepatocellular carcinoma (HCC) in an AAV vector-treated patient with hemophilia B was determined highly unlikely associated with the vector, because the patient had multiple known clinical risk factors for HCC.…”