The Immune Response to the fVIII Gene Therapy in Preclinical Models

Abstract

Neutralizing antibodies to factor VIII (fVIII), referred to as "inhibitors," remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product-and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liverspecific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pretransplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, Patel et al. Preclinical Gene Therapy fVIII Immunology respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors.

Novel Severe Hemophilia A Mouse Model with Factor VIII Intron 22 Inversion

“…However, HA patients with FVIII inhibitors or AAV-capsid antibodies may experience a reduction in the efficacy of the therapy. Additional drawbacks remain, such as unknown side effects due to random AAV integration [ 25 , 26 ]. Therefore, to permanently rescue hemophilia, gene correction treatment can be considered as the best option for patients with F8I22I-mediated HA.…”

Gene Therapy for Inherited Bleeding Disorders

“…Strategies being investigated to enhance FVIII HSC-directed gene transfer may inform on how to avoid the problems with conditioning in GT gene therapy, including nongenotoxic bone marrow conditioning regimens 105 and in vivo LV HSC-directed approaches. 106 A better understanding the immune response to transgenes after gene therapy 36,107 may similarly inform on the risk of transgene α IIb β 3 . The availability of small-and large-animal models of GT will facilitate the adoption of gene therapy breakthroughs for GT gene therapy.…”

Haemophilia: factoring in new therapies

“…Preclinical studies are ongoing to evaluate the targeting of different cell types and utilisation of less genotoxic conditioning regimens, results of which will be critically important to determine the most effective approach while minimising potential toxicity. 75,76 Gene editing is yet another method in preclinical exploration for therapeutic indications in haemophilia. Genome editing involves targeting and correcting the gene using techniques like zinc-finger nucleases (ZFN) or clustered regularly interspaced short palindromic repeats associated with Cas9 endonuclease (CRISPR/Cas9) technology to restore FVIII or FIX protein expression.…”

“…Important considerations include long‐term genotoxicity risk from the integration event and the potential need for pre‐treatment myeloablation. Preclinical studies are ongoing to evaluate the targeting of different cell types and utilisation of less genotoxic conditioning regimens, results of which will be critically important to determine the most effective approach while minimising potential toxicity 75,76 …”