Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs

Callan, Mary Beth; Haskins, Mark E.; Wang, Ping; Zhou, Shangzhen; High, Katherine A.; Arruda, Valder R.

doi:10.1371/journal.pone.0151800

Cited by 27 publications

(22 citation statements)

References 39 publications

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“…Interestingly, when the same experimental setup was replicated in hemophilia A mice on a mixed S129-C57BL/6 background, AAV8-hAAT-hfVIII gene therapy only resulted in a significantly diminished inhibitor response following subsequent challenge with recombinant human fVIII. The differential outcome observed between both strains of hemophilia A mice is similar to the disparate immune response to recombinant human fVIII that is observed in different background strains of hemophilia A mice (90). These results highlight the potential role of genetics, in particular immune polymorphisms, on whether a patient will respond to AAV-fVIII gene therapy.…”

Section: Murine Preclinical Studiessupporting

confidence: 55%

The Immune Response to the fVIII Gene Therapy in Preclinical Models

et al. 2020

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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.

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Section: Murine Preclinical Studiessupporting

confidence: 55%

The Immune Response to the fVIII Gene Therapy in Preclinical Models

et al. 2020

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“…The hemophilia gene therapy arena also has good examples of long-term delivery with AAV for up to a decade of Factor IX in hemophilic humans (59) and of Factor VIII in hemophilic dogs (60). For more examples on long-term delivery with AAV of hemophilia factors, please see the following references (61)(62)(63)(64)(65)(66)(67)(68). The long-term delivery described in our report here is significant as the first such report for very long-term delivery of an antibody, particularly given the serious difficulties that have been encountered when AAV has been used to deliver antibodies that are significantly diverged from germ line or contain unusual features (1,14,28,30,31).…”

Section: Discussionmentioning

confidence: 99%

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV

et al. 2020

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Long-term delivery of anti-HIV monoclonal antibodies using adeno-associated virus (AAV) holds promise for the prevention and treatment of HIV infection. We previously reported that after receiving a single administration of AAV vector coding for anti-SIV antibody 5L7, monkey 84-05 achieved high levels of AAV-delivered 5L7 IgG1 in vivo which conferred sterile protection against six successive, escalating dose, intravenous challenges with highly infectious, highly pathogenic SIVmac239, including a final challenge with 10 animal infectious doses (1). Here we report that monkey 84-05 has successfully maintained 240-350 µg/ml of anti-SIV antibody 5L7 for over 6 years. Approximately 2% of the circulating IgG in this monkey is this one monoclonal antibody. This monkey generated little or no anti-drug antibodies (ADA) to the AAV-delivered antibody for the duration of the study. Due to the nature of the high-dose challenge used and in order to rule out a potential low-level infection not detected by regular viral loads, we have used ultrasensitive techniques to detect cell-associated viral DNA and RNA in PBMCs from this animal. In addition, we have tested serum from 84-05 by ELISA against overlapping peptides spanning the whole envelope sequence for SIVmac239 (PepScan) and against recombinant p27 and gp41 proteins. No reactivity has been detected in the ELISAs indicating the absence of naturally arising anti-SIV antibodies; moreover, the ultrasensitive cell-associated viral tests yielded no positive reaction. We conclude that macaque 84-05 was effectively protected and remained uninfected. Our data show that durable, continuous antibody expression can be achieved after one single administration of AAV and support the potential for lifelong protection against HIV from a single vector administration.

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“…They are large outbred animals that exhibit the severe bleeding phenotype including spontaneous bleeds that is the sine qua non of hemophilia. The immunogenicity of novel therapeutics can also be evaluated in a species-specific system (41,65,66). Dose, safety, and efficacy can all be evaluated in the canine model.…”

Section: Discussionmentioning

confidence: 99%

“…Two HA dogs received AAV8-cFVIII-ΔF with a liver-specific promoter via the cephalic vein diluted in PBS over a 30-minute period. Prior to gene therapy, both animals were confirmed to have anti-AAV8 neutralizing antibody titers less than 1:3 (65). WBCT and cFVIII antigen and activity were assayed as previously described (41).…”

Section: Methodsmentioning

confidence: 99%

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

et al. 2016

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Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645-1648) in the design of B-domain-deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector-based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.

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Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs

Cited by 27 publications

References 39 publications

The Immune Response to the fVIII Gene Therapy in Preclinical Models

The Immune Response to the fVIII Gene Therapy in Preclinical Models

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

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