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Factor VIII (FVIII) is a large glycoprotein that is challenging to express both and . Several studies suggest that high levels of FVIII expression can lead to cellular stress. After gene transfer, transgene expression is restricted to a subset of cells and the increased FVIII load per cell may impact activation of the unfolded protein response. We sought to determine whether increased FVIII expression in mice after adeno-associated viral liver gene transfer would affect the unfolded protein response and/or immune response to the transgene. The FVIII gene was delivered as B-domain deleted single chain or two chain (light and heavy chains) at a range of doses in hemophilia A mice. A correlation between FVIII expression and anti-FVIII antibody titers was observed. Analysis of key components of the unfolded protein response, binding immunoglobulin protein (BiP), and C/EBP homologous protein (CHOP), showed transient unfolded protein response activation in the single chain treated group expressing >200% of FVIII but not after two chain delivery. These studies suggest that supraphysiological single chain FVIII expression may increase the likelihood of a cellular stress response but does not alter liver function. These data are in agreement with the observed long-term expression of FVIII at therapeutic levels after adeno-associated viral delivery in hemophilia A dogs without evidence of cellular toxicity.

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9–11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity. AAV therapy in dogs leads to clonal expansions of transduced cells.

Developing adeno-associated viral (AAV)–mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose–dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.

The major complication associated with protein replacement therapy currently used in the treatment of hemophilia B (HB) is the development of antibodies to the infused human Factor IX (hF.IX). We hypothesized that vector-mediated expression of hF.IX, either at a prenatal stage or early in life may lead to tolerance to hF.IX and long-term transgene expression. Fetal, neonatal, and adult F.IX-deficient mice were injected with AAV-1-hF.IX, and the hF.IX levels as well as antibodies to hF.IX in the circulation were assayed. In utero injection followed by postnatal re-administration of adeno-associated virus 1 (AAV-1) vector achieved persistent expression of hF.IX in all animals, with no cellular or humoral immune response to F.IX. Similar results were seen after initial injection in neonatal mice followed by re-administration, whereas all mice injected at the adult stage developed antibodies to hF.IX. In contrast, after administration of AAV-2-hF.IX in the neonatal period, antibodies to hF.IX were formed in all the injected animals. We conclude that in utero or neonatal-stage injection of AAV-1-hF.IX can lead to long-term expression and absence of immune response. The differences in immune response between the AAV-1 and AAV-2 groups suggests that tolerance may be related to differences in bio-distribution, timing of expression, and/or the initial levels of hF.IX expression. This supports the concept of a narrow “window of opportunity” for tolerance induction.

Hepatic adeno-associated virus (AAV)-serotype 2 mediatedgene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid–specific memory CD8 + T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid–specific CD8 + T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.

Adeno-associated virus has been developed for use as a gene transfer vector. To understand the impact of AAV capsid-specific CD8(+) T cells on AAV-mediated gene transfer, we identified CD8(+) T cell epitopes for AAV-2 and AAV-8 capsid in C57BL/6 (H-2(b) MHC haplotype) and BALB/c (H-2(d) MHC haplotype) mice. Mice of both the H-2(b) and the H-2(d) haplotypes recognized epitopes on AAV-2 and AAV-8 capsid. T cells from H-2(b) mice recognized an epitope that was conserved between AAV-2 and AAV-8 capsid. Cross-reactivity of AAV-specific CD8(+) T cells induced by different AAV serotypes may have important implications for gene transfer. Identification of these epitopes will facilitate studies of immune response to AAV capsid in mouse models.

Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked γ -globin gene in transgenic mice. We inserted the Ank/Aγ -globin gene into retrovirus vectors that could transfer one or two copies of the Ank/Aγ -globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/Aγ -globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse α -globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe β -thalassemia if the level of expression can be further increased.

Nine hemophilia A dogs were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9%−11.3% of normal FVIII levels. In two of nine dogs, FVIII activity increased gradually starting about four years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of these integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A while emphasizing the importance of long-term monitoring for potential genotoxicity. An AAV gene therapy study in hemophilia A dogs finds clonal expansions of transduced cells.

In a Phase I/II study of gene transfer for hemophilia B, an adeno-associated viral vector serotype 2 (AAV-2) was introduced into the liver of human subjects and therapeutic levels of transgene expression (up to 11% of normal) were reached. However, the duration of gene expression was limited; four weeks after infusion, levels of circulating factor IX (F.IX) began to decline, and returned to baseline by week 10. This phenomenon was accompanied by a mild, self-limited, increase in liver enzymes. After a similar phenomenon was observed in another patient, the clinical trial was halted. We hypothesized that T cells directed towards AAV capsid antigens harbored by transduced hepatocytes were activated and these mediated destruction of the transduced hepaotocytes, thereby causing loss of transgene expression and a transient transaminitis. We used IFNγ ELISpot analysis of peripheral blood mononuclear cells (PBMCs) from a subject in the clinical study, combined with bioinformatics tools for the prediction of MHC class I binders, to define an MHC Class I-restricted T cell epitope for an HLA type common in the general population (B*0702). We then designed pentamers in which five peptide-loaded MHC class I molecules are linked to a fluorochrome to detect and study AAV-specific B*0702-restricted CD8+ T cells. Indeed, in the HLA B*0702 subject who developed transaminitis after vector infusion, AAV-specific CD8+ T cells were detected by pentamer staining of PBMCs up to two years later. In contrast, we have not detected AAV-specific CD8+ T cells in the peripheral blood of normal donors, though after several rounds of ex vivo stimulation with AAV capsid or peptide epitopes, we have been able to enrich a population of AAV-specific T cells, suggesting that AAV-specific T cells exist in the T cell memory pool. Functional assays on expanded AAV-specific T cells have demonstrated that these T cells specifically produced IFNγ upon detection of AAV antigen and specifically lyse target cells presenting antigen in the context of the HLA B*0702. These data provide direct evidence that humans mount a cytotoxic immune response to AAV capsid proteins, and that a contracted pool of AAV-specific T cells can remain as memory T cells in normal donors. Thus, re-activation of an AAV-specific T cell response may account for the limited duration of transgene expression that we observed in our clinical study. Moreover, the use of alternate serotypes may not easily avoid this immune response, as T cells from subjects infused with AAV-2 showed functional responses to AAV-1 and AAV-8-derived peptides by both cytokine production and killing assays, and T cells from normal donors that were expanded with an AAV-2 derived epitope also cross-react with the homologous epitope from AAV-5 by pentamer staining. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.