Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

Division of Hematology and Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Blood (Impact Factor: 10.45). 01/2012; 119(13):3038-41. DOI: 10.1182/blood-2011-09-382317
Source: PubMed


In previous work we transferred a human factor IX-encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

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    • "Thus, by inducing higher constitutive SMAD3 levels in cardiovascular tissues, in particular, aortic smooth muscle cells (the main target of adeno-associated virus in arteries, AAV) [17], we should be able to enhance the effects of endogenous secreted TGFβ1. Adeno-associated virus (AAV), first investigated in 1984, is a useful tool for gene delivery to study gene function / therapeutic effect [18-21], and its expression is known to last at least 10 years in clinical trials [22]. The predicted amino acid (aa) sequence homology of mouse and human (h) SMAD3 is 99%, thus, hSMAD3 was the choice as a therapeutic gene. "
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    ABSTRACT: Background Inflammation is a key etiologic component in atherogenesis and transforming growth factor beta 1 (TGFß1) is a well known anti-inflammatory cytokine which potentially might be used to limit it. Yet TGFß1 is pleiomorphic, causing fibrosis, cell taxis, and under certain circumstances, can even worsen inflammation. SMAD3 is an important member of TGFß1¿s signal transduction pathway, but is a fully intracellular protein.Objectives With the hope of attenuating TGFß1¿s adverse systemic effects (eg. fibrosis) and accentuating its anti-inflammatory activity, we proposed the use of human (h)SMAD3 as an intracellular substitute for TGFß1.Study designTo test this hypothesis adeno-associated virus type 2/8 (AAV)/hSMAD3 or AAV/Neo (control) was tail vein injected into the low density lipoprotein receptor knockout (LDLR-KO) mice, then placed on a high-cholesterol diet (HCD).ResultsThe hSMAD3 delivery was associated with significantly lower atherogenesis as measured by larger aortic cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-treated controls. HSMAD3 delivery also resulted in fewer aortic macrophages by immunohistochemistry for CD68 and ITGAM, and quantitative reverse transcriptase polymerase chain reaction analysis of EMR and ITGAM. Overall, aortic cytokine expression showed an enhancement of Th2 response (higher IL-4 and IL-10); while Th1 response (IL-12) was lower with hSMAD3 delivery. While TGFß1 is often associated with increased fibrosis, AAV/hSMAD3 delivery exhibited no increase of collagen 1A2 or significantly lower 2A1 expression in the aorta compared with Neo-delivery. Connective tissue growth factor (CTGF), a mediator of TGFß1/SMAD3-induced fibrosis, was unchanged in hSMAD3-delivered aortas. In the liver, all three of these genes were down-regulated by hSMAD3 gene delivery.Conclusion These data strongly suggest that AAV/hSMAD3 delivery gave anti-atherosclerosis therapeutic effect without the expected undesirable effect of TGFß1-associated fibrosis.
    Full-text · Article · Sep 2014 · Journal of Translational Medicine
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    • "Therefore, as UGT1A1 is expressed at high levels in the liver but it is also expressed at lower levels in other organs such as intestine and kidney (Buckley and Klaassen, 2007), we considered the alternative possibility of expressing UGT1A1 in a surrogate tissue such as skeletal muscle, with the aim of improving the efficiency of the therapy. As mentioned above, targeting the skeletal muscle for gene therapy offers a series of advantages over a liver-directed gene therapy approach (Mingozzi and High, 2011; Buchlis et al., 2012). However, when we treated in parallel mutant mice with CMV-hUGT1A1 and AAT-hUGT1A1 AAV vectors , plasma bilirubin levels were much higher in CMV- hUGT1A1-treated mice than in AAT-hUGT1A1-treated ones, despite of the 4–6-fold higher muscle expression of hUGT1a1. "
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    ABSTRACT: Null mutations in the UGT1A1 gene result in Crigler-Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies we applied AAV-liver specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months post-injection. The therapeutic effect was mediated by the presence of transcriptionally active double stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver vs. skeletal muscle transgene expression. We observed that 5%-8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1±1.5 mg/dL). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4±2.0 mg/dL), despite 20-30% of hUgt1a1 expression levels, compared to normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters.
    Full-text · Article · Jul 2014 · Human Gene Therapy
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    • "Upon gene transfer myofibers are capable of producing biologically active material, and the first clinical trial on AAV-F.IX gene transfer utilized intramuscular injections at multiple skeletal muscle sites as the route of vector administration [16-19]. F.IX-expressing muscle fibers may persist in humans for at least 10 years after initial gene transfer [20]. However, a concern about muscle-directed gene transfer is the increased risk of immune responses against F.IX. Hence, in this study we chose the more immunogenic intramuscular route to assess the potential for B and T cell responses against F.IX as a function of the vector genome (scAAV vs ssAAV) and the underlying F9 gene mutation. "
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    ABSTRACT: Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX). Hemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The outcome of gene transfer was assessed, including transgene expression as well as antibody and CD8+ T cell responses to hF.IX. Self-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX expression) but stronger CD8+ T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a result, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9 nonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression regardless of the vector genome. The nature of the AAV genome can impact the CD8+ T cell response to the therapeutic transgene product. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break tolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific immunity than the molecular form of the AAV genome.
    Full-text · Article · Jan 2014 · Journal of Translational Medicine
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