Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy

Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, AL, USA.
Blood (Impact Factor: 10.45). 11/2008; 113(4):797-806. DOI: 10.1182/blood-2008-10-181479
Source: PubMed


Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.

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    • "Unlike the AAV vector capsid, which is not synthesized by infected cells, the transgene product is expressed for a long time after target tissue transduction (37–39). Vector-encoded transgene product may be recognized as a foreign antigen, especially if the recipient of gene transfer is not tolerant to the protein encoded by the vector, thus triggering immune responses that can result in production of transgene-specific neutralizing antibodies (40) or triggering of T cell responses directed against transgene-expressing transduced cells (41). "
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    ABSTRACT: Adeno-associated virus (AAV) vectors are one of the most efficient in vivo gene delivery platforms. Over the past decade, clinical trials of AAV vector-mediated gene transfer led to some of the most exciting results in the field of gene therapy and, recently, to the market approval of an AAV-based drug in Europe. With clinical development, however, it became obvious that the host immune system represents an important obstacle to successful gene transfer with AAV vectors. In this review article, we will discuss the issue of cytotoxic T cell responses directed against the AAV capsid encountered on human studies. While over the past several years the field has acquired a tremendous amount of information on the interactions of AAV vectors with the immune system, a lot of questions are still unanswered. Novel concepts are emerging, such as the relationship between the total capsid dose and the T cell-mediated clearance of transduced cells, the potential role of innate immunity in vector immunogenicity highlighted in preclinical studies, and the cross talk between regulatory and effector T cells in the determination of the outcome of gene transfer. There is still a lot to learn about immune responses in AAV gene transfer, for example, it is not well understood what are the determinants of the kinetics of activation of T cells in response to vector administration, why not all subjects develop detrimental T cell responses following gene transfer, and whether the intervention strategies currently in use to block T cell-mediated clearance of transduced cells will be safe and effective for all gene therapy indications. Results from novel preclinical models and clinical studies will help to address these points and to reach the important goal of developing safe and effective gene therapy protocols to treat human diseases.
    Frontiers in Immunology 07/2014; 5:350. DOI:10.3389/fimmu.2014.00350
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    • "Since mice and dogs do not have pre-existing immunity to AAV, correction in coagulation actor IX (FIX) deficient animal studies have been sustained without concern of immune responses (5–7). Unfortunately, the lack of immunity in pre-clinical studies did not fully reflect clinical results. "
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    ABSTRACT: Transitioning to human trials from preclinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL) responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity, and explored methods for bypassing these responses. Many efforts towards measuring innate immunity have utilized Toll-Like Receptor (TLR) deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody (NAb) assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T cells as well as cytotoxicity towards AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T cell proliferation, but actual transgene level reduction and parameters of cytotoxicity towards transduced target cells has only been shown in one model. The model utilized adoptive transfer of capsid specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells has also been explored as well as modulating t
    Frontiers in Immunology 02/2014; 5:28. DOI:10.3389/fimmu.2014.00028
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    • "Importantly for application for gene therapies, recombinant AAV6 (rAAV6) and in particular rAAV8 and 9 vectors have been shown to transduce both cardiac and skeletal muscle at high efficiencies after systemic gene transfer, allowing the potential for bodywide gene transfer (Gregorevic et al., 2004; Wang et al., 2005; Foster et al., 2008; Zincarelli et al., 2008). A number of studies have also demonstrated very stable gene expression: over 8 years in canine models after systemic injection of rAAV2 (Niemeyer et al., 2009) and 6 years in nonhuman primate models after intramuscular injection of rAAV2/1 (Rivera et al., 2005). These factors, along with the apparent lack of immunogenicity to both transgene and virus in animal models, have made rAAV vectors very promising facilitators of genetic delivery in gene therapy. "
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    ABSTRACT: Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle following systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by DMD gene mutations leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (11.1 kb). A series of truncated micro-dystrophin cDNAs (delivered via a single AAV) and mini-dystrophin cDNAs (delivered via dual AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein (DAP) complex, such as neuronal nitric oxide synthase (nNOS), syntrophin and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilise membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single AAV packaging capacity (4.7 kb). Here we develop a novel method for the delivery of full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple AAV trans-splicing vector system. We report for the first time that three independent AAV vectors carrying 'in tandem' sequential exonic parts of the human DMD coding sequence, enable the expression of the full-length protein as a result of trans-splicing events co-joining three vectors via their inverted terminal repeat (ITR) sequences. This method of triple AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (≥11kb ORF) into post-mitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases.
    Human gene therapy 11/2013; 25(2). DOI:10.1089/hum.2013.164 · 3.76 Impact Factor
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