Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy.
ABSTRACT 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.
- SourceAvailable from: George Dickson[Show abstract] [Hide abstract]
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; DOI:10.1089/hum.2013.164 · 3.62 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Formation of pathogenic antibodies is a major problem in replacement therapies for inherited protein deficiencies. For example, antibodies to coagulation factors ('inhibitors') seriously complicate treatment of haemophilia. While immune tolerance induction (ITI) protocols have been developed, inhibitors against factor IX (FIX) are difficult to eradicate due to anaphylactic reactions and nephrotic syndrome and thus substantially elevate risks for morbidity and mortality. However, hepatic gene transfer with an adeno-associated virus (AAV) serotype 8 vector expressing FIX (at levels of ≥4% of normal) rapidly reversed pre-existing high-titre inhibitors in haemophilia B mice, eliminated antibody production by B cells, desensitized from anaphylaxis (even if protein therapy was resumed) and provided long-term correction. High levels of FIX protein suppressed memory B cells and increased Treg induction, indicating direct and indirect mechanisms of suppression of inhibitor formation. Persistent presence of Treg was required to prevent relapse of antibodies. Together, these data suggest that hepatic gene transfer-based ITI provides a safe and effective alternative to eradicate inhibitors. This strategy may be broadly applicable to reversal of antibodies in different genetic diseases.EMBO Molecular Medicine 11/2013; 5(11). DOI:10.1002/emmm.201302859 · 8.25 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05ug DNA per ml of media (21ug DNA/15cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×10(8) to 2×10(9) IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×10(12) to 6×10(13) vg/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer.Journal of virological methods 06/2013; 193(2). DOI:10.1016/j.jviromet.2013.06.008 · 1.88 Impact Factor