• Liver-restricted expression of FIX-Padua induces immune tolerance to the transgene in hemophilia B inhibitor dog models.• Long-term toxicity studies show no increased risk of thrombogenicity of FIX-Padua in mice and dogs.Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose dependent. To decrease the vector dose, we explored AAV-8 (1-3 3 10 12 vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine)in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8-to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB. (Blood. 2015;125(10):1553-1561

Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients. (Blood. 2010;116(26): 5842-5848) IntroductionThe development of neutralizing antibodies to replacement protein is a major complication of protein and enzyme replacement therapies for several genetic diseases. Hemophilia A is an X-linked bleeding disorder characterized by deficiency in the activity of factor VIII (FVIII), a key component of the coagulation cascade. The disease occurs in approximately 1 in 10 000 live births worldwide, and Ͼ 40% of these patients have severe disease, with FVIII activity Ͻ 1% of normal. 1 Infusion of plasma-derived or recombinant FVIII is the standard treatment. Alloantibodies (inhibitors) that neutralize the protein-replacement therapy develop in 20% to 30% of young patients with severe and moderate hemophilia A, resulting in high morbidity and mortality, 2,3 and this is a growing problem for adults as well. 4,5 Risk factors for inhibitor formation include both genetic and environmental factors. Underlying mutations in the FVIII gene, such as large gene deletions, nonsense mutations, and the most common mutation in severe hemophilia A patients, the inversion of intron 22, are all associated with inhibitor formation; however, it is not possible to predict with certainty which patients will develop inhibitors. For this reason, preventive strategies are not currently feasible. [6][7][8] Patients with high titers of inhibitors, defined as Ͼ 5 Bethesda units (BU), cannot usually be treated with FVIII replacement, necessitating the use of products that bypass the procoagulant effect of FVIII and are extremely expensive. 1 Thus, strategies for the eradication of inhibitors are of fundamental clinical relevance.Currently, the only pro...

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a genome-editing technology1,2 that utilizes archaeal and bacterial Cas9 nucleases to introduce double-stranded breaks in DNA at targeted sites. These breaks can be used to remove, replace, or add pieces of DNA. While not the first genome editor, CRISPR-Cas9 is efficient and cost-effective because cutting is guided by a strand of RNA rather than a protein. The potential uses in health care are plentiful, from disrupting dominant genes that cause cancer3 to repairing mutated genes that cause genetic diseases, such as muscular dystrophy4. Therapeutic approaches based on this technology fill the preclinical pipeline, and rely on the use of viral vectors to deliver the Cas9 gene and guide RNA to a gene of interest. However, concerns regarding the safety and efficacy of CRISPR-Cas9 use in gene therapy remain. A pre-print released prior to peer review has recently underlined the question of whether immunological responses to Cas9 may negatively impact its clinical use5. Here we discuss the implications of this finding for the application of CRISPR/Cas in gene therapy.

Muscular dystrophy (MD) is a group of progressive genetic diseases affecting the musculature that are characterized by inflammatory infiltrates, necrosis and connective tissue and fat replacement of the affected muscles. Unfortunately, treatments do not exist for the vast majority of MD patients. Adeno-associated viral vector (AAV)-based gene therapy is thus emerging as a potential treatment for many types of MD. Treatments strategies based on AAV are being adapted for replacement of mutant disease-causing genes, knockdown of dominant disease-causing genes using antisense oligonucleotides or inhibitory RNAs, delivery of gene editing tools such as clustered regularly interspaced short palindromic repeats/Cas9 and effecting alterations in pre-mRNA splicing and by manipulating expression levels of modifier genes. Translational and clinical trial work focused on these types of AAV treatments for Duchenne MD, various limb girdle MDs, myotonic dystrophy 1, facioscapulohumeral MD, dysferlinopathies and congenital MDs are discussed here, with a focus on recent studies, pre-clinical large animal work and many promising ongoing and upcoming AAV clinical trials.