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.
Abstract
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.
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 × 1012 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.
•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.
Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle ...fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6–8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients.
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Comparisons of CRISPR/Cas9 gene editing and micro-dystrophin gene replacement in old dystrophic dogs highlight the promise and limitations of AAV-mediated gene therapy in older subjects with advanced muscle pathology.
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.
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the
gene. Although mouse models of DMD provide ...insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (μDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups control, 1 × 10
vector genomes per kilogram (vg/kg), 1 × 10
vg/kg, and 2 × 10
vg/kg;
= 3 each, treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-μDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; μDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.
Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This ...belief prompted the retention of the furin recognition motif (amino acids 1645-1648) in the design of B-domain-deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector-based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.
Recent success in AAV liver gene therapy for hemophilia A (HA) and hemophilia B (HB) has demonstrated that the lower the vector dose, the better the safety profile. Strategies to enhance vector ...performance currently being investigated in clinical trials include the use of gain of function clotting factor variants such as Factor IX (FIX) Padua (R338L), a hyperactive variant with an activity to antigen ratio ~8 fold higher than FIX wild type (WT). However, early studies in HB mice suggested that immune tolerance induction following AAV liver gene therapy required FIX antigen levels above a certain threshold (Mingozzi et al . JCI 2003). These results raise the concern that lower antigen levels with gain of function FIX variants could increase the risk of an anti-FIX immune response, including inhibitor development. Inhibitor development is associated with a substantial increase in mortality and morbidity and remains a major safety concern for any new HB therapeutic.
To evaluate this potential risk in a large animal model, we have undertaken a study in adult dogs with severe HB. Two canine (c) models with different underlying F9 gene mutations (missense (MS) and null) and distinct risks of inhibitor development received AAV liver- or skeletal muscle-mediated expression of cFIX Padua. A naïve MS HB dog was treated with a sub-therapeutic dose of AAV-cFIX-Padua for skeletal muscle gene therapy to investigate the role of antigen level in transgene immune tolerance. This strategy resulted in cFIX activity level of only ~1% and undetectable cFIX antigen levels (assay sensitivity ~0.3%, 15 ng/mL). However, even this minute antigen level was sufficient to maintain immune tolerance despite several treatments with cFIX-WT protein for bleeding. In addition, we have previously treated 3 MS HB dogs from the same colony with higher vector doses and achieved cFIX activity levels of 3.5-8% and antigen levels of 0.4-1.5%. No FIX antibody was detected in these animals, even after further challenges with cFIX-WT protein.
We have reported that liver-directed AAV-cFIX-Padua gene therapy can eradicate a pre-existing anti-cFIX immune response and induce immune tolerance in an inhibitor-prone null HB dog (Wiley) (Crudele et al . Blood 2015). This immune tolerance has been maintained despite multiple challenges with recombinant cFIX protein. Here we report eradication of a pre-existing anti-cFIX immune response in a second inhibitor-prone HB dog (Otis) with similar gene therapy. Both dogs had detectable cFIX antigen levels within 1 week of vector delivery (1-1.5%), but low activity to antigen ratios for cFIX Padua (<1), consistent with circulating immune complexes of FIX and antibody, as is observed in HB inhibitor patients (Nilsson et al. PNAS 1986). Wiley's activity to antigen ratio only normalized after inhibitor eradication. Interestingly, Otis was challenged with recombinant cFIX-WT protein after his inhibitor titer was ≤0.6 BU and his FIX-specific IgG1 and IgG2 were baseline, but his cFIX activity to antigen ratio was still <1. This resulted in an anamnestic response with substantial increases in the cFIX-IgG1, but not IgG2 (equivalent to human IgG4). Importantly, continued expression of cFIX Padua ultimately resulted in the disappearance of the cFIX-IgG1, as well as the normalization of his cFIX Padua activity to antigen ratio. Neither Wiley nor Otis has bled since gene therapy. The experience of Otis suggests that decreased activity to antigen ratio is a marker for a continued anti-FIX immune response, even in the absence of other laboratory findings, likely due to immune complexes. After eradication, these dogs expressed cFIX-Padua antigen at ~10% with the expected activity to antigen ratio of ~8 for ≥3.5 years.
Combined these results support the concept that the continuous uninterrupted transgene expression after gene therapy, even at undetectable antigen levels, can prevent and/or eradicate inhibitors. These data are in agreement with the results of the International Immune Tolerance Induction trial for HA patients with inhibitors, where the rates of inhibitor eradication at a 3 year period were similar between high- and low-dose cohorts (Hay et al . Blood 2012). Overall, this work supports the emerging understanding that gene therapy utilizing gain of function variants for inherited protein deficiencies can simultaneously promote immune tolerance and provide therapeutic activity levels.
No relevant conflicts of interest to declare.
The paired basic amino acid cleaving enzyme (PACE)/Furin is a protein convertase system that plays a vital role in several biological processes, including coagulation. The propeptide processing of ...human FIX by PACE/Furin is a critical posttranslational modification, so cells co-expressing PACE/Furin and FIX are used for production of clinical recombinant protein. In the development of recombinant B-domain deleted (BDD) FVIII for hemophilia A (HA), a 14 amino acid B-domain sequence containing a putative cleavage site for PACE/Furin was retained because it was believed to be critical for intracellular processing and secretion. In contrast to FIX, we report here a surprising detrimental effect of PACE/Furin in FVIII activity and intracellular processing and secretion.
We engineered a human FVIII variant where the PACE/Furin site at residues 1645-1648 was deleted from FVIII-BDD (FVIII-ΔP/F). Notably, FVIII-ΔP/F exhibits a 3-fold increased activity over FVIII-BDD (p=0.0004) in a 2-stage APTT assay. Moreover, the A2-domain dissociation of activated FVIII-ΔP/F was also 3-fold longer compared to FVIII-BDD, suggesting a more stable activated FVIII molecule. The amount of FVIII secreted from stably transduced BHK cells was about 3-fold higher for FVIII-ΔP/F than for FVIII-BDD. Conversely, the amount of intracellular FVIII antigen was lower for FVIII-ΔP/F than for FVIII-BDD.
To confirm that PACE/Furin was implicated in the underlying mechanisms for the observed differences in FVIII secretion, we inhibited PACE/Furin in FVIII-BDD producing BHKs by transducing them with vectors expressing an engineered α1-antitrypsin variant (haat-PDX) that specifically inhibits PACE/Furin. This resulted in a 40% increase in FVIII secretion (p=0.017) and a decrease in intracellular FVIII-BDD, whereas transduction with the haat-wild type control, which does not inhibit PACE/Furin, did not significantly change the amount of FVIII secreted (p=0.32). Importantly, the secretion and intracellular levels of FVIII-ΔP/F were not affected by the inhibition of PACE/Furin by haat-PDX, indicating that the secretion of this FVIII variant does not benefit from further inhibition of PACE/Furin cleavage. Together these data suggest that the increased secretion of FVIII-ΔP/F compared to FVIII-BDD is due to the former circumventing PACE/Furin.
Furin is ubiquitously expressed in mammal tissues. In a stringent cellular model, we used LoVo, a unique human cell line that lacks functional Furin to determine whether expression of FVIII-ΔP/F and FVIII-BDD would differ, as we have observed in cells expressing Furin. Interestingly, the secretion of FVIII-ΔP/F and FVIII-BDD were comparable. This result confirms that FVIII-BDD is secreted better in the absence of Furin. In summary, our novel variant FVIII-ΔP/F exhibits enhanced secretion primarily by bypassing PACE/Furin cleavage; inhibiting this cellular process also enhances the secretion of FVIII. Futhermore in vivo experiments also demonstrated a beneficial effect of FVIII-ΔP/F: HA mice (n=4-7/dose) given adeno-associated viral 8 (AAV8) vectors for liver gene expression of FVIII-ΔP/F resulted in a 3-fold higher circulating FVIII levels than FVIII-BDD-expressing mice (p=0.025).
These exciting results from human FVIII-ΔP/F prompt us to test this variant HA canine model. First we found that recombinant canine FVIII with the entire PACE/Furin site deleted (cFVIII-ΔP/F) had increased activity in a 2-stage aPTT assay compared to wild-type cFVIII-BDD. Injection of cFVIII-ΔP/F effectively corrects the hemophilia coagulopathy in two HA dogs. Further, AAV8 liver gene therapy with cFVIII-ΔP/F in additional two HA dogs at doses of ~6 x 1012 vg/kg, a log lower than previously used for canine FVIII-BDD AAV8 gene therapy, resulted in therapeutic levels of cFVIII and shortening of clotting times. Preliminary data on injection of cFVIII-BDD protein was well tolerated in cFVIII-ΔP/F-expressing dogs.
In conclusion, these data suggest that PACE/Furin cleavage of FVIII hampers protein biological activity. FVIII variants lacking PACE/Furin recognition sequences are secreted more efficiently and exhibit improved hemostatic effects in both protein- and gene-based strategies. Inhibition of PACE/Furin in manufacturing systems for recombinant human FVIII may increase the yields of protein production. Thus these strategies have a strong rationale for translation to HA therapy.
No relevant conflicts of interest to declare.
Emerging data from early phase clinical studies of AAV gene therapy for hemophilia B (HB) (factor IX FIX deficiency) show sustained expression of therapeutic levels of FIX and phenotypic improvement. ...However, the safety and efficacy of in vivo gene therapy is limited by the vector dose. Recently, we reported a naturally occurring, hyperfunctional FIX (FIX Padua) caused by a single amino acid change of arginine 338 to leucine that exhibits an 8-fold increase in specific activity in humans (N Engl J Med 2009), making it a potential candidate for HB gene therapy with reduced vector doses. However, to take advantage of FIX Padua for HB gene therapy, it is critical to first define the risk of immunogenicity of this variant in preclinical models of severe HB.
We have previously shown that delivery of AAV-cFIX-Padua to skeletal muscle in HB dogs with a missense mutation in the canine (c) F9 gene resulted in no anti-FIX neutralizing antibodies (inhibitors), non-neutralizing antibodies (IgG) or FIX-specific T-cell response (Blood 2012). While promising, these dogs express FIX RNA and have a pre-existing tolerance to cFIX due to the nature of their mutation, and so do not represent the most rigorous model for immunogenicity studies.
Here, we tested the efficacy and immunogenicity of cFIX Padua in a severe HB dog colony with an early stop codon mutation. This mutation results in no FIX RNA transcript, and the dogs are prone to develop cFIX inhibitors upon exposure to protein concentrates. Three dogs were infused peripherally with a liver-specific AAV8-cFIX-Padua at two different doses, and monitored for cFIX antigen and activity levels and inhibitors. The first dog, which received 3 x 1012 vg/kg, showed average plateaued expression levels of 3.98 ± 1.44% antigen and 24.5 ± 4.1% activity, with no development of anti-cFIX inhibitors or IgG antibodies. Whole blood clotting time (WBCT) and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >20 months (ongoing observations). A second dog was treated with a lower dose of 1 x 1012 vg/kg and showed average plateaued expression levels of 2.41 ± 0.05% antigen and 22.0 ± 0.4% activity, with no development of anti-cFIX inhibitors or IgG antibodies. WBCT and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >3 months.
An additional dog, upon previous exposure to recombinant human (h) FIX protein, had developed inhibitors that cross-reacted with cFIX. This immune response was ongoing at the time of vector administration (3 x 1012 vg/kg). Anti-cFIX antibodies peaked at day 14 post-AAV, with 4.7 BUs and 3643 ng/mL IgG2, but dropped to undetectable levels by day 70. There was a concurrent rise in cFIX Padua expression levels, suggesting successful tolerization to the cFIX Padua. Antigen levels plateaued at 14.6 ± 4.3% and activity at 51.7 ± 23.5%, with ongoing normalization of WBCT and aPTTs for >18 months. In all three dogs, cholesterol, albumin and total protein were within normal limits with no clinical or laboratory evidence of nephrotic syndrome (a potential complication in FIX inhibitor patients that have undergone immune tolerance induction with frequent FIX protein injections).
The safety of FIX Padua was further confirmed using a mouse model of HB. Mice (n=8-12/group) were treated with 5 x 1010 vg/kg liver-directed AAV8-hFIX-WT or AAV8-hFIX-Padua, resulting in expression levels of 1076 ± 343 ng/mL (21.5 ± 6.9% antigen, 67.5 ± 10.1% activity) and 797 ± 255 ng/mL (15.9 ± 5.1% antigen, 274.8 ± 73.8% activity), respectively. In cross-over experiments, the mice were then were immunologically challenged 10-15 weeks after gene delivery with 100 ug/kg of the reciprocal recombinant protein (ie mice expressing hFIX Padua were challenged with hFIX WT, and visa versa). Challenges were administered subcutaneously alone or with adjuvant (CFA) weekly for 4 weeks. In no instance did mice develop antibodies to either FIX, suggesting that tolerance was successfully induced in all cases.
Together, these date show that FIX Padua shows no increase in immunogenicity compared to FIX WT and is capable not only of preventing inhibitor formation, but also of eradicating pre-existing inhibitory antibodies to FIX in an inhibitor-prone HB dog model. Thus, FIX Padua is an attractive transgene that will allow for decreased vector doses in human HB gene therapy, improving the safety profile of AAV liver gene therapy without increased immunogenicity.
High:Alnylam Pharmaceuticals: Consultancy; BioMarin: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity's Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy.
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