Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.

Safety of Same-Eye Subretinal Sequential Readministration of AAV2-hRPE65v2 in Non-human Primates.

We have demonstrated safe and effective subretinal readministration of recombinant adeno-associated virus serotype (rAAV) to the contralateral eye in large animals and humans even in the setting of preexisting neutralizing antibodies (NAbs). Readministration of AAV to the same retina may be desirable in order to treat additional areas of the retina not targeted initially or to boost transgene expression levels at a later time point. To better understand the immune and structural consequences of subretinal rAAV readministration to the same eye, we administered bilateral subretinal injections of rAAV2-hRPE65v2 to three unaffected non-human primates (NHPs) and repeated the injections in those same eyes 2 months later. Ophthalmic exams and retinal imaging were performed after the first and second injections. Peripheral blood monocytes, serum, and intraocular fluids were collected at baseline and post-injection time points to characterize the cellular and humoral immune responses. Histopathologic and immunohistochemical studies were carried out on the treated retinas. Ipsilateral readministration of AAV2-hRPE65v2 in NHPs did not threaten the ocular or systemic health through the time span of the study. The repeat injections were immunologically and structurally well tolerated, even in the setting of preexisting serum NAbs. Localized structural abnormalities confined to the outer retina and retinal pigmented epithelium (RPE) after readministration of the treatment do not differ from those observed after single or contralateral administration of an AAV carrying a non-therapeutic transgene in NHPs and were not observed in a patient treated with the nearly identical, FDA-approved, AAV2-hRPE65v2 vector (voretigene neparvovec-rzyl), suggesting NHP-specific abnormalities.

AKT3 Gene Transfer Promotes Anabolic Reprogramming and Photoreceptor Neuroprotection in a Pre-clinical Model of Retinitis Pigmentosa.

Mutations within over 250 known genes are associated with inherited retinal degeneration. Clinical success following gene-replacement therapy for congenital blindness due to RPE65 mutations establishes a platform for the development of downstream treatments targeting other forms of inherited ocular disease. Unfortunately, several challenges relevant to complex disease pathology and limitations of current gene-transfer technologies impede the development of related strategies for each specific form of inherited retinal degeneration. Here, we describe a gene-augmentation strategy that delays retinal degeneration by stimulating features of anabolic metabolism necessary for survival and structural maintenance of photoreceptors. We targeted two critical points of regulation in the canonical insulin/AKT/mammalian target of rapamycin (mTOR) pathway with AAV-mediated gene augmentation in a mouse model of retinitis pigmentosa. AAV vectors expressing the serine/threonine kinase, AKT3, promote dramatic preservation of photoreceptor numbers, structure, and partial visual function. This protective effect was associated with successful reprogramming of photoreceptor metabolism toward pathways associated with cell growth and survival. Collectively, these findings underscore the importance of AKT activity and downstream pathways associated with anabolic metabolism in photoreceptor survival and maintenance.

CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters.

Validation of gene transfer vectors containing tissue-specific promoters in cell-based functional assays poses a formidable challenge for gene therapy product development. Here, we describe a novel approach based on CRISPR/dCas9 transcriptional activation to achieve robust transgene expression from transgene cassettes containing tissue or cell type-specific promoters after infection with AAV vectors in cell-based systems. Guide RNA sequences targeting two promoters that are highly active within mammalian photoreceptors were screened in a novel promoter activation assay. Using this screen, we generated and characterized stable cell lines that co-express dCas9.VPR and top-performing guide RNA candidates. These cells exhibit potent activation of proviral plasmids after transfection or after infection with AAV vectors delivering transgene cassettes carrying photoreceptor-specific promoters. In addition, we interrogated mechanisms to optimize this platform through the addition of multiple guide RNA sequences and co-expression of the universal adeno-associated virus receptor (AAVR). Collectively, this investigation identifies a rapid and broadly applicable strategy to enhance in vitro expression and to evaluate potency of AAV vectors that rely upon cell or tissue-specific regulatory elements.

Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1-9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons.

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs.

Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1-2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs.

Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII.

Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (>10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have <1% FVII activity. By western blot, we show that they have undetectable plasmatic antigen, thus representing the most prevalent type of human FVII deficiency (low antigen/activity). In these dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for >1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, d-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only large-animal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency.

In vivo genome editing of the albumin locus as a platform for protein replacement therapy.

Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice.

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 × 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.

CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome.

Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8+ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells. Our results clearly show that depending on the vectors' genome, CD8+ T cells can detect such epitopes on AAV8's capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.

Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery.

Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

Robust ZFN-mediated genome editing in adult hemophilic mice.

Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.

Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

Overcoming preexisting humoral immunity to AAV using capsid decoys.

Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models.

Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.

AAV-mediated expression of an ADAMTS13 variant prevents shigatoxin-induced thrombotic thrombocytopenic purpura.

Severe deficiency of plasma ADAMTS13 activity causes thrombotic thrombocytopenic purpura (TTP), a life-threatening syndrome for which plasma is the only effective therapy currently available. As much as 5% of TTP cases are hereditary, resulting from mutations of the ADAMTS13 gene. Here, we report the efficacy and safety of recombinant adeno-associated virus serotype 8 (AAV8)-mediated expression of a murine ADAMTS13 variant (MDTCS), truncated after the spacer domain, in a murine model of TTP. Administration of AAV8-hAAT-mdtcs at doses greater than 2.6 × 10(11) vg/kg body weight resulted in sustained expression of plasma ADAMTS13 activity at therapeutic levels. Expression of the truncated ADAMTS13 variant eliminated circulating ultralarge von Willebrand factor multimers, prevented severe thrombocytopenia, and reduced mortality in Adamts13(-/-) disease-prone mice triggered by shigatoxin-2. These data support AAV vector-mediated expression of a comparable truncated ADAMTS13 variant as a novel therapeutic approach for hereditary TTP in humans.

Treatment of diabetes and long-term survival after insulin and glucokinase gene therapy.

Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a "glucose sensor" in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant.

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.