High value of 64Cu as a tool to evaluate the restoration of physiological copper excretion after gene therapy in Wilson's disease.

Wilson's disease (WD) is an inherited disorder of copper metabolism associated with mutations in ATP7B gene. We have shown that the administration of an adeno-associated vector (AAV) encoding a mini version of human ATP7B (VTX-801) provides long-term correction of copper metabolism in a murine WD model. In preparation of a future clinical trial, we have evaluated by positron emission tomography (PET) the value of 64Cu biodistribution, excretion pattern, and blood kinetics as pharmacodynamic biomarkers of VTX-801 effects. Six-week-old WD mice were injected intravenously with increasing doses of VTX-801 and 3 weeks or 3 months later with [64Cu]CuCl2. Untreated WD and wild-type (WT) mice were included as controls. Control WD mice showed increased hepatic 64Cu retention, reduced fecal excretion of the radiotracer, and altered 64Cu blood kinetics (BK) compared with WT mice. VTX-801 treatment in WD mice resulted in a significant reduction of hepatic 64Cu accumulation, the restoration of fecal 64Cu excretion, and the correction of 64Cu BK. This study showed that VTX-801 restores physiological copper metabolism in WD mice, confirming the mechanism of action of VTX-801, and demonstrated the translational potential of [64Cu]CuCl2-PET to explore VTX-801 pharmacodynamics in a minimally invasive and sensitive manner in WD patients.

Optimising the IgG-degrading enzyme treatment regimen for enhanced adeno-associated virus transduction in the presence of neutralising antibodies.

OBJECTIVE: Pre-existing neutralising antibodies (NAbs) to adeno-associated viruses (AAVs) remain an impediment for systemically administered AAV-mediated gene therapy treatment in many patients, and various strategies are under investigation to overcome this limitation. Here, IgG-degrading enzymes (Ides) derived from bacteria of the genus Streptococcus were tested for their ability to cleave human IgG and allow AAV-mediated transduction in individuals with pre-existing NAbs. METHODS: Cleavage activity of three different Ides was evaluated in vitro in serum from different species. Passively immunised mice or non-human primates (NHP) with naturally occurring anti-AAV NAbs were used to define the optimal IdeS dose and administration window for AAVAnc80 and AAV8 vectors in mice and AAV3B in NHPs. RESULTS: The selected candidate, IdeS, was found to be highly efficient at cleaving human IgG, less efficient against NHP IgG and inefficient against mouse IgG. In vivo, we observed differences in how IdeS affected liver transduction in the presence of NAbs depending on the AAV serotype. For AAVAnc80 and AAV3B, the best transduction levels were achieved when the vector was administered after IgG digestion products were cleared from circulation. However, for AAV8 we only observed a modest and transient inhibition of transduction by IdeS cleavage products. CONCLUSION: Preconditioning with IdeS represents a unique treatment opportunity for patients primarily excluded from participation in gene therapy clinical trials because of elevated circulating anti-AAV NAb levels. However, careful determination of the optimal IdeS dose and timing for the administration of each AAV serotype is essential for optimal transduction.

Use of an adeno-associated virus serotype Anc80 to provide durable cure of phenylketonuria in a mouse model.

Phenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno-associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon-optimized human PAH gene, with or without an intron spacer, to the Pahenu2 mouse model of PKU. Dose-dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pahenu2 mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 × 1012 VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV-dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki-67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration.

Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates.

Vision restoration is an ideal medical application for optogenetics, because the eye provides direct optical access to the retina for stimulation. Optogenetic therapy could be used for diseases involving photoreceptor degeneration, such as retinitis pigmentosa or age-related macular degeneration. We describe here the selection, in non-human primates, of a specific optogenetic construct currently tested in a clinical trial. We used the microbial opsin ChrimsonR, and showed that the AAV2.7m8 vector had a higher transfection efficiency than AAV2 in retinal ganglion cells (RGCs) and that ChrimsonR fused to tdTomato (ChR-tdT) was expressed more efficiently than ChrimsonR. Light at 600 nm activated RGCs transfected with AAV2.7m8 ChR-tdT, from an irradiance of 1015 photons.cm-2.s-1. Vector doses of 5 × 1010 and 5 × 1011 vg/eye transfected up to 7000 RGCs/mm2 in the perifovea, with no significant immune reaction. We recorded RGC responses from a stimulus duration of 1 ms upwards. When using the recorded activity to decode stimulus information, we obtained an estimated visual acuity of 20/249, above the level of legal blindness (20/400). These results lay the groundwork for the ongoing clinical trial with the AAV2.7m8 - ChR-tdT vector for vision restoration in patients with retinitis pigmentosa.

Gene therapy for progressive familial intrahepatic cholestasis type 3 in a clinically relevant mouse model.

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare monogenic disease caused by mutations in the ABCB4 gene, resulting in a reduction in biliary phosphatidylcholine. Reduced biliary phosphatidylcholine cannot counteract the detergent effects of bile salts, leading to cholestasis, cholangitis, cirrhosis and ultimately liver failure. Here, we report results from treating two- or five-week-old Abcb4-/- mice with an AAV vector expressing human ABCB4, resulting in significant decreases of PFIC3 disease biomarkers. All male mice achieved a sustained therapeutic effect up through 12 weeks, but the effect was achieved in only 50% of females. However, two-week-old females receiving a second inoculation three weeks later maintained the therapeutic effect. Upon sacrifice, markers of PFIC3 disease such as, hepatosplenomegaly, biliary phosphatidylcholine and liver histology were significantly improved. Thus, AAV-mediated gene therapy successfully prevented PFIC3 symptoms in a clinically relevant mouse model, representing a step forward in improving potential therapy options for PFIC3 patients.

Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹° vector genomes/ml; 95% CI, 2.70 x 10¹° to 4.75 x 10¹° vector genomes/ml), transducing units ({X}, 5.09 x 108 transducing units/ml; 95% CI, 2.00 x 108 to 9.60 x 108 transducing units/ml), and infectious units ({X}, 4.37 x 109 TCID50 IU/ml; 95% CI, 2.06 x 109 to 9.26 x 109 TCID50 IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.

Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.

Therapeutic gene delivery to the whole spinal cord is a major challenge for the treatment of motor neuron (MN) diseases. Systemic administration of viral gene vectors would provide an optimal means for the long-term delivery of therapeutic molecules from blood to the spinal cord but this approach is hindered by the presence of the blood-brain barrier (BBB). Here, we describe the first successful study of MN transduction in adult animals following intravenous (i.v.) delivery of self-complementary (sc) AAV9 vectors (up to 28% in mice). Intravenous MN transduction was achieved in adults without pharmacological disruption of the BBB and transgene expression lasted at least 5 months. Importantly, this finding was successfully translated to large animals, with the demonstration of an efficient systemic scAAV9 gene delivery to the neonate and adult cat spinal cord. This new and noninvasive procedure raises the hope of whole spinal cord correction of MN diseases and may lead to the development of new gene therapy protocols in patients.

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis.

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

Major subsets of human dendritic cells are efficiently transduced by self-complementary adeno-associated virus vectors 1 and 2.

Dendritic cells (DC) are antigen-presenting cells pivotal for inducing immunity or tolerance. Gene transfer into DC is an important strategy for developing immunotherapeutic approaches against infectious pathogens and cancers. One of the vectors previously described for the transduction of human monocytes or DC is the recombinant adeno-associated virus (rAAV), with a genome conventionally packaged as a single-stranded (ss) molecule. Nevertheless, its use is limited by the poor and variable transduction efficiency of DC. In this study, AAV type 1 (AAV1) and AAV2 vectors, which expressed the enhanced green fluorescent protein and were packaged as ss or self-complementary (sc) duplex strands, were used to transduce different DC subsets generated ex vivo and the immunophenotypes, states of differentiation, and functions of the subsets were carefully examined. We show here for the first time that a single exposure of monocytes (M(o)) or CD34(+) progenitors (CD34) to sc rAAV1 or sc rAAV2 leads to high transduction levels (5 to 59%) of differentiated M(o)-DC, M(o)-Langerhans cells (LC), CD34-LC, or CD34-plasmacytoid DC (pDC), with no impact on their phenotypes and functional maturation of these cells, compared to those of exposure to ss rAAV. Moreover, we show that all these DC subpopulations can also be efficiently transduced after commitment to their differentiation pathways. Furthermore, these DC subsets transduced with sc rAAV1 expressing a tumor antigen were potent activators of a CD8(+)-T-cell clone. Altogether, these results show the high potential of sc AAV1 and sc AAV2 vectors to transduce ex vivo conventional DC, LC, or pDC or to directly target them in vivo for the design of new DC-based immunotherapies.

Reversal of type 1 diabetes by engineering a glucose sensor in skeletal muscle.

Type 1 diabetic patients develop severe secondary complications because insulin treatment does not guarantee normoglycemia. Thus, efficient regulation of glucose homeostasis is a major challenge in diabetes therapy. Skeletal muscle is the most important tissue for glucose disposal after a meal. However, the lack of insulin during diabetes impairs glucose uptake. To increase glucose removal from blood, skeletal muscle of transgenic mice was engineered both to produce basal levels of insulin and to express the liver enzyme glucokinase. After streptozotozin (STZ) administration of double-transgenic mice, a synergic action in skeletal muscle between the insulin produced and the increased glucose phosphorylation by glucokinase was established, preventing hyperglycemia and metabolic alterations. These findings suggested that insulin and glucokinase might be expressed in skeletal muscle, using adeno-associated viral 1 (AAV1) vectors as a new gene therapy approach for diabetes. AAV1-Ins+GK-treated diabetic mice restored and maintained normoglycemia in fed and fasted conditions for >4 months after STZ administration. Furthermore, these mice showed normalization of metabolic parameters, glucose tolerance, and food and fluid intake. Therefore, the joint action of basal insulin production and glucokinase activity may generate a "glucose sensor" in skeletal muscle that allows proper regulation of glycemia in diabetic animals and thus prevents secondary complications.

Comparative efficacy of intratracheal adeno-associated virus administration to newborn rats.

Transient local overexpression of genes that promote lung defense or repair may help to protect or promote alveolar development in premature neonates. We showed that the use of adenoviral vectors in neonates was limited by the induction of lung growth disorders. In the present work we compare the efficiency of gene transfer to the neonatal lung by three adeno-associated viral vectors: rAAV1, rAAV2, and rAAV5. Transduction efficiency was first measured in vitro, by infecting A549 immortalized human lung epithelial cells, and primary epithelial and mesenchymal cells isolated from human fetal lung. AAV vectors yielded similar low levels of luciferase gene expression in the different cell types. In vivo transduction efficiency was evaluated in newborn rats, with AAV-LacZ vectors being intratracheally instilled at 3 days of age. Both rAAV5 and rAAV1, but not rAAV2, induced significant lung beta-galactosidase expression, which persisted on day 35. Highest beta- galactosidase levels were measured with rAAV5, but remained far lower than those obtained with adenoviral vectors. A transient increase in alveolar macrophages was observed on day 6, but not on day 8, after rAAV5-LacZ instillation. Morphometric evaluation of lung structures was performed on day 21, and showed no altered lung growth. We conclude that rAAV1 or rAAV5 was more efficient at mediating gene transfer in the neonatal lung than was rAAV2, without adversely affecting lung development. However, in vivo transgene expression was relatively low, and needs to be improved for future therapeutic use of these adeno-associated vectors.

Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid.

A permissive site for insertion of heterologous peptide sequences has been identified in the capsid proteins of AAV2. While attempting to use this site for insertion of a nuclear localization sequence, we have observed a drastic reduction in the yield of DNA-containing particles. ELISA analysis showed that capsid assembly was modestly affected, whereas genome encapsidation was more profoundly altered, a phenomenon we did not observed when a RGD peptide was inserted at the same location. Furthermore, the NLS viruses displayed poor transduction efficiency on HeLa and 293 cells. Altogether, these results indicate that the nature of the peptide inserted at position 587 in the capsid may have important consequences on both particle formation and infectivity.

Gene therapy for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a severe autoimmune systemic disease. Chronic synovial inflammation results in destruction of the joints. No conventional treatment is efficient in RA. Gene therapy of RA targets mainly the players of inflammation or articular destruction: TNF-alpha or IL-1 blocking agents (such as anti-TNF-alpha monoclonal antibodies, soluble TNF-alpha receptor, type II soluble receptor of IL-1, IL-1 receptor antagonist), antiinflammatory cytokines (such as IL-4, IL-10, IL-1), and growth factors. In this polyarticular disease, the vector expressing the therapeutic protein can be administered as a local (intra-articular injection) or a systemic treatment (extra-articular injection). All the main vectors have been used in experimental models, including the more recent lentivirus and adeno-associated virus. Ex vivo gene transfer was performed with synovial cells, fibroblasts, T cells, dendritic cells, and different cells from xenogeneic origin. In vivo gene therapy is simpler, although a less controlled method. Clinical trials in human RA have started with ex vivo retrovirus-expressing IL-1 receptor antagonists and have demonstrated the feasibility of the strategy of gene therapy. The best target remains to be determined and extensive research has to be conducted in preclinical studies.

Sustained delivery of therapeutic concentrations of human clotting factor IX--a comparison of adenoviral and AAV vectors administered in utero.

BACKGROUND: Prenatal somatic gene therapy has been considered for genetic disorders presenting with morbidity at birth. Haemophilia is associated with an increased risk of catastrophic perinatal bleeding complications such as intracranial haemorrhage, which could be prevented by gene transfer in utero. Prenatal gene therapy may be more promising than postnatal treatment, as the fetus may be more amenable to uptake and integration of therapeutic DNA and the immaturity of its immune system may permit life-long immune tolerance of the transgenic protein, thus avoiding the dominant problem in haemophilia treatment, the formation of inhibitory antibodies. METHODS: Adenovirus serotype 5-derived or AAV serotype 2-derived vectors carrying human clotting factor IX (hfIX) cDNA or a reporter gene were administered intramuscularly, intraperitoneally or intravascularly to late-gestation mouse fetuses. Both vector types were evaluated with respect to the kinetics of hfIX delivery to the systemic circulation and possible immune responses against the vector or the transgene product. RESULTS: Mice treated in utero by intramuscular injection of an adenoviral vector carrying hfIX cDNA exhibited high-level gene expression at birth and therapeutic--albeit continuously decreasing--plasma concentrations of hfIX over the entire 6 months of the study. Adenoviral vector spread to multiple organs was detected by polymerase chain reaction (PCR). Intramuscular, intraperitoneal or intravascular application of AAV vectors carrying hfIX cDNA led to much lower plasma concentrations of hfIX shortly after birth, which appeared to decline during the first month of life but stabilized in some of the mice at detectable levels. No signs of immune responses were found, either against the different viral vectors or against hfIX. CONCLUSION: This study demonstrates for the first time that sustained systemic delivery of a therapeutic protein can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy in utero and provides a basis for considering this concept as a preventive therapeutic strategy for haemophilia and perhaps also for other plasma protein deficiencies.