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A common feature of diverse brain disorders is the alteration of GABA-mediated inhibition because of aberrant, intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and in some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of gamma-aminobutyric acid (GABA)-mediated inhibition, and neuronal network dynamics in vitro and ex vivo. Importantly, adeno-associated virus (AAV)-mediated, neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach., Pompe disease is currently treated with enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (GAA). Here, the authors show hepatic-directed gene therapy with AAV vectors enhances GAA bioavailability compared with ERT, resulting in improved rescue of the disease phenotype in mice and broad enzyme distribution in mice and non-human primates.

Description, A new therapeutic approach for the treatment of a skeletal disorder with AAVs bypasses bone resistance to gene transfer., Adeno-associated virus (AAV) vectors are a well-established gene transfer approach for rare genetic diseases. Nonetheless, some tissues, such as bone, remain refractory to AAV. X-linked hypophosphatemia (XLH) is a rare skeletal disorder associated with increased levels of fibroblast growth factor 23 (FGF23), resulting in skeletal deformities and short stature. The conventional treatment for XLH, lifelong phosphate and active vitamin D analogs supplementation, partially improves quality of life and is associated with severe long-term side effects. Recently, a monoclonal antibody against FGF23 has been approved for XLH but remains a high-cost lifelong therapy. We developed a liver-targeting AAV vector to inhibit FGF23 signaling. We showed that hepatic expression of the C-terminal tail of FGF23 corrected skeletal manifestations and osteomalacia in a XLH mouse model. Our data provide proof of concept for AAV gene transfer to treat XLH, a prototypical bone disease, further expanding the use of this modality to treat skeletal disorders.

International audience

Pompe disease (PD) is a neuromuscular disorder caused by deficiency of acidalpha-glucosidase (GAA), leading to motor and respiratory dysfunctions. Available Gaa knock-out (KO) mouse models do not accurately mimic PD, particularly its highly impaired respiratory phenotype. Here we developed a new mouse model of PD crossing Gaa KOB6;129 with DBA2/J mice. We subsequently treated Gaa KODBA2/J mice with adeno-associated virus (AAV) vectors expressing a secretable form of GAA (secGAA). Male Gaa KODBA2/J mice present most of the key features of the human disease, including early lethality, severe respiratory impairment, cardiac hypertrophy and muscle weakness. Transcriptome analyses of Gaa KODBA2/J, compared to the parental Gaa KOB6;129 mice, revealed a profoundly impaired gene signature in the spinal cord and a similarly deregulated gene expression in skeletal muscle. Muscle and spinal cord transcriptome changes, biochemical defects, respiratory and muscle function in the Gaa KODBA2/J model were significantly improved upon gene therapy with AAV vectors expressing secGAA. These data show that the genetic background impacts on the severity of respiratory function and neuroglial spinal cord defects in the Gaa KO mouse model of PD. Our findings have implications for PD prognosis and treatment, show novel molecular pathophysiology mechanisms of the disease and provide a unique model to study PD respiratory defects, which majorly affect patients. This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics. This work was supported by Genethon and the French Muscular Dystrophy Association (AFM, to F.M.). It was also supported by the European Union’s research and innovation program under grant agreement no. 667751 (to F.M.), the European Research Council Consolidator Grant under grant agreement no. 617432 (to F.M.), Marie Skodowska-Curie Actions Individual Fellowship (MSCA-IF) grant agreement no. 797144 (to U.C.), and by Spark Therapeutics under a sponsored research agreement. Sí

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa−/−) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa−/− mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa−/− mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa−/− mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa−/− mouse model via liver gene transfer of secGAA., Graphical Abstract, AAV vector-mediated hepatic expression of secretable GAA drives clearance of glycogen from multiple tissues, resulting in rescue of muscle function in Pompe mice with advanced pathology. Reversal of the Pompe phenotype in mice includes normalization of lysosomal parameters and mitochondrial homeostasis, which is also reflected in the muscle transcriptomics.

International audience; Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans.

International audience; Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.

Pre-existing immunity to adeno-associated virus (AAV) is highly prevalent in humans and can profoundly impact transduction efficiency. Despite the relevance to AAV-mediated gene transfer, relatively little is known about the fate of AAV vectors in the presence of neutralizing antibodies (NAbs). Similarly, the effect of binding antibodies (BAbs), with no detectable neutralizing activity, on AAV transduction is ill defined. Here, we delivered AAV8 vectors to mice carrying NAbs and demonstrated that AAV particles are taken up by both liver parenchymal and non-parenchymal cells; viral particles are then rapidly cleared, without resulting in transgene expression. In vitro, imaging of hepatocytes exposed to AAV vectors pre-incubated with either NAbs or BAbs revealed that virus is taken up by cells in both cases. Whereas no successful transduction was observed when AAV was pre-incubated with NAbs, an increased capsid internalization and transgene expression was observed in the presence of BAbs. Accordingly, AAV8 vectors administered to mice passively immunized with anti-AAV8 BAbs showed a more efficient liver transduction and a unique vector biodistribution profile compared to mice immunized with NAbs. These results highlight a virtually opposite effect of neutralizing and binding antibodies on AAV vectors transduction., Graphical Abstract

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8+ T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25+ T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration., Immunogenicity of AAV vectors renders repeated AAV dosing ineffective. Here the authors show that coadministration of nanoparticle-encapsulated rapamycin overcomes AAV immunogenicity through Treg induction, enabling efficient AAV redosing in mice and nonhuman primates.

Results from clinical trials of liver gene transfer for hemophilia demonstrate the potential of the adeno-associated virus (AAV) vector platform. However, to achieve therapeutic transgene expression, in some cases high vector doses are required, which are associated with a higher risk of triggering anti-capsid cytotoxic T-cell responses. Additionally, anti-AAV preexisting immunity can prevent liver transduction even at low neutralizing antibody (NAb) titers. Here, we describe the use of exosome-associated AAV (exo-AAV) vectors as a robust liver gene delivery system that allows the therapeutic vector dose to be decreased while protecting from preexisting humoral immunity to the capsid. The in vivo efficiency of liver targeting of standard AAV8 or AAV5 and exo-AAV8 or exo-AAV5 vectors expressing human coagulation factor IX (hF.IX) was evaluated. A significant enhancement of transduction efficiency was observed, and in hemophilia B mice treated with 4 × 1010 vector genomes per kilogram of exo-AAV8 vectors, a staggering ∼1 log increase in hF.IX transgene expression was observed, leading to superior correction of clotting time. Enhanced liver expression was also associated with an increase in the frequency of regulatory T cells in lymph nodes. The efficiency of exo- and standard AAV8 vectors in evading preexisting NAbs to the capsid was then evaluated in a passive immunization mouse model and in human sera. Exo-AAV8 gene delivery allowed for efficient transduction even in the presence of moderate NAb titers, thus potentially extending the proportion of subjects eligible for liver gene transfer. Exo-AAV vectors therefore represent a platform to improve the safety and efficacy of liver-directed gene transfer.

Adeno-associated virus (AAV) based vectors are ideal tools for in vivo gene transfer. The excellent safety profile and the highly efficient targeting of hepatocytes in vivo have been demonstrated in both preclinical studies and clinical trials. Although AAV vectors are not strongly immunogenic, they can give rise to both cellular and humoral immune responses. Additionally, as a result of exposure to wild-type AAV, an important fraction of humans harbor pre-existing neutralizing antibodies against the viral capsid, which can prevent successful transduction by AAV vectors. This pose a serious obstacle to the widespread use of AAV vectors in gene therapy. Recently, we have shown that AAV can associate with exosomes (exo-AAV) ultracentrifuged media samples from 293T producer cells. Exosome-associated AAV1, 2, and 9 serotype capsids were tested and found to have enhanced transduction and antibody evasion capabilities compared to conventional AAV vectors. With the goal of developing an enhanced vector for liver directed gene therapy, we sought to characterize the in vivo transduction and biodistribution profile of exo-AAV8 compared to conventional cell-lysated harvested, iodixanol-gradient purified AAV8 vectors purified from the same preparation. The efficiency of liver targeting of conventional AAV8 vs. exo-AAV8 expressing human factor IX (F.IX) under the control of liver specific promotor were tested in naive and pre-immunized animals. C57BL/6 mice (n=5/group) were passively immunized with intravenous human immunoglobulin (IVIg) intraperitoneally, followed 24h later by the intravenous administration of either conventional AAV8 or exo-AAV8 vectors expressing coagulation factor IX (F.IX, 5×1010 vg/mouse). exo-AAV8 completely shielded the capsid vector from neutralizing antibodies at IVIg doses between 0.5mg and 2mg/mouse (NAb titer ~ 1:3.16), resulting in equivalent levels of F.IX transgene expression to naive animals treated with conventional AAV8 vector. At the highest IVIg dose tested (8mg/mouse), residual levels of F.IX transgene expression were about 30% of naive animals treated with conventional AAV8 vectors. We next evaluated the efficiency of exo-AAV8 vs. AAV8 vectors in naive animals. In male mice, no statistically significant difference in F.IX transgene expression levels was observed between the two vector types; also indicated by the similar pattern of vector genome biodistribution. Interestingly, female mice (in which the efficiency of liver transduction with AAV is extremely low compared with male animals) treated with exo-AAV8 showed a dramatic increase in transgene expression, comparable to that of male mice receiving conventional AAV8 vectors. In conclusion, exo-AAV8 vectors present an enhanced liver transduction profile compared with conventionally purified AAV vectors, both in naive female animals and in animals carrying anti-capsid neutralizing antibodies.

Crigler-Najjar syndrome (CN) is an autosomal recessive rare disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene. In severe CN, lack or reduced activity of UGT1A1 results in high levels of serum unconjugated bilirubin (UCB), which can lead to brain damage and death. Treatment of CN consists of phototherapy for 10-12 hours per day to convert UCB into soluble photoisomers without the need of conjugation, which presents several limitations and has a major impact on life quality of the patients. Liver transplantation is the only curative option for CN. The limited therapeutic options available prompted us to develop a new therapy for CN based on the transfer of a corrected copy of the UGT1A1 gene to hepatocytes.We developed an AAV8 vector optimized for the liver expression of the hUGT1A1 transgene (AAV8-hUGT1A1). Safety and efficacy of correction of total serum bilirubin (TB) levels with AAV-hUGT1A1 were demonstrated in mouse and rat models of CN at vector doses as low as 5×10 11 vector genomes (vg)/kg, a result confirmed also by the detection of conjugated bilirubin in bile of treated rats. In juvenile CN rats, long-term correction of total bilirubin levels were observed for more than 8 months following AAV8-hUGT1A1 gene transfer. In neonate Ugt1a1–/– mice, intraperitoneal delivery of the vector at doses as low as 1×10 9 vg/mouse resulted in correction of TB at 4 weeks. However, vector delivery in P2 and P4 animals resulted in lower efficiency of correction when compared with P11-transduced mice, a finding likely due to the more advanced development of the liver at later time points.Based on these data, dose finding studies were performed in CN rats, which showed a profile of liver transduction with AAV8 vectors similar to humans, i.e. lower efficiency of transduction than mice. No differences in efficacy of correction of TB were observed in male vs. female rats.A scalable process for the production of AAV8-hUGT1A1 production was established based on a protocol of triple transfection of suspension HEK293 cells, leading to high yield vector preparations with excellent purity profile. In vitro transduction assays in human hepatocytes based on Western blot for hUGT1A1 protein and in vivo studies in CN rats demonstrated that vectors produced with the suspension process have potency characteristics undistinguishable from research grade vectors produced by triple transfection of adherent cells and purified by cesium chloride gradient centrifugation.In conclusion, our data demonstrate the safety and efficacy of gene transfer for Crigler-Najjar syndrome in two relevant animal models of the disease and provide tools and a strong rationale for the translation of these results in human subjects.

Optimization of the transgene expression cassette for adeno-associated virus (AAV) vector-mediated gene therapy has been mainly focused on the design of efficient promoter/enhancers and on the optimization of the transgene codon usage. Addition of intron sequences to expression cassette of a given transgene has also been shown to be beneficial in terms of expression levels.In an effort to develop a liver gene transfer approach to treat Crigler-Najjar (CN) syndrome, an autosomal recessive disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene and associated with toxic accumulation of unconjugated bilirubin in serum, we designed several UGT1A1 transgene expression cassettes and we packaged them into serotype 8 AAV vectors.We first performed the codon optimization of the UGT1A1 wildtype (w.t.) cDNA following two algorithms (v1 and v2), which led to cDNAs with nucleotide sequences significantly different from each other (nucleotide sequence homology of 77.5% for v1 and 78.3% for v2 vs. w.t.). Surprisingly, the sequence with a codon adaptation index closer to that of w.t. (w.t.=0.76, v1=0.76, v2=0.96) thus predicted to be the least efficient in terms of expression, showed the best performance in vitro in human hepatocyte cell lines, both at the RNA and protein levels. These results were confirmed in vivo in Ugt1a1-/- mice, a model of CN syndrome, where gene transfer with the optimized UGT1A1 resulted in better reduction of total bilirubin in serum. Next, we analyzed the 5’ untranslated region (UTR) of the expression cassette for sequences that may affect the efficacy of the transgene expression levels. Aside from the introduction of an optimized Kozak consensus sequence, the human beta2-globin (hBB2) intron sequence associated with the promoter revealed the presence of out of frame and in frame ATG codons before the start site of the UGT1A1 transgene. Removal of these cryptic ATGs from the intron sequence enhanced transgene expression, which resulted in stable, long-term correction of the disease phenotype following in vivo gene transfer. This demonstrated that the intron sequence optimization increases mRNA and protein levels in vitro and enhance the efficacy of the vector in vivo. Similar analysis was conducted in other common promoter/intron combinations used in AAV vector gene transfer.In conclusion, our results provide evidence that the combination of a potent liver specific promoter, codon optimization, and optimized untranslated region elements allow for decreasing the therapeutic dose of AAV vectors.

Adeno-associated virus vectors based on serotype 8 (AAV8) have demonstrated superior efficiency of transduction of hepatocytes in vivo in animal models and in humans. Recently, we described extracellular vesicle (exosomes) -associated AAV vectors termed vexosomes (vAAV). We tested vAAV1, 2, and 9 and found them to enhance transduction and antibody evasion capabilities compared to conventional AAV vectors.With the goal of developing an enhanced vector for liver gene therapy, we sought to characterize the in vitro and in vivo transduction, biodistribution, and immunogenicity profile of vAAV8 vectors compared to conventional AAV8 vectors purified from the same preparation.Several cell lines were transduced with conventional AAV8 or vAAV8 vectors expressing luciferase, at multiplicity of infection (MOI) ranging from 250 to 25000, and luciferase expression was measured after an overnight incubation. In HeLa and HEK293 cells, levels of luciferase up to 1 log higher were observed with vAAV8 compared with AAV8 vectors, with detectable levels of luciferase at MOIs as low as 250. In hepatocyte cell lines, the magnitude of enhancement was about 3-fold, with lower max relative light unit signal measured compared to the other cell lines. Notably, although at low levels, transduction with vAAV8 vectors was also observed in a Jurkat T cell line, in which no transduction was observed with conventional AAV8 vectors.We then evaluated the efficiency of liver targeting in vivo with this vector system using the secreted factor IX (F.IX) transgene. Conventional or vAAV8 vectors expressing human F.IX under the control of a liver-specific promoter were administered i.v. to C57BL/6J male mice at 5×1010 vg/mouse. In naive animals, no difference in F.IX transgene expression levels was observed between the two vector types; similarly, vector genome were found at similar levels in liver, spleen, muscle, lungs, heart, and kidneys. In these animals, i.v. vector administration also resulted in identical levels of anti-AAV antibody formation. We next evaluated the effect of vAAV8 on liver transduction in the presence of antibodies. Animals were passively immunized with IVIg intraperitoneally (0.5, 5.0, and 15 mg/mouse) followed by administration of vector (5×1010 vg) intravenously 24 hours later. At a neutralizing antibody titer of~1:10 (0.5mg IVIg), vAAV8 vector-injected mice had levels of expression identical to those in naive animals. At higher titers (>1:100, 5.0 mg), low levels of residual expression of F.IX were measure with vAAV8 vectors and no expression was measured with conventional AAV. No expression was detected with either AAV8 or vAAV8 vectors in animals immunized with the highest dose of IVIg.In conclusion, vAAV8 vectors have unique transduction characteristics that may be exploited in vitro to transduce various cell types. In vivo, these vectors showed identical levels of liver transduction in naive male mice and provided some level of protection from antibody-mediated vector neutralization.

Inclusion of synthetic introns in adeno-associated virus (AAV) vector expression cassettes is a commonly used maneuver to drive increased transgene expression, possibly via an increased stability and/or increased nuclear translocation of mRNA. Although the positive effect of intronic sequences on transgene expression has been demonstrated in several models, less clear is whether splicing in these synthetic expression cassettes occurs with 100% efficiency and if the presence of the unspliced intron can influence the efficiency of translation of the transgene. In an effort to optimize a liver gene transfer approach to treat Crigler-Najjar (CN) syndrome, an autosomal recessive disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene, we analyzed the sequence of a synthetic intron derived from human beta-globin intron 2 (hBB2) for motifs that may affect the transgene expression levels. This analysis revealed the presence of out of frame and in frame ATG codons before the start site of the UGT1A1 transgene. Unexpectedly, the removal of these cryptic ATGs from the intronic sequence enhanced transgene expression, which resulted in stable, long-term correction of serum bilirubin levels in animal models of CN syndrome. These results demonstrate that intron optimization increases mRNA and protein levels in vitro and enhance the efficacy of the vector in vivo. Next, we conducted a similar analysis on other intronic sequences commonly used in AAV-mediated gene transfer, showing that the removal of cryptic ATGs in the intron at the 5’ of a transgene generally increases its expression. The scanning ribosome model may provide a solid explanation for our findings given that the intronic sequence is not completely removed by splicing. We thus analyzed the structure of the region at the 5’ of the transgene by RT-PCR and primer elongation assay and we observed the accumulation of unspliced forms of the mRNA. We also confirmed that the presence of cryptic ATGs has a critical impact on the translation efficiency of the transgene. In conclusion, our results confirm the positive effect of an intron fused to the 5’ of a transgene on its expression levels. On the other hand, our results provide evidence that the synthetic introns used in gene therapy are not spliced with 100% efficiency, thus the optimization of the sequence of the intron by removal of cryptic ATGs is important to increase the transgene expression in AAV vector-mediated gene transfer.