Giuseppe Ronzitti

Emerging Issues in AAV-Mediated In Vivo Gene Therapy

In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, has established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective “therapeutic products” is, however, a long process that involves several cycles of iterations from bench to bedside that are required to address issues encountered during drug development. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from preclinical and clinical studies.

A translationally optimized AAV-UGT1A1 vector drives safe and long-lasting correction of Crigler-Najjar syndrome

Crigler-Najjar syndrome is a severe metabolic disease of the liver due to a reduced activity of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme. In an effort to translate to the clinic an adeno-associated virus vector mediated liver gene transfer approach to treat Crigler-Najjar syndrome, we developed and optimized a vector expressing the UGT1A1 transgene. For this purpose, we designed and tested in vitro and in vivo multiple codon-optimized UGT1A1 transgene cDNAs. We also optimized noncoding sequences in the transgene expression cassette. Our results indicate that transgene codon-optimization is a strategy that can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo. Additionally, while inclusion of introns can enhance gene expression, optimization of these introns, and in particular removal of cryptic ATGs and splice sites, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, using a translationally optimized adeno-associated virus vector expressing the UGT1A1 transgene, we demonstrated rescue of the phenotype of Crigler-Najjar syndrome in two animal models of the disease, Gunn rats and Ugt1a1-/- mice. We also showed long-term (>1 year) correction of the disease in Gunn rats. These results support further translation of the approach to humans.

Low-dose liver targeted gene therapy for Pompe disease enhances therapeutic efficacy of ERT via immune tolerance induction

Pompe disease results from acid α-glucosidase (GAA) deficiency, and enzyme replacement therapy (ERT) with recombinant human (rh) GAA has clinical benefits, although its limitations include the short half-life of GAA and the formation of antibody responses. The present study compared the efficacy of ERT against gene transfer with an adeno-associated viral (AAV) vector containing a liver-specific promoter. GAA knockout (KO) mice were administered either a weekly injection of rhGAA (20 mg/kg) or a single injection of AAV2/8-LSPhGAA (8 × 1011 vector genomes [vg]/kg). Both treatments significantly reduced glycogen content of the heart and diaphragm. Although ERT triggered anti-GAA antibody formation, there was no detectable antibody response following AAV vector administration. The efficacy of three lower dosages of AAV2/8-LSPhGAA was evaluated in GAA-KO mice, either alone or in combination with ERT. The minimum effective dose (MED) identified was 8 × 1010 vg/kg to reduce glycogen content in the heart and diaphragm of GAA-KO mice. A 3-fold higher dose was required to suppress antibody responses to ERT. Efficacy from liver gene therapy was slightly greater in male mice than in female mice. Vector dose correlated inversely with anti-GAA antibody formation, whereas higher vector doses suppressed previously formed anti-GAA antibodies as late as 25 weeks after the start of ERT and achieved biochemical correction of glycogen accumulation. In conclusion, we identified the MED for effective AAV2/8-LSPhGAA-mediated tolerogenic gene therapy in Pompe disease mice.

Autophagy determines efficiency of liver‐directed gene therapy with adeno‐associated viral vectors

Use of adeno‐associated viral (AAV) vectors for liver‐directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)–dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV‐mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short‐term treatment with a single dose of torin 1 significantly increased vector‐mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid‐α‐glucosidase in nonhuman primates. Conclusion: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver‐directed gene therapy using AAV vectors and showed in a proof‐of‐principle study how this virus–host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252–265).

Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors

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.

Long-term exposure to Myozyme results in a decrease of anti-drug antibodies in late-onset Pompe disease patients

Immunogenicity of recombinant human acid-alpha glucosidase (rhGAA) in enzyme replacement therapy (ERT) is a safety and efficacy concern in the management of late-onset Pompe disease (LOPD). However, long-term effects of ERT on humoral and cellular responses to rhGAA are still poorly understood. To better understand the impact of immunogenicity of rhGAA on the efficacy of ERT, clinical data and blood samples from LOPD patients undergoing ERT for >4 years (n = 28) or untreated (n = 10) were collected and analyzed. In treated LOPD patients, anti-rhGAA antibodies peaked within the first 1000 days of ERT, while long-term exposure to rhGAA resulted in clearance of antibodies with residual production of non-neutralizing IgG. Analysis of  T cell responses to rhGAA showed detectable T cell reactivity only after in vitro restimulation. Upregulation of several cytokines and chemokines was detectable in both treated and untreated LOPD subjects, while IL2 secretion was detectable only in subjects who received ERT. These results indicate that long-term ERT in LOPD patients results in a decrease in antibody titers and residual production of non-inhibitory IgGs. Immune responses to GAA following long-term ERT do not seem to affect efficacy of ERT and are consistent with an immunomodulatory effect possibly mediated by regulatory T cells.

77. Antigen-Specific Modulation of Capsid Immunogenicity with Tolerogenic Nanoparticles Results in Successful AAV Vector Readministration

Gene transfer approaches based on the adeno-associated virus (AAV) vector platform have shown great therapeutic potential in both preclinical studies and clinical trials. Neutralizing immune responses to AAV, however, are an important limitation to the use of AAV vectors as therapeutic tools, as even low-titer anti-capsid neutralizing antibodies (NAb) can lead to vector clearance and lack of efficacy. In particular, anti-AAV NAbs develop at high titers following vector administration and persist for several years after AAV vector administration, making vector re-administration hard if not impossible. Here we tested a novel strategy to modulate immune responses directed against AAV vectors based on the co-administration of biodegradable tolerogenic poly(lactic acid co-glycolytic acid) (PLGA) nanoparticles (tNP) containing rapamycin at the time of vector administration. C57BL/6 mice (n=5/group) received an AAV8 vector encoding for luciferase (AAV8-Luc) at a dose of 4×1012 vg/kg injected intravenously alone, or formulated with empty PLGA nanoparticles (NP), or formulated with tNP containing 100 µg of rapamycin. Three weeks after treatment, anti-AAV8 antibodies were measured and animals received a second intravenous infusion with an AAV8 vector encoding for human coagulation factor IX (AAV8-hFIX) at a dose of 4×1012 vg/kg formulated with NP or tNP. An anti-AAV8 antibody ELISA and an in vitro neutralization assay was used to follow humoral immune responses to the vector. While no development of anti-AAV8 antibodies was observed in the tNP-treated animals after the first and second vector administration, control groups developed robust humoral immune responses to the AAV8 capsid, which prevented vector readministration. Consequently, efficient hFIX transgene expression deriving from the second AAV8 vector administration was observed only when tNP were used, at levels identical to animals that received only a single administration of AAV8-hFIX. Lack of antibody formation against the AAV8 capsid in animals treated with tNP was also accompanied by a downregulation of both CD4+ and CD8+ T cell responses in the liver. A mild decrease in the frequency of CD4+ T cells in spleen, with no change in frequency of regulatory T cells, were also noted. In a separate set of experiments, we tested the antigen-specificity of the treatment of tNP with AAV8 administration. Mice (n=5/group) received 4×1012 vg/kg of an AAV8-luc vector with tNP followed by either challenge with hFIX protein in complete Freunds adjuvant, AAV5-hFIX vector intravenous injection, or AAV8-hFIX vector intravenous injection. All animals pretreated with AAV8-luc and tNP developed antibodies against the hFIX and the AAV5 antigens, while they anti-AAV8 antibody titers were significantly decreased. In conclusion, tNP administration together withAAV vector prevents anti-capsid immune responses in an antigen-specific manner and allows for AAV vector readministration, addressing one of the most important challenges of the in vivo gene transfer field.

Influence of Pre-existing Anti-capsid Neutralizing and Binding Antibodies on AAV Vector Transduction

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.

Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidase

Liver delivery of engineered GAA transgenes to mice with Pompe disease rescued glycogen accumulation in multiple tissues. Revealing a secretable GAA for Pompe disease Pompe disease is a genetic disorder caused by mutations in the acid α-glucosidase (GAA) gene, leading to glycogen accumulation in all cells of the body. This accumulation leads to severe neuromuscular disabilities that can be life-threatening. Puzzo et al. used bioinformatic analysis, protein engineering, and gene therapy to develop and deliver a GAA transgene encoding a secretable GAA. Liver-specific, adeno-associated virus (AAV) vector–mediated GAA delivery rescued the Pompe disease phenotype in a mouse model and increased GAA expression in healthy monkeys, opening possibilities for future translation of this approach for treating Pompe disease. Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa−/−) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa−/− mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector–mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.

Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration

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.