Cell-Based Tracers as Trojan Horses for Image-Guided Surgery.

Surgeons rely almost completely on their own vision and palpation to recognize affected tissues during surgery. Consequently, they are often unable to distinguish between different cells and tissue types. This makes accurate and complete resection cumbersome. Targeted image-guided surgery (IGS) provides a solution by enabling real-time tissue recognition. Most current targeting agents (tracers) consist of antibodies or peptides equipped with a radiolabel for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), magnetic resonance imaging (MRI) labels, or a near-infrared fluorescent (NIRF) dye. These tracers are preoperatively administered to patients, home in on targeted cells or tissues, and are visualized in the operating room via dedicated imaging systems. Instead of using these 'passive' tracers, there are other, more 'active' approaches of probe delivery conceivable by using living cells (macrophages/monocytes, neutrophils, T cells, mesenchymal stromal cells), cell(-derived) fragments (platelets, extracellular vesicles (exosomes)), and microorganisms (bacteria, viruses) or, alternatively, 'humanized' nanoparticles. Compared with current tracers, these active contrast agents might be more efficient for the specific targeting of tumors or other pathological tissues (e.g., atherosclerotic plaques). This review provides an overview of the arsenal of possibilities applicable for the concept of cell-based tracers for IGS.

Successful Repeated Hepatic Gene Delivery in Mice and Non-human Primates Achieved by Sequential Administration of AAV5ch and AAV1.

In the gene therapy field, re-administration of adeno-associated virus (AAV) is an important topic because a decrease in therapeutic protein expression might occur over time. However, an efficient re-administration with the same AAV serotype is impossible due to serotype-specific, anti-AAV neutralizing antibodies (NABs) that are produced after initial AAV treatment. To address this issue, we explored the feasibility of using chimeric AAV serotype 5 (AAV5ch) and AAV1 for repeated liver-targeted gene delivery. To develop a relevant model, we immunized animals with a high dose of AAV5ch-human secreted embryonic alkaline phosphatase (hSEAP) that generates high levels of anti-AAV5ch NAB. Secondary liver transduction with the same dose of AAV1-human factor IX (hFIX) in the presence of high levels of anti-AAV5ch NAB proved to be successful because expression/activity of both reporter transgenes was observed. This is the first time that two different transgenes are shown to be produced by non-human primate (NHP) liver after sequential administration of clinically relevant doses of both AAV5ch and AAV1. The levels of transgene proteins achieved after delivery with AAV5ch and AAV1 illustrate the possibility of both serotypes for liver targeting. Furthermore, transgene DNA and RNA biodistribution patterns provided insight into the potential cause of decrease or loss of transgene protein expression over time in NHPs.

Recombinant AAV Integration Is Not Associated With Hepatic Genotoxicity in Nonhuman Primates and Patients.

Recombinant adeno-associated viral vectors (rAAV) currently constitute a real therapeutic strategy for the sustained correction of diverse genetic conditions. Though a wealth of preclinical and clinical studies have been conducted with rAAV, the oncogenic potential of these vectors is still controversial, particularly when considering liver-directed gene therapy. Few preclinical studies and the recent discovery of incomplete wild-type AAV2 genomes integrated in human hepatocellular carcinoma biopsies have raised concerns on rAAV safety. In the present study, we have characterized the integration of both complete and partial rAAV2/5 genomes in nonhuman primate tissues and clinical liver biopsies from a trial aimed to treat acute intermittent porphyria. We applied a new multiplex linear amplification-mediated polymerase chain reaction (PCR) assay capable of detecting integration events that are originated throughout the rAAV genome. The integration rate was low both in nonhuman primates and patient's samples. Importantly, no integration clusters or events were found in genes previously reported to link rAAV integration with hepatocellular carcinoma development, thus showing the absence of genotoxicity of a systemically administered rAAV2/5 in a large animal model and in the clinical context.

Mir-142-3p target sequences reduce transgene-directed immunogenicity following intramuscular adeno-associated virus 1 vector-mediated gene delivery.

BACKGROUND: Muscle represents an important tissue target for adeno-associated virus (AAV) vector-mediated gene transfer in muscular, metabolic or blood-related genetic disorders. However, several studies have demonstrated the appearance of immune responses against the transgene product after intramuscular AAV vector delivery that resulted in a limited efficacy of the treatment. Use of microRNAs that are specifically expressed in antigen-presenting cells (APCs) is a promising approach for avoiding those immune responses. Cellular mir-142-3p, which is APC-specific, is able to repress the translation of its target cellular transcripts by binding to a specific target sequences. METHODS: In the present study, we explored the potential of mir-142-3p specific target sequences with respect to reducing or abolishing immune responses directed against ovalbumin (OVA), a highly immunogenic protein, expressed as transgene and delivered by AAV1 vector administered intramuscularly. RESULTS: The occurrence of immune responses against OVA transgene following intramuscular delivery by AAV have been described previously and resulted in the loss of OVA protein expression. In the present study, we demonstrate that OVA protein expression was maintained when mir-142-3pT sequences were incorporated into the expression cassette. The sustained expression of OVA protein over time correlated with a reduced increase in anti-OVA antibody levels. Furthermore, no cellular infiltrates were observed in the muscle tissue when AAV1 vectors containing four or eight repeats of mir-142-3p target sequences after the OVA sequence were used. CONCLUSIONS: The rising humoral and cellular immune responses against OVA protein after intramuscular delivery can be efficiently reduced by the use of mir-142-3p target sequences.

Murine CD4⁺CD25⁻ cells activated in vitro with PMA/ionomycin and anti-CD3 acquire regulatory function and ameliorate experimental colitis in vivo.

BACKGROUND: Induced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance. METHODS: Using phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4⁺CD25⁻ cells we generated in vitro functional CD4⁺CD25⁻ iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model. RESULTS: TregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 ß and higher levels of colonic IL-17 when compared to diseased control group. CONCLUSIONS: This study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo.

Activin A induces a non-fibrotic phenotype in smooth muscle cells in contrast to TGF-ß.

AIMS: Activin A and transforming growth factor-ß1 (TGF-ß1) belong to the same family of growth and differentiation factors that modulate vascular lesion formation in distinct ways, which we wish to understand mechanistically. METHODS AND RESULTS: We investigated the expression of cell-surface receptors and activation of Smads in human vascular smooth muscle cells (SMCs) and demonstrated that activin receptor-like kinase-1 (ALK-1), ALK-4, ALK-5 and endoglin are expressed in human SMCs. As expected, TGF-ß1 activates Smad1 and Smad2 in these cells. Interestingly, activin A also induces phosphorylation of both Smads, which has not been reported for Smad1 before. Transcriptome analyses of activin A and TGF-ß1 treated SMCs with subsequent Gene-Set Enrichment Analyses revealed that many downstream gene networks are induced by both factors. However, the effect of activin A on expression kinetics of individual genes is less pronounced than for TGF-ß1, which is explained by a more rapid dephosphorylation of Smads and p38-MAPK in response to activin A. Substantial differences in expression of fibronectin, alpha-V integrin and total extracellular collagen synthesis were observed. CONCLUSIONS: Genome-wide mRNA expression analyses clarify the distinct modulation of vascular lesion formation by activin A and TGF-ß1, most significantly because activin A is non-fibrotic.

miRNA-Mediated Knockdown of ATXN3 Alleviates Molecular Disease Hallmarks in a Mouse Model for Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the ATXN3 gene. This mutation leads to a toxic gain of function of the ataxin-3 protein, resulting in neuronal dysfunction and atrophy of specific brain regions over time. As ataxin-3 is a dispensable protein in rodents, ataxin-3 knockdown by gene therapy may be a powerful approach for the treatment of SCA3. In this study, we tested the feasibility of an adeno-associated viral (AAV) vector carrying a previously described artificial microRNA against ATXN3 in a striatal mouse model of SCA3. Striatal injection of the AAV resulted in good distribution throughout the striatum, with strong dose-dependent ataxin-3 knockdown. The hallmark intracellular ataxin-3 inclusions were almost completely alleviated by the microRNA-induced ATXN3 knockdown. In addition, the striatal lesion of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) in the SCA3 mice was rescued by ATXN3 knockdown, indicating functional rescue of neuronal signaling and health upon AAV treatment. Together, these data suggest that microRNA-induced ataxin-3 knockdown is a promising therapeutic strategy in the treatment of SCA3.

Clinical Considerations for Capsid Choice in the Development of Liver-Targeted AAV-Based Gene Transfer.

As gene transfer with adeno-associated virus (AAV) vectors is starting to enter clinical practice, this review examines the impact of vector capsid choice in liver-directed gene transfer for hemophilia. Given that there are multiple clinical trials completed and ongoing in this field, it is important to review the clinical evidence, particularly as a range of AAV-vector serotypes including AAV2, AAV5, AAV8, and AAV10 have been tested. Although there have been a number of successful trials, the development of two investigational AAV vectors for hemophilia B has been discontinued because they did not meet efficacy and/or safety expectations. Whether this difference between success and failure of gene transfer approaches reflects capsid choice, vector design, manufacturing system, or other variables is a question of great interest. Here, we examine the body of evidence across trials to determine the possible influences of serotype choice on key clinical outcomes such as safety, vector clearance, treatment eligibility, occurrence of transaminase elevations, activation of capsid-directed cytotoxic T cell responses, and clinical efficacy. In summary, gene transfer requires a balance between achieving sufficient transgene expression and minimizing destructive immune responses, which may be affected by AAV-vector serotype choice.

Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates.

Adeno-associated virus (AAV)-based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

Enhanced Factor IX Activity following Administration of AAV5-R338L "Padua" Factor IX versus AAV5 WT Human Factor IX in NHPs.

Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%-13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L "Padua" FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile.

Therapeutic hFIX Activity Achieved after Single AAV5-hFIX Treatment in Hemophilia B Patients and NHPs with Pre-existing Anti-AAV5 NABs.

Currently, individuals with pre-existing neutralizing antibodies (NABs) against adeno-associated virus (AAV) above titer of 5 are excluded from systemic AAV-based clinical trials. In this study we explored the impact of pre-existing anti-AAV5 NABs on the efficacy of AAV5-based gene therapy. AMT-060 (AAV5-human FIX) was evaluated in 10 adults with hemophilia B who tested negative for pre-existing anti-AAV5 NABs using a GFP-based assay. In this study, using a more sensitive luciferase-based assay, we show that 3 of those 10 patients tested positive for anti-AAV5 NABs. However, no relationship was observed between the presence of pre-treatment anti-AAV5 NABs and the therapeutic efficacy of AMT-060. Further studies in non-human primates (NHPs) showed that AAV5 transduction efficacy was similar following AMT-060 treatment, irrespective of the pre-existing anti-AAV5 NABs titers. We show that therapeutic efficacy of AAV5-mediated gene therapy was achieved in humans with pre-existing anti-AAV5 NABs titers up to 340. Whereas in NHPs circulating human factor IX (hFIX) protein was achieved, at a level therapeutic in humans, with pre-existing anti-AAV5 NABs up to 1030. Based on those results, no patients were excluded from the AMT-061 (AAV5-hFIX-Padua) phase IIb clinical trial (n = 3). All three subjects presented pre-existing anti-AAV5 NABs, yet had therapeutic hFIX activity after AMT-061 administration.

Endothelium specific matrilysin (MMP-7) expression in human cancers.

Over-expression of matrilysin (MMP-7) is predominantly associated with epithelial (pre)malignant cells. In the present study MMP-7 expression is also found in endothelial cells in various human cancer types. Endothelial MMP-7 was associated with CD34 and/or CD105 expression. These immunohistochemical data were confirmed by RT-PCR on VEGF-stimulated endothelial cells. In addition, MMP-7 was also identified in sprouting endothelial cells in vitro. The potential clinical relevance of endothelial MMP-7 was assessed for cervical cancer patients by evaluating the association with overall survival. In contrast to MMP-7 in malignant epithelial cells, MMP-7 expression in endothelial cells showed a significant association with poor survival (LR 5.12, P=0.02, n=30). Our data suggest that MMP-7 is involved in tumor angiogenesis, thereby contributing to malignant growth and hence associated with decreased survival.

Macrophage-specific inhibition of NF-kappaB activation reduces foam-cell formation.

Accumulation of lipid-laden macrophages is a hallmark of atherosclerosis. The relevance of the key transcription factor nuclear factor kappaB (NF-kappaB) for macrophage-derived foam-cell formation has not been unequivocally resolved. Transgenic mice lines were generated in which NF-kappaB activation is specifically inhibited in macrophages by overexpressing a trans-dominant, non-degradable form of IkappaBalpha (IkappaBalpha (32A/36A)) under control of the macrophage-specific SR-A promoter. Alanine substitution of serines 32 and 36 prevents degradation and retains the inactive NF-kappaB/IkappaBalpha (32A/36A) complex in the cytoplasm. Similarly, stable human THP1 monocytic cell lines were generated with integrated copies of IkappaBalpha (32A/36A) cDNA. Upon treatment with oxidized low-density lipoprotein (ox-LDL), murine peritoneal macrophages from transgenic IkappaBalpha (32A/36A) mice, as well as THP1/IkappaBalpha (32A/36A) clones, display decreased lipid loading after differentiation into macrophages. This is accompanied by increased expression of the transcription factors PPARgamma and LXRalpha as well as of the major cholesterol-efflux transporter ABCA1. Paradoxically, mRNA expression of the 'lipid-uptake' receptor CD36 is also increased. Since the net result of these changes is reduction of foam-cell formation, it is proposed that under specific inhibition of NF-kappaB activation, ABCA1-mediated cholesterol efflux prevails over CD36-mediated lipid influx.

Nuclear receptors Nur77, Nurr1, and NOR-1 expressed in atherosclerotic lesion macrophages reduce lipid loading and inflammatory responses.

OBJECTIVE: Atherosclerosis is an inflammatory disease in which macrophage activation and lipid loading play a crucial role. In this study, we investigated expression and function of the NR4A nuclear receptor family, comprising Nur77 (NR4A1, TR3), Nurr1 (NR4A2), and NOR-1 (NR4A3) in human macrophages. METHODS AND RESULTS: Nur77, Nurr1, and NOR-1 are expressed in early and advanced human atherosclerotic lesion macrophages primarily in areas of plaque activation/progression as detected by in situ-hybridization and immunohistochemistry. Protein expression localizes to the nucleus. Primary and THP-1 macrophages transiently express NR4A-factors in response to lipopolysaccharide and tumor necrosis factor alpha. Lentiviral overexpression of Nur77, Nurr1, or NOR-1 reduces expression and production of interleukin (IL)-1beta and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1alpha and -1beta and monocyte chemoattractant protein-1 chemokines. In addition, NR4A-factors reduce oxidized-low-density lipoprotein uptake, consistent with downregulation of scavenger receptor-A, CD36, and CD11b macrophage marker genes. Knockdown of Nur77 or NOR-1 with gene-specific lentiviral short-hairpin RNAs resulted in enhanced cytokine and chemokine synthesis, increased lipid loading, and augmented CD11b expression, demonstrating endogenous NR4A-factors to inhibit macrophage activation, foam-cell formation, and differentiation. CONCLUSIONS: NR4A-factors are expressed in human atherosclerotic lesion macrophages and reduce human macrophage lipid loading and inflammatory responses, providing further evidence for a protective role of NR4A-factors in atherogenesis.

Improvement of Adeno-Associated Virus-Mediated Liver Transduction Efficacy by Regional Administration in Macaca fascicularis.

The liver is a central organ in metabolism and can be affected by numerous inherited metabolic disorders. Recombinant adeno-associated virus (AAV)-based gene therapy represents a promising therapeutic approach for such diseases. AAVs have been demonstrated to be safe, and resulted in high and long-term expression in preclinical and clinical studies. However, there are still some concerns regarding the expression levels that can be achieved and the percentage of hepatocytes that can be transduced. Because of the cell-autonomous nature of most metabolic liver disorders, a high percentage of hepatocytes needs to be corrected in order to achieve a therapeutic effect. The goal of our work was to improve transduction efficacy of the liver by conveying the vector directly to hepatic tissue. Interventional radiology procedures were used to administer an AAV5 vector expressing a secreted form of human embryonic alkaline phosphatase (hSEAP) under the control of a liver-specific promoter to a clinically relevant animal model, Macaca fascicularis. Balloon occlusion of the regional hepatic venous flow was performed while injecting the vector either into the hepatic artery (HA) or, against flow, via the suprahepatic vein (SHV). In both cases the vector was injected into the right hepatic lobules, and the two routes were compared with conventional intravenous administration. Higher hSEAP levels were obtained when the vector was administered via SHV or HA than after intravenous injection. Furthermore, higher expression levels correlated with a higher number of vector genomes in the injected lobules. In conclusion, direct administration of AAV vectors via the hepatic blood flow with simultaneous balloon occlusion of the hepatic outflow increases liver transduction efficacy in comparison with systemic delivery and can be further improved in bigger animals or humans, where it would be technically feasible to inject the vector into the hepatic vasculature in the generality of lobules.

Expression and localization of tissue factor pathway inhibitor-2 in normal and atherosclerotic human vessels.

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type, serine protease inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor:activated factor VII complex. In this study, we investigated TFPI-2 expression and localization in normal and atherosclerotic human arteries by using in situ hybridization and immunohistochemical techniques. In healthy human blood vessels, TFPI-2 was detected in the vascular endothelium alone. In human atherosclerotic tissues, TFPI-2 expression was assigned to macrophages, T cells, endothelial cells, and smooth muscle cells. Western blot analysis for TFPI-2 confirmed its production by cultured human aortic smooth muscle cells, U937 cells (monocytes), and Jurkat (T cell) cell lines. Reverse transcription-polymerase chain reaction revealed similar TFPI-2 expression levels in both monocytes and macrophages in culture. Electron microscopic study with immunogold labeling revealed the association of TFPI-2 antigen with both the extracellular matrix and plasma membranes. TFPI-2 antigen was detected in some areas of atheroma that also stained positively for both tissue factor and factor VII. Moreover, detection of TFPI-2 in close spatial proximity to plasmin/plasminogen on macrophages, on endothelial cells, and in matrix-rich areas highlighted its possible functional significance in the regulation of plasmin activity and downstream proteolytic mechanisms that occur in the atherosclerotic lesion.

Immune Responses to AAV-Vectors, the Glybera Example from Bench to Bedside.

Alipogene tiparvovec (Glybera(®)) is an adeno-associated virus serotype 1 (AAV1)-based gene therapy that has been developed for the treatment of patients with lipoprotein lipase (LPL) deficiency. Alipogene tiparvovec contains the human LPL naturally occurring gene variant LPL(S447X) in a non-replicating viral vector based on AAV1. Such virus-derived vectors administered to humans elicit immune responses against the viral capsid protein and immune responses, especially cellular, mounted against the protein expressed from the administered gene have been linked to attenuated transgene expression and loss of efficacy. Therefore, a potential concern about the use of AAV-based vectors for gene therapy is that they may induce humoral and cellular immune responses in the recipient that may impact on efficacy and safety. In this paper, we review the current understanding of immune responses against AAV-based vectors and their impact on clinical efficacy and safety. In particular, the immunogenicity findings from the clinical development of alipogene tiparvovec up to licensing in Europe will be discussed demonstrating that systemic and local immune responses induced by intra-muscular injection of alipogene tiparvovec have no deleterious effects on clinical efficacy and safety. These findings show that muscle-directed AAV-based gene therapy remains a promising approach for the treatment of human diseases.

Immune responses to intramuscular administration of alipogene tiparvovec (AAV1-LPL(S447X)) in a phase II clinical trial of lipoprotein lipase deficiency gene therapy.

Cellular immune responses to adeno-associated viral (AAV) vectors used for gene therapy have been linked to attenuated transgene expression and loss of efficacy. The impact of such cellular immune responses on the clinical efficacy of alipogene tiparvovec (Glybera; AAV1-LPL(S447X); uniQure), a gene therapy consisting of intramuscular administration of a recombinant AAV1 mediating muscle-directed expression of lipoprotein lipase (LPL), was investigated. Five subjects with LPL deficiency (LPLD) were administered intramuscularly with a dose of 1 × 10(12) gc/kg alipogene tiparvovec. All subjects were treated with immune suppression starting shortly before administration of alipogene tiparvovec and maintained until 12 weeks after administration. Systemic antibody and T cell responses against AAV1 and LPL(S447X), as well as local cellular immune responses in the injected muscle, were investigated in five LPLD subjects. Long-term transgene expression was demonstrated despite a transient systemic cellular response and a stable humoral immune response against the AAV1 capsid protein. Cellular infiltrates were found in four of the five subjects but were not associated with adverse clinical events or elevation of inflammation markers. Consistent herewith, CD8+ T cells in the infiltrates lacked cytotoxic potential. Furthermore, FoxP3+/CD4+ T cells were found in the infiltrates, suggesting that multiple mechanisms contribute to local tolerance. Systemic and local immune responses induced by intramuscular injection of alipogene tiparvovec did not appear to have an impact on safety and did not prevent LPL transgene expression. These findings support the use of alipogene tiparvovec in individuals with LPLD and indicate that muscle-directed AAV-based gene therapy remains a promising approach for the treatment of human diseases.

Teaching tolerance: New approaches to enzyme replacement therapy for Pompe disease.

Babies born with Pompe disease require life-long treatment with enzyme-replacement therapy (ERT). Despite the human origin of the therapy, recombinant human lysosomal acid α glucosidase (GAA, rhGAA), ERT unfortunately leads to the development of high titers of anti-rhGAA antibody, decreased effectiveness of ERT, and a fatal outcome for a significant number of children who have Pompe disease. The severity of disease, anti-drug antibody (ADA) development, and the consequences thereof are directly related to the degree of the enzyme deficiency. Babies born with a complete deficiency GAA are said to have cross-reactive immunologic material (CRIM)-negative Pompe disease and are highly likely to develop GAA ADA. Less frequently, GAA ADA develop in CRIM-positive individuals. Currently, GAA-ADA sero-positive babies are treated with a combination of immunosuppressive drugs to induce immunological tolerance to ERT, but the long-term effect of these regimens is unknown. Alternative approaches that might redirect the immune response toward antigen-specific tolerance without immunosuppressive agents are needed. Methods leading to the induction of antigen-specific regulatory T cells (Tregs), using peptides such as Tregitopes (T regulatory cell epitopes) are under consideration for the future treatment of CRIM-negative Pompe disease. Tregitopes are natural T cell epitopes derived from immunoglobulin G (IgG) that cause the expansion and activation of regulatory T cells (Treg). Teaching the immune system to tolerate GAA by co-delivering GAA with Tregitope peptides might dramatically improve the lives of CRIM-negative babies and could be applied to other enzyme replacement therapies to which ADA have been induced.

Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis.

AIM: To explore the anti-inflammatory potential of adeno-associated virus-mediated delivery of Tregitope 167 in an experimental colitis model. METHODS: The trinitrobenzene sulfonate (TNBS) model of induced colitis was used in Balb/c mice. Subsequently after intravenous adeno-associated virus-mediated regulatory T-cell epitopes (Tregitope) delivery, acute colitis was initiated by intra-rectal administration of 1.5 mg TNBS in 40% ethanol followed by a second treatment with TNBS (0.75 mg in 20% ethanol) 8 d later. Control groups included mice not treated with TNBS (healthy control group) and mice treated by TNBS only (diseased group). At the time of sacrifice colon weight, the disease activity index and histology damage score were determined. Immunohistochemical staining of the colonic tissues was performed to asses the cellular infiltrate and the presence of transcription factor forkhead Box-P3 (Foxp3). Thymus, mesenteric lymph nodes, liver and spleen tissue were collected and the corresponding lymphocyte populations were further assessed by flow cytometry analysis for the expression of CD4+ T cell and regulatory T cell associated markers. RESULTS: The Tregitope 167 treated mice gained an average of 4% over their initial body weight at the time of sacrifice. In contrast, the mice treated with TNBS alone (no Tregitope) developed colitis, and lost 4% of their initial body weight at the time of sacrifice (P < 0.01). The body weight increase that had been observed in the mice pre-treated with Tregitope 167 was substantiated by a lower disease activity index and a decreased colon weight as compared to the diseased control group (P < 0.01 and P < 0.001, respectively). Immunohistochemical staining of the colonic tissues for CD4+ showed that inflammatory cell infiltrates were present in TNBS treated mice with or without administration with tregitope 167 and that these cellular infiltrates consisted mainly of CD4+ cells. For both TNBS treated groups CD4+ T cell infiltrates were observed in the sub-epithelial layer and the lamina propria. CD4+ T cell infiltrates were also present in the muscularis mucosa layer of the diseased control mice, but were absent in the Tregitope 167 treated group. Numerous Foxp3 positive cells were detected in the lamina propria and sub-epithelium of the colon sections from mice treated with Tregitope 167. Furthermore, the Foxp3 and glycoprotein A repetitions predominant markers were significantly increased in the CD4+ T lymphocyte population in the thymus of the mice pre-treated with adeno-associated virus serotype 5 (cytomegalovirus promoter-Tregitope 167), as cytomegalovirus promoter compared to lymphocyte populations in the thymus of diseased and the healthy control mice (P < 0.05 and P < 0.001, respectively). CONCLUSION: This study identifies adeno-associated virus-mediated delivery of regulatory T-cell epitope 167 as a novel anti-inflammatory approach with the capacity to decrease intestinal inflammation and induce long-term remission in inflammatory bowel disease.