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.

Complement System Response to Adeno-Associated Virus Vector Gene Therapy.

Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.

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.

Pre-existing humoral immunity and complement pathway contribute to immunogenicity of adeno-associated virus (AAV) vector in human blood.

AAV gene transfer is a promising treatment for many patients with life-threatening genetic diseases. However, host immune response to the vector poses a significant challenge for the durability and safety of AAV-mediated gene therapy. Here, we characterize the innate immune response to AAV in human whole blood. We identified neutrophils, monocyte-related dendritic cells, and monocytes as the most prevalent cell subsets able to internalize AAV particles, while conventional dendritic cells were the most activated in terms of the CD86 co-stimulatory molecule upregulation. Although low titers (≤1:10) of AAV neutralizing antibodies (NAb) in blood did not have profound effects on the innate immune response to AAV, higher NAb titers (≥1:100) significantly increased pro-inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. Interestingly, both full and empty viral particles were equally potent in inducing complement activation and cytokine secretion. By using a compstatin-based C3 and C3b inhibitor, APL-9, we demonstrated that complement pathway inhibition lowered CD86 levels on APCs, AAV uptake, and cytokine/chemokine secretion in response to AAV. Together these results suggest that the pre-existing humoral immunity to AAV may contribute to trigger adverse immune responses observed in AAV-based gene therapy, and that blockade of complement pathway may warrant further investigation as a potential strategy for decreasing immunogenicity of AAV-based therapeutics.

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.

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.

Gene and cell therapy based treatment strategies for inflammatory bowel diseases.

Inflammatory bowel diseases (IBD) are a group of chronic inflammatory disorders most commonly affecting young adults. Currently available therapies can result in induction and maintenance of remission, but are not curative and have sometimes important side effects. Advances in basic research in IBD have provided new therapeutic opportunities to target the inflammatory process involved. Gene and cell therapy approaches are suitable to prevent inflammation in the gastrointestinal tract and show therefore potential in the treatment of IBD. In this review, we present the current progress in the field of both gene and cell therapy and future prospects in the context of IBD. Regarding gene therapy, we focus on viral vectors and their applications in preclinical models. The focus for cell therapy is on regulatory T lymphocytes and mesenchymal stromal cells, their potential for the treatment of IBD and the progress made in both preclinical models and clinical trials.