23 research outputs found
Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B
BACKGROUND: Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX). METHODS: Hemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The outcome of gene transfer was assessed, including transgene expression as well as antibody and CD8(+) T cell responses to hF.IX. RESULTS: Self-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX expression) but stronger CD8(+) T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a result, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9 nonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression regardless of the vector genome. CONCLUSIONS: The nature of the AAV genome can impact the CD8(+) T cell response to the therapeutic transgene product. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break tolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific immunity than the molecular form of the AAV genome
Common Security of the Czech Republic and Slovakia Airspace by JAS-39 GRIPEN Fighter Aircrafts
Diplomová práce se zabývá problematikou ochrany vzdušného prostoru České republiky a Slovenska nadzvukovými letouny JAS-39 Gripen. První část práce je zaměřena na teoretickou rovinu obrany jako služby v obecném zájmu nehospodářské povahy a na vybrané pojmy z oblasti obrany a bezpečnosti státu. Druhá část se zabývá komparací Armády České republiky a Ozbrojených sil Slovenské republiky se zaměřením na vzdušné síly a možnosti ochrany vzdušného prostoru pomoci nadzvukových bojových letounů obou zemí. Třetí částí je zhodnocení možnosti pořízení a provozování nadzvukových letounů JAS-39 Gripen Vzdušnými silami Ozbrojených sil Slovenské republiky, které vychází z předchozího zkoumání a zároveň přibližuje možnosti zabezpečení společného vzdušného prostoru obou zemí. V poslední části jsou doporučení pro společné zabezpečení vzdušného prostoru obou zemí, vycházející z jednotlivých možností, ale i se stávající bezpečnostní a ekonomické situace.This thesis deals with the airspace protection of Czech Republic and Slovakia by JAS-39 Gripen supersonic aircrafts. The first part is focused on the theoretical level of defense as a service of general non-economic interest and certain terms of defense and national security. The second part deals with the comparsion of the Czech Army and Slovak Army with focuse on options of airspace protection by supersonic fighter aircrafts of both countries. The third part evaluates the possibility of acquisition and usage JAS-39 supersonic aircraft by the Slovak Air Force which is based on previous research and shows how to secure the common airspace of both countries. In the final chapter are recommendations for common airspace security of both countries based on each option, but also on current security and economic situation.153 - Katedra veřejné ekonomikyvelmi dobř
Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity
Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies
Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy
Adeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector's DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs
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Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer
The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8) vector expressing cytoplasmic ovalbumin (OVA) into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal) are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages) and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products
Evaluation of engineered AAV capsids for hepatic factor IX gene transfer in murine and canine models
Cellular Proteins Required for Adeno-Associated Virus DNA Replication in the Absence of Adenovirus Coinfection
We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3′ primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase α-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase δ and ɛ, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase δ or polymerase ɛ for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete
MOESM1 of Evaluation of engineered AAV capsids for hepatic factor IX gene transfer in murine and canine models
Additional file 1: Table S1. Clinical chemistry panel for hemophilia B dog O19. Baseline values obtained prior to vector. Pre refers to blood collected on the day of vector administrations and post and onward refer to time points after vector delivery. Low values are colored blue, values within normal ranges are black, and values above normal ranges are red