Selection of an Efficient AAV Vector for Robust CNS Transgene Expression.
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ABSTRACT:
SUBMITTER: Hanlon KS
PROVIDER: S-EPMC6881693 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.
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Project description:The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene expression cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic expression cassette. As with the minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene expression compared with minicircle vectors containing the same expression cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches.
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Project description:Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill and time-intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with standard AAV. Here, we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood-brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV-transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.
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Project description:Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137) in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities.
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Project description:Recombinant adeno-associated viral (rAAV) vectors exhibit interesting properties as vaccine carriers for their ability to induce long-lasting antibody responses. However, rAAV-based vaccines have been suggested to trigger functionally impaired long-term memory CD8+ T cell responses, in part due to poor dendritic cell (DC) transduction. Such results, albeit limited to intramuscular immunization, undermined the use of rAAV as vaccine vehicles against intracellular pathogens. We report here that intradermal immunization with a model rAAV2/1-based vaccine drives the development of bona fide long-term memory CD8+ T cell responses. The intradermal route of immunization and the presence of potent major histocompatibility complex (MHC) class II responses showed synergistic effects on the overall quantity and quality of systemic long-term effector memory transgene-specific CD8+ T cells being generated against the transgene. Of key interest, we found that the induction of memory cytotoxic T lymphocytes (CTLs) following intradermal immunization was solely dependent on the cross-presentation of skin-expressed transgene products, which appeared highly enhanced as compared to muscle-expressed transgene products. Overall our results highlight key tissue-specific differences in transgene presentation pathway requirements of importance for the design of rAAV-based T cell-inducing vaccines.
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Project description:Adeno-associated virus (AAV) vectors can deliver transgenes to diverse cell types and are therefore useful for basic research and gene therapy. Although AAV has many advantages over other viral vectors, its relatively small packaging capacity limits its use for delivering large genes. The available transgene size is further limited by the existence of additional elements in the expression cassette without which the gene expression level becomes much lower. By using alternative combinations of shorter elements, we generated a series of AAV expression cassettes and systematically evaluated their expression efficiency in neurons to maximize the transgene size available within the AAV packaging capacity while not compromising the transgene expression. We found that the newly developed smaller expression cassette shows comparable expression efficiency with an efficient vector generally used for strong gene expression. This new expression cassette will allow us to package larger transgenes without compromising expression efficiency.
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Project description:The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic.
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Project description:To better understand and exploit microRNA (miR) regulation, a more precise characterization of miR expression patterns within a tissue or a lineage during development, differentiation, and homeostasis is needed. We previously showed that lentiviral vectors (LV) can be made responsive to miR to stringently control transgene expression as well as to report miR activity "live" and at the single-cell level. Although very useful, this approach reports miR activity by transgene suppression, hampering the direct identification and selection of miR-expressing cells. Here, we describe a strategy to couple transgene expression to the activity of the miR of interest. To this aim, we generated LV encoding two in-series OFF switches: a transcriptional repressor tagged with miR target sequences and a reporter cassette under the control of the repressor. Reporter expression is ON only when the miR is active and represses translation of the transcriptional repressor. We successfully applied this design to different types of repressors, multiple gene encoding vectors and delivered the system either by two separate or a self-contained vector. We demonstrated its performance by live monitoring of two miRs in different stages of human primary hematopoietic stem/progenitor cell differentiation in vivo. Further applications of this approach include imaging of rare miR-expressing cells and positive regulation of a therapeutic or selector gene in target cells identified by the expression of selected miRs.
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Project description:Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6?vg/cell). After 24, 48, 72, and 96?h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6?vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.
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Project description:Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-? production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents.
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