Microvesicle-associated AAV vector as a novel gene delivery system.
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ABSTRACT:
SUBMITTER: Maguire CA
PROVIDER: S-EPMC3345986 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cystic fibrosis is an autosomal-recessive disease that is caused by a mutant CFTR (cystic fibrosis transmembrane conductance regulator) gene and is characterized by chronic bacterial lung infections and inflammation. Complementation with functional CFTR normalizes anion transport across the airway surface. Adeno-associated virus (AAV) is a useful vector for gene therapy because of its low immunogenicity and ability to persist for months to years. However, because its episomal expression may decrease after cell division, readministration of the AAV vector may be required. To overcome this, we designed an integrating AAV-based CFTR-expressing vector, termed piggyBac (PB)/AAV, carrying CFTR flanked by the terminal repeats of the piggyBac transposon. With codelivery of the piggyBac transposase, PB/AAV can integrate into the host genome. Because of the packaging constraints of AAV, careful consideration was required to ensure that the vector would package and express its CFTR cDNA cargo. In this short-term study, PB/AAV-CFTR was aerosolized to the airways of CF pigs in the absence of the transposase. Two weeks later, transepithelial Cl- current was restored in freshly excised tracheal and bronchial tissue. Additionally, we observed an increase in tracheal airway surface liquid pH and bacterial killing in comparison with untreated CF pigs. Airway surface liquid from primary airway cells cultured from treated CF pigs exhibited increased pH correlating with decreased viscosity. Together, these results show that complementing CFTR in CF pigs with PB/AAV rescues the anion transport defect in a large-animal CF model. Delivery of this integrating viral vector system to airway progenitor cells could lead to persistent, life-long expression in vivo.
| S-EPMC6890402 | biostudies-literature
Project description:Lung carcinoma is the most common type of cancer and the leading cause of cancer-related death worldwide. Among the numerous therapeutic strategies for the treatment of lung cancer, adeno-associated virus (AAV)-mediated gene transfer has been demonstrated to have the potential to effectively suppress tumor growth or reverse the progression of the disease in a number of preclinical studies. AAV vector has a safety profile; however, the relatively low delivery efficacy to particular subtypes of lung carcinoma has limited its prospective clinical translation. Exosomes are nanosized extracellular vesicles secreted from nearly all known cell types. Exosomes have a membrane-enclosed structure carrying a range of cargo molecules for efficient intercellular transfer of functional entities, thus are considered as a superior vector for drug delivery. In the present study, we developed a novel strategy to produce and purify AAV-containing exosomes (AAVExo) from AAV-packaging HEK 293T cells. The cellular uptake capacity of exosomes assisted and enhanced AAV entry into cells and protected AAV from antibody neutralization, which was a serious challenge for AAV in vivo application. We tested a list of lung cancer cell lines representing non-small-cell lung cancer and small-cell lung cancer and found that AAVExo apparently improved the gene transfer efficiency compared to conventional AAV vector. Our in vitro results were supported in vivo in a lung cancer xenograft rodent model. Additionally, we evaluated the gene delivery efficiency in the presence of neutralizing antibody on lung cancer cells. The results demonstrated that AAVExo-mediated gene transfer was not impacted, while the AAV vectors were significantly blocked by the neutralizing antibody. Taken together, we established an efficient methodology for AAVExo purification, and the purified AAVExo largely enhanced gene delivery to lung cancer cells with remarkable resistance to antibody neutralization.
| S-EPMC8414974 | biostudies-literature
Project description:There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies. The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.
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Project description:Accumulation of amyloid-? peptide (A?) in the brain is closely associated with cognitive decline in Alzheimer's disease (AD). Stereotaxic infusion of neprilysin-encoding viral vectors into the hippocampus has been shown to decrease A? in AD-model mice, but more efficient and global delivery is necessary to treat the broadly distributed burden in AD. Here we developed an adeno-associated virus (AAV) vector capable of providing neuronal gene expression throughout the brains after peripheral administration. A single intracardiac administration of the vector carrying neprilysin gene in AD-model mice elevated neprilysin activity broadly in the brain, and reduced A? oligomers, with concurrent alleviation of abnormal learning and memory function and improvement of amyloid burden. The exogenous neprilysin was localized mainly in endosomes, thereby effectively excluding A? oligomers from the brain. AAV vector-mediated gene transfer may provide a therapeutic strategy for neurodegenerative diseases, where global transduction of a therapeutic gene into the brain is necessary.
| S-EPMC3600598 | biostudies-literature
Project description:Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness by middle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab escort protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no established treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-associated virus (AAV) encoding the CHM gene.
| S-EPMC4355255 | biostudies-literature
Project description:Gene delivery using adeno-associated virus (AAV) vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson's disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson's disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson's disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson's disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV-a-synuclein (a-syn) to target substantia nigra dopamine neurons to produce an ?-syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP), which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in the substantia nigra.
| S-EPMC5333052 | biostudies-other
Project description:The Adeno-associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ?40-70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded ?-barrel core and ?A helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral twofold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ?61%, ?81%, and ?63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system.
| S-EPMC4085184 | biostudies-literature
Project description:In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8(+) T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγ(null) (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45(+) cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8(+) T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8(+) T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting.
| S-EPMC3916402 | biostudies-literature
Project description:Human gene therapy has advanced from twentieth-century conception to twenty-first-century reality. The recombinant Adeno-Associated Virus (rAAV) is a major gene therapy vector. Research continues to improve rAAV safety and efficacy using a variety of AAV capsid modification strategies. Significant factors influencing rAAV transduction efficiency include neutralizing antibodies, attachment factor interactions and receptor binding. Advances in understanding the molecular interactions during rAAV cell entry combined with improved capsid modulation strategies will help guide the design and engineering of safer and more efficient rAAV gene therapy vectors.
| S-EPMC8310307 | biostudies-literature
Project description:Gene therapy to the gastrointestinal tract has remarkable potential for treating gastrointestinal disorders that currently lack effective treatments. Adeno-associated viral vectors (AAVs) have been extensively applied to the central nervous system, and have repeatedly demonstrated safety and efficacy in animal models. The enteric nervous system (ENS) represents a vast collection of neurons and glial cells that may also be subject to treatment by AAV, however little work has been conducted on AAV delivery to the ENS. Challenges for gastrointestinal gene therapy include identifying gene targets, optimizing gene delivery, and target cell selection. Researchers are now beginning to tackle the later of the two challenges with AAV, and the same AAV technology can be used to identify novel gene targets in the future. Continued efforts to understand AAV delivery and improve vector design are essential for therapeutic development. This review summarizes the current knowledge about AAV delivery to the ENS.
| S-EPMC5001153 | biostudies-other