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PubMed2006

Metabolic biotinylation provides a unique platform for the purification and targeting of multiple AAV vector serotypes.

The development of rationally designed targeted gene delivery vectors is an important focus for gene therapy. While genetic modification of AAV can produce vectors with modified tropism, incorporation of targeting peptides into the structural context of the AAV virion often results in loss of function or loss of virion integrity. To address this issue, we have developed a targeting system using metabolically biotinylated AAV. We generated serotype 1, 2, 3, 4, and 5 AAV capsids with small peptide insertions that are metabolically biotinylated in packaging cells during vector production by coexpression of the Escherichia coli BirA, biotin ligase, gene. Biotin moieties are exposed on the surface of assembled AAV particles and can interact with avidin. Metabolically biotinylated AAV vectors produced in this manner maintained endogenous titer and tissue tropism, could be purified on monomeric avidin resin, and could be retargeted to cells engineered to express an artificial avidin-biotin receptor. This technology provides not only a single platform for the purification of multiple AAV vector serotypes, but also a means for the development of multiple targeted AAV vectors utilizing a single capsid modification via straightforward avidin-biotin ligand coupling.

Amino Acid Sequence↗
PubMed1999

Recombinant adeno-associated virus (AAV) drives constitutive production of glutamate decarboxylase in neural cell lines.

Many neurological disorders result directly or indirectly from the loss of inhibitory function. Engineering the production of GABA, an inhibitory neurotransmitter, may therefore be able at least partly to restore the lost inhibition seen in epilepsy, Parkinson's disease, or Huntington's disease. In this article, we describe a set of recombinant adeno-associated viruses (AAVs) that can deliver cDNAs encoding the GABA-producing enzyme, glutamate decarboxylase (GAD), directly into neural cells. We have characterized these recombinant AAVs in several cell lines derived from the CNS. These recombinant AAVs effectively transduced all neural cell lines, although with different efficiencies. Transduction occurred in both proliferating and nonproliferating cells, but actively proliferating cell lines had approximately six times greater transduction efficiency than nonproliferating cells. Furthermore, these AAVs maintained long-term expression of GAD in an astrocytic cell line for at least seven passages. These recombinant AAVs are promising vehicles for investigating the potential therapeutic effects of GABA in animal models of epilepsy and neurodegenerative diseases.

Animals↗
PubMed2010

Sites in the AAV5 capsid tolerant to deletions and tandem duplications.

Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >10(5) members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands.

Amino Acid Sequence↗
PubMed2005

Genetically modified dendritic cells for cancer immunotherapy.

The ability to grow and differentiate dendritic cells (DC) ex vivo has allowed their genetic manipulation to enhance immune activation against tumor antigens. Gene engineering of DC can be achieved with a variety of physical methods and using different viral vectors. RNA or DNA transfection, either alone (naked), coated with liposomes or using electroporation or gene guns leads to T cell activation while transgene expression is frequently undetectable. Adenoviral and retroviral vectors have proven to be highly efficient in DC genetic modification, and have been widely used in preclinical models. Other vectors like lentivirus, poxvirus, herpes virus and adeno-associated virus (AAV) can also lead to foreign transgene expression in DC leading to immune cell activation. DC have been genetically engineered to provide constitutive and high level of tumor antigen expression or to introduce genes that further enhance their immune stimulatory ability. The promising results from preclinical animal models and from in vitro human immune cell culture systems have provided a strong rationale to initiate pilot clinical trials. Recently published or communicated clinical experiences and ongoing trials have used defined tumor antigen RNA transfection for prostate carcinoma and melanoma, liposome-encoated DNA transfection for breast or pancreatic cancer, adenoviral vector tumor antigen gene modification for melanoma and small cell lung cancer, and poxvirus-mediated expression of costimulatory molecules for colon carcinoma. These preliminary experiences suggest that genetically modified DC can safely induce T cell responses but few clinical responses.

Animals↗
PubMed2004

Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD).

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in the dystrophin gene. The size of the gene (2.4 Mb) and mRNA (14 kb) in addition to immunogenicity problems and inefficient transduction of mature myofibres by currently available vector systems are formidable obstacles to the development of efficient gene therapy approaches. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems (nonpathogenicity and minimal immunogenicity, extensive cell and tissue tropism) but accommodate limited transgene capacity (<5 kb). As a result of these observations, a number of laboratories worldwide have engineered a series of microdystrophin cDNAs based on genotype-phenotype relationship in Duchenne (DMD) and Becker (BMD) dystrophic patients, and transgenic studies in mdx mice. Recent progress in characterization of AAV serotypes from various species has demonstrated that alternative AAV serotypes are far more efficient in transducing muscle than the traditionally used AAV2. This article summarizes the current progress in the field of recombinant adeno-associated viral (rAAV) delivery for DMD, including optimization of recombinant AAV-microdystrophin vector systems/cassettes targeting the skeletal and cardiac musculature.

Animals↗
PubMed2012

The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting.

Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord-derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3'-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3'-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications.

Blotting, Western↗
PubMed2002

The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.

The structure of the adeno-associated virus (AAV-2) has been determined to 3-A resolution by x-ray crystallography. AAV is being developed as a vector for gene therapy to treat diseases including hemophilia, cancer, and cystic fibrosis. As in the distantly related autonomous parvoviruses, the capsid protein has a beta-barrel fold, but long loops between the beta-strands share little structural homology with other parvoviruses, leading to unique surface features. Most prominent are groups of threefold-related peaks, each an intimate association of loops from two neighboring subunits. Mutations affecting cell entry and receptor binding are clustered near the positively charged side of each peak, implicating the region in attachment to the cellular receptor, heparan sulfate proteoglycan. Amino acids involved in antibody binding are in the same general vicinity. The structure will guide rational engineering of vector capsids to tailor cellular targeting and to avoid immediate neutralization by an immune system sensitized by prior exposure to AAV.

Amino Acid Sequence↗
PubMed2021

AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset.

Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood-brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.

Administration, Intravenous↗
PubMed2002

Tropism-modified adenoviral and adeno-associated viral vectors for gene therapy.

One of the most rapidly advancing areas of gene therapy is vector development. For the majority of gene therapy procedures, efficient and selective transduction would provide safe and more effective treatments at optimal vector doses. Advances in vector targeting strategies have been rapid within the field of DNA-based viruses, particularly adenovirus (Ad) and more recently adeno-associated virus (AAV) based vectors. Vector targeting at the level of virus: cell interaction can be achieved using both non-genetic and genetic methodology. Non-genetic approaches typically utilise bispecific antibodies that both neutralise wild-type virus tropism and provide a new cell binding capacity. For genetic targeting strategies, the virus capsid can be engineered to express foreign ligands that target selected receptors in the absence or presence of additional modification to ablate the virus' natural tropism. This review covers technological advances that have led to targeting of Ad and AAV and highlights the potential for these 'designer' viruses for future gene-based therapeutics.

Adenoviridae↗
PubMed2006

Viral gene therapy.

Cancer is a multigenic disorder involving mutations of both tumor suppressor genes and oncogenes. A large body of preclinical data, however, has suggested that cancer growth can be arrested or reversed by treatment with gene transfer vectors that carry a single growth inhibitory or pro-apoptotic gene or a gene that can recruit immune responses against the tumor. Many of these gene transfer vectors are modified viruses. The ability for the delivery of therapeutic genes, made them desirable for engineering virus vector systems. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Because these vector systems have unique advantages and limitations, each has applications for which it is best suited. Retroviral vectors can permanently integrate into the genome of the infected cell, but require mitotic cell division for transduction. Adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types, but immune elimination of infected cells often limits gene expression in vivo. Herpes simplex virus can deliver large amounts of exogenous DNA; however, cytotoxicity and maintenance of transgene expression remain as obstacles. AAV also infects many non-dividing and dividing cell types, but has a limited DNA capacity. This review discusses current and emerging virusbased genetic engineering strategies for the delivery of therapeutic molecules or several approaches for cancer treatment.

Antineoplastic Agents↗
PubMed2013

A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

BACKGROUND: The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. METHODS: Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . RESULTS: We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. CONCLUSIONS: In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

Animals↗
PubMed2010

AAV's anatomy: roadmap for optimizing vectors for translational success.

Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success.

Cystic Fibrosis↗
PubMed2002

Replication competent helper functions for recombinant AAV vector generation.

Adeno-associated virus (AAV) is a promising gene transfer vector tested in both animal studies and human clinical trials. However, current production methods are generally inefficient and require improvements to meet the increasing clinical need for economical, high titer and high quality rAAV vectors. The inefficiency of the current systems largely arises from the AAV helper function, which contains only the AAV coding region but lacks inverted terminal repeats. The terminal repeats were originally removed to prevent replication competent AAV contamination. Here we designed a novel and highly efficient rAAV helper function containing AAV terminal repeats. The new helper function not only mimics the wild-type AAV growth as it replicates along with the vector plasmid, but also restores the cis regulating function of the AAV terminal repeats. Addition of heterologous introns to the helper genome and use of a mutant AAV terminal repeat defective in packaging effectively controls the contamination of replication competent AAV particles. This new strategy also performs better in AAV producing cell lines than those based on non-replicating AAV rep and cap genome.

Cell Line↗
PubMed2005

Therapeutic benefit of TH-engineered mesenchymal stem cells for Parkinson's disease.

The present study was designed to assess the potential of marrow stromal cells (MSCs) to deliver therapeutic genes to the brain and result in biologically significant functional recovery. The tyrosine hydroxylase (TH) gene was transfected to MSCs with an adeno-associated virus (AAV) vector. MSCs expressing TH gene were transplanted into the striatum of Parkinson's disease (PD) rat. The asymmetric rotation of these models after apomorphine administration was detected every week after transplantation. Six weeks after grafting, animals were sacrificed. Some brains were sectioned to do TH immunohistochemistry. The others were used to detect the dopamine levels by high-performance liquid chromatograph and electrochemical detection (HPLC-ECD). The results showed that MSCs multiply rapidly and formed fibroblast colony-forming units in primary culture. The gene expression efficiency was about 75%. The rounds of asymmetric rotation after apomorphine administration decreased after TH-engineered MSCs were grafted. Histological examination showed that TH gene was expressed around the transplantation points. The dopamine level in the lesioned striatum of rats injected with TH-MSCs was significantly greater than that in rats treated with LacZ-MSCs (P < 0.05). All the data demonstrated that MSCs could readily be genetically engineered. Therefore, MSCs could be useful gene delivery vehicles of gene therapy for Parkinson's disease.

Animals↗
PubMed1997

Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson's disease.

Current approaches to gene therapy of CNS disorders include grafting genetically modified autologous cells or introducing genetic material into cells in situ using a variety of viral or synthetic vectors to produce and deliver therapeutic substances to specific sites within the brain. Here we discuss issues related to the application of ex-vivo and in-vivo gene therapies as possible treatments for Parkinson's disease. Autologous monkey fibroblasts engineered ex-vivo to express tyrosine hydroxylase were grafted into MPTP-treated monkeys and found to express for up to 4 months. Adeno-associated (AAV) viral vectors expressing beta-galactosidase or tyrosine hydroxylase were introduced into monkey brains to determine the extent of infection and the types of cells infected by the vector at 21 days and 3 months. Gene expression was detected at both time points and was restricted to neurons in the striatum. These experiments demonstrate that two different approaches can be used to deliver proteins into the CNS. However, further technological advances are required to optimize gene delivery, regulation of gene expression, and testing in appropriate functional models before gene therapy can be considered for treating human disease.

Animals↗
PubMed2025

Gene regulation technologies for gene and cell therapy.

Gene therapy stands at the forefront of medical innovation, offering unique potential to treat the underlying causes of genetic disorders and broadly enable regenerative medicine. However, unregulated production of therapeutic genes can lead to decreased clinical utility due to various complications. Thus, many technologies for controlled gene expression are under development, including regulated transgenes, modulation of endogenous genes to leverage native biological regulation, mapping and repurposing of transcriptional regulatory networks, and engineered systems that dynamically react to cell state changes. Transformative therapies enabled by advances in tissue-specific promoters, inducible systems, and targeted delivery have already entered clinical testing and demonstrated significantly improved specificity and efficacy. This review highlights next-generation technologies under development to expand the reach of gene therapies by enabling precise modulation of gene expression. These technologies, including epigenome editing, antisense oligonucleotides, RNA editing, transcription factor-mediated reprogramming, and synthetic genetic circuits, have the potential to provide powerful control over cellular functions. Despite these remarkable achievements, challenges remain in optimizing delivery, minimizing off-target effects, and addressing regulatory hurdles. However, the ongoing integration of biological insights with engineering innovations promises to expand the potential for gene therapy, offering hope for treating not only rare genetic disorders but also complex multifactorial diseases.

Humans↗
PubMed2017

Future of rAAV Gene Therapy: Platform for RNAi, Gene Editing, and Beyond.

The use of recombinant adeno-associated viruses (rAAVs) ushered in a new millennium of gene transfer for therapeutic treatment of a number of conditions, including congenital blindness, hemophilia, and spinal muscular atrophy. rAAV vectors have remarkable staying power from a therapeutic standpoint, withstanding several ebbs and flows. As new technologies such as clustered regularly interspaced short palindromic repeat genome editing emerge, it is now the delivery tool-the AAV vector-that is the stalwart. The long-standing safety of this vector in a multitude of clinical settings makes rAAV a selling point in the advancement of approaches for gene replacement, gene knockdown, gene editing, and genome modification/engineering. The research community is building on these advances to develop more tailored delivery approaches and to tweak the genome in new and unique ways. Intertwining these approaches with newly engineered rAAV vectors is greatly expanding the available tools to manipulate gene expression with a therapeutic intent.

Dependovirus↗