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Front Matter |
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Abstract
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Abstract
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Small Scale Production of Recombinant Adeno-Associated Viral Vectors for Gene Delivery to the Nervou |
Joost Verhaagen,Barbara Hobo,Erich M. E. Ehlert,Ruben Eggers,Joanna A. Korecka,Stefan A. Hoyng,Calla |
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Abstract
Adeno-associated viral vectors have numerous applications in neuroscience, including the study of gene function in health and disease, targeting of light-sensitive proteins to anatomically distinct sets of neurons to manipulate neuronal activity (optogenetics), and the delivery of fluorescent protein to study anatomical connectivity in the brain. Moreover several phase I/II clinical trials for gene therapy of eye and brain diseases with adeno-associated viral vectors have shown that these vectors are well tolerated by human patients. In this chapter we describe a detailed protocol for the small scale production of recombinant adeno-associated viral vectors. This protocol can be executed by investigators with experience in cell culture and molecular biological techniques in any well-equipped molecular neurobiology laboratory. With this protocol we typically obtain research batches of 100–200 μL that range in titer from 5 × 10. to 2 × 10. genomic copies/mL.
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Small and Micro-Scale Recombinant Adeno-Associated Virus Production and Purification for Ocular Gene |
Christopher A. Reid,Daniel M. Lipinski |
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Abstract
Over the past two decades recombinant adeno-associated virus (rAAV) vectors have emerged as the gold standard for transferring genetic material to cells of the retina. The ability to effectively produce small batches of rAAV vector at high enough purity for in vitro and in vivo applications in a cost-effective manner is paramount. This is particularly the case when conducting preclinical experiments to screen novel serotypes, promoters or transgenes, where production of numerous vector batches is required. Current vector production methods often produce large quantities of vector, limiting the cost-effectiveness and practicality of such screening experiments, which often require only small volumes of vector to carry out. Herein, we describe a method to produce high titer (10.–10. vector genomes (vg)/mL) rAAV vector on small (~100 μL) or micro (~15 μL) scale for in vitro and in vivo applications.
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Design and Development of AAV-based Gene Supplementation Therapies for Achromatopsia and Retinitis P |
Christian Schön,Elvir Becirovic,Martin Biel,Stylianos Michalakis |
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Abstract
Achromatopsia (ACHM) and retinitis pigmentosa (RP) are inherited disorders caused by mutations in cone and rod photoreceptor-specific genes, respectively. ACHM strongly impairs daylight vision, whereas RP initially affects night vision and daylight vision at later stages. Currently, gene supplementation therapies utilizing recombinant adeno-associated virus (rAAV) vectors are being developed for various forms of ACHM and RP. In this chapter, we describe the procedure of designing and developing specific and efficient rAAV vectors for cone- and rod-specific gene supplementation.
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Development of Multigenic Lentiviral Vectors for Cell-Specific Expression of Antiangiogenic miRNAs a |
Anne Louise Askou,Thomas J. Corydon |
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Abstract
Generation of lentivirus (LV)-based vectors holding multiple gene cassettes for coexpression of several therapeutic factors provides potent tools in both gene delivery studies as well as in gene therapy. Here we describe the development of such multigenic LV gene delivery vectors enabling cell-specific coexpression of antiangiogenic microRNA (miRNA) and protein factors and, if preferred, a fluorescent reporter, from RNApol(II)-driven expression cassettes orientated in a back-to-back fashion. This configuration may contribute to the development of new combination therapies for amelioration of diseases involving intraocular neovascularization including exudative age-related macular degeneration (AMD).
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Design and In Vitro Use of Antisense Oligonucleotides to Correct Pre-mRNA Splicing Defects in Inheri |
Alejandro Garanto,Rob W. J. Collin |
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Abstract
Antisense oligonucleotides (AONs) are small molecules able to bind to the pre-mRNA and modulate splicing. The increasing amount of intronic mutations leading to pseudoexon insertion in genes underlying inherited retinal dystrophies (IRDs) has highlighted the potential of AONs as a therapeutic tool for these disorders. Here we describe how to design and test AON molecules in vitro in order to correct pre-mRNA splicing defects involved in IRDs.
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Three-Dimensional Co-Culture Bioassay for Screening of Retinal Gene Delivery Systems |
Ding Wen Chen,Kathleen Pauloff,Marianna Foldvari D.Pharm.Sci., Ph.D. |
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Abstract
Herein we describe a three-dimensional co-culture bioassay protocol designed to assess the therapeutic potential of the proteins expressed from gene delivery transfected cells through the evaluation of expressed protein bioavailability and bioactivity. Using a combination of enzyme-linked immunosorbent assay (ELISA) and immunofluorescent-based neurite length profiling methodologies, the bioavailability of the secreted therapeutic protein in the medium can be quantitated, and the bioactivity of the secreted therapeutic protein can also be evaluated through neurite length profiling, respectively. The versatility and rationale of this bioassay could serve as a useful screening tool in the development of retinal gene delivery systems.
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Retinal Gene Therapy for Choroideremia: In Vitro Testing for Gene Augmentation Using an Adeno-Associ |
Maria I. Patrício,Robert E. MacLaren |
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Abstract
As gene therapy of choroideremia is becoming a clinical reality, there is a need for reliable and sensitive assays to determine the expression of exogenously delivered Rab Escort Protein-1 (REP1), in particular to test new gene therapy vectors and as a quality control screen for clinical vector stocks. Here we describe an in vitro protocol to test transgene expression following AAV2/2-REP1 transduction of a human cell line. Gene augmentation can be confirmed by western blot and quantification of the fold-increase of human REP1 levels over untransduced controls.
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Abstract
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In Vivo Electroporation of Developing Mouse Retina |
Jimmy de Melo,Seth Blackshaw |
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Abstract
In vivo electroporation enables the transformation of retinal tissue with engineered DNA plasmids, facilitating the selective expression of desired gene products. This method achieves plasmid transfer via the application of an external electrical field, which both generates a transient increase in the permeability of cell plasma membranes, and promotes the incorporation of DNA plasmids by electrophoretic transfer through the permeabilized membranes. Here we describe a method for the preparation, injection, and electroporation of DNA plasmids into neonatal mouse retinal tissue. This method can be utilized to perform gain of function or loss of function studies in the mouse. Experimental design is limited only by construct availability.
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Methods for In Vivo CRISPR/Cas Editing of the Adult Murine Retina |
Sandy S. Hung,Fan Li,Jiang-Hui Wang,Anna E. King,Bang V. Bui,Guei-Sheung Liu,Alex W. Hewitt |
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Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) is used by some bacteria and most archaea to protect against viral phage intrusion and has recently been adapted to allow for efficient editing of the mammalian genome. Whilst CRISPR/Cas-based technology has been used to modify genes in mammalian cells in vitro, delivery of CRISPR/Cas system into mammalian tissue and/or organs is more difficult and often requires additional vectors. With the use of adeno-associated virus (AAV) gene delivery system, active CRISPR/Cas enzyme can be maintained for an extended period of time and enable efficient editing of genome in the retina in vivo. Herein we outline the method to edit the genome in mouse retina using a dual AAV vector-mediated CRISPR/Cas9 system.
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AAV Gene Augmentation Therapy for ,-Associated Retinitis Pigmentosa |
C. Henrique Alves,Jan Wijnholds |
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Abstract
Mutations in the . gene account for around 10,000 persons with Leber congenital amaurosis (LCA) and 70,000 persons with retinitis pigmentosa (RP) worldwide. Therefore, the . gene is a key target in the fight against blindness. A proof-of-concept for an adeno-associated virus (AAV)-mediated . gene augmentation therapy for .-RP was recently described. Preclinical studies using animal models such as knockout or mutant mice are crucial to obtain such proof-of-concept. In this chapter we describe a technique to deliver AAV vectors, into the murine retinas, via the subretinal route. We also present protocols to detect expression of the therapeutic protein by fluorescence immunohistochemistry and to perform histological studies using ultra-thin sections stained with toluidine blue. These techniques in combination with electroretinography and visual behavior tests are in principle sufficient to obtain proof-of-concept for new gene therapies.
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Dual AAV Vectors for Stargardt Disease |
Ivana Trapani |
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Abstract
Stargardt disease (STGD1), due to mutations in the large . gene, is the most common inherited macular degeneration in humans. Attempts at developing gene therapy approaches for treatment of STGD1 are currently ongoing. Among all the vectors available for gene therapy of inherited retinal diseases, those based on adeno-associated viruses (AAV) are the most promising given the efficacy shown in various animal models and their excellent safety profile in humans, as confirmed in many ongoing clinical trials. However, one of the main obstacles for the use of AAV is their limited effective packaging capacity of about 5 kb. Taking advantage of the AAV genome’s ability to concatemerize, others and we have recently developed dual AAV vectors to overcome this limit. We tested dual AAV vectors for . delivery, and found that they transduce efficiently both mouse and pig photoreceptors, and rescue the . mouse retinal phenotype, indicating their potential for gene therapy of STGD1. This chapter details how we designed dual AAV vectors for the delivery of the . gene and describes the techniques that can be explored to evaluate dual AAV transduction efficiency in vitro and in the retina, and their
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Optogenetic Retinal Gene Therapy with the Light Gated GPCR Vertebrate Rhodopsin |
Benjamin M. Gaub,Michael H. Berry,Meike Visel,Amy Holt,Ehud Y. Isacoff,John G. Flannery |
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Abstract
In retinal disease, despite the loss of light sensitivity as photoreceptors die, many retinal interneurons survive in a physiologically and metabolically functional state for long periods. This provides an opportunity for treatment by genetically adding a light sensitive function to these cells. Optogenetic therapies are in development, but, to date, they have suffered from low light sensitivity and narrow dynamic response range of microbial opsins. Expression of light-sensitive G protein coupled receptors (GPCRs), such as vertebrate rhodopsin, can increase sensitivity by signal amplification, as shown by several groups. Here, we describe the methods to (1) express light gated GPCRs in retinal neurons, (2) record light responses in retinal explants in vitro, (3) record cortical light responses in vivo, and (4) test visually guided behavior in treated mice.
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CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa: A Brief Met |
Wen-Hsuan Wu,Yi-Ting Tsai,Sally Justus,Galaxy Y. Cho,Jesse D. Sengillo,Yu Xu,Thiago Cabral,Chyuan-Sh |
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Abstract
CRISPR/Cas9 genome engineering is currently the leading genome surgery technology in most genetics laboratories. Combined with other complementary techniques, it serves as a powerful tool for uncovering genotype–phenotype correlations. Here, we describe a simplified protocol that was used in our publication, ., providing an overview of each section of the experimental process.
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In-Depth Functional Analysis of Rodents by Full-Field Electroretinography |
Vithiyanjali Sothilingam,Regine Mühlfriedel,Naoyuki Tanimoto,Mathias W. Seeliger |
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Abstract
Full-field electroretinography (ERG) belongs to the gold-standard of electrophysiological test systems in ophthalmology and reflects the sum response of the entire retina to light stimulation. The assessment of the retinal function is a fundamental diagnostic technique not only in the clinical ophthalmology it is also indispensable in the ophthalmic research, in particular, in therapeutic approaches where the in vivo follow up of the benefit after treatment is absolutely necessary. Several current therapeutic approaches have demonstrated long-lasting amelioration in respective disease models and show promise for a successful translation to human patients. In this chapter we provide electroretinography protocols of experimental data which may serve as informative features for upcoming gene therapeutic approaches and clinical trials.
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Advanced Ocular Injection Techniques for Therapy Approaches |
Regine Mühlfriedel,Marina Garcia Garrido,Christine Wallrapp,Mathias W. Seeliger |
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Abstract
Treatment approaches for inherited eye diseases require local therapeutic molecule delivery by intraocular injection. One important factor that can influence the study outcome is the quality of intraocular administration. The intracompartmental structure (e.g., vitreous) of the eye allows a sustainable release of therapeutic biologicals using an intravitreal delivery. The protocol described here aims at providing the details relevant to perform a transscleral . intravitreal transfer in small eyes using a genetically modified stem cell system. The fact that cells and therewith visually distinct particles are implanted, allows for the assessment of the implantation site and the distribution, and possibilities for temporal follow up studies—hence, valuable information becomes available which can be used to fine-tune the intravitreal delivery technique.
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Neutralizing Antibodies Against Adeno-Associated Virus (AAV): Measurement and Influence on Retinal G |
Mélissa Desrosiers,Deniz Dalkara |
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Abstract
Adeno-associated viral vectors have become widely used in the clinic for retinal gene therapy. Thanks to AAVs impeccable safety profile and positive functional outcomes in its clinical application, interest in retinal gene therapy has increased exponentially over the past decade. Although early clinical trials have shown there is little influence of neutralizing antibodies on the performance of AAV when vector is administered into the subretinal space, recent findings suggest neutralizing antibodies may play a role when AAV is delivered via the intravitreal route. These findings highlight the importance of microenvironment on gene delivery and stress the need for a versatile assay to screen subjects for the presence of AAV-neutralizing antibodies. Measuring NAb titers against AAV prior and after gene therapy will help us better understand the impact of preexisting immunity on gene transfer, especially when the vector is administered intravitreally.
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Screening for Neutralizing Antibodies Against Natural and Engineered AAV Capsids in Nonhuman Primate |
Timothy P. Day,Leah C. Byrne,John G. Flannery,David V. Schaffer |
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Abstract
Adeno-associated virus (AAV) has shown promise as a therapeutic gene delivery vector for inherited retinal degenerations in both preclinical disease models and human clinical trials. The retinas of nonhuman primates (NHPs) share many anatomical similarities to humans and are an important model for evaluating AAV gene delivery. Recent evidence has shown that preexisting immunity in the form of neutralizing antibodies (NABs) in NHPs strongly correlates with weak or lack of AAV transduction in the retina when administered intravitreally, work with translational implications. This necessitates prescreening of NHPs before intravitreal delivery of AAV. In this chapter, we describe a method for screening NHP serum for preexisting NABs.
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发表于 2025-3-21 20:03:49
