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Science Translational Medicine Mar 2022Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in...
Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced Cas9 prime editor (PE) lacking the RNaseH domain (PE2) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the locus and were further elevated to 58% by delivering unsplit PE2 via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.
Topics: Animals; Dependovirus; Gene Editing; Liver Diseases; Mice; Phenylketonurias
PubMed: 35294257
DOI: 10.1126/scitranslmed.abl9238 -
Nature Communications Oct 2023Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and...
Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.
Topics: Humans; Animals; Mice; Trans-Splicing; Gene Editing; Genetic Therapy; Stargardt Disease; Genetic Vectors; Dependovirus; ATP-Binding Cassette Transporters
PubMed: 37852949
DOI: 10.1038/s41467-023-42386-0 -
Cell Reports Jun 2019Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging,...
Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.
Topics: Animals; Clustered Regularly Interspaced Short Palindromic Repeats; Dependovirus; Electroporation; Female; Gene Knock-In Techniques; Mice; Mice, Transgenic; Parvoviridae Infections; Ribonucleoproteins
PubMed: 31242412
DOI: 10.1016/j.celrep.2019.05.103 -
Annual Review of Virology Sep 2019The recent market approvals of recombinant adeno-associated virus (rAAV) gene therapies in Europe and the United States are landmark achievements in the history of...
The recent market approvals of recombinant adeno-associated virus (rAAV) gene therapies in Europe and the United States are landmark achievements in the history of modern science. These approvals are also anticipated to herald the emergence of a new class of therapies for monogenic disorders, which had hitherto been considered untreatable. These events can be viewed as stemming from the convergence of several important historical trends: the study of basic virology, the development of genomic technologies, the imperative for translational impact of National Institutes of Health-funded research, and the development of economic models for commercialization of rare disease therapies. In this review, these historical trends are described and the key developments that have enabled clinical rAAV gene therapies are discussed, along with an overview of the current state of the field and future directions.
Topics: Animals; Clinical Trials as Topic; Dependovirus; Genetic Therapy; Genetic Vectors; History, 20th Century; History, 21st Century; Humans
PubMed: 31283441
DOI: 10.1146/annurev-virology-092818-015530 -
Proceedings of the National Academy of... Aug 2023Tissue macrophages, including microglia, are notoriously resistant to genetic manipulation. Here, we report the creation of Adeno-associated viruses (AAV) variants that...
Tissue macrophages, including microglia, are notoriously resistant to genetic manipulation. Here, we report the creation of Adeno-associated viruses (AAV) variants that efficiently and widely transduce microglia and tissue macrophages in vivo following intravenous delivery, with transgene expression of up to 80%. We use this technology to demonstrate manipulation of microglia gene expression and microglial ablation, thereby providing invaluable research tools for the study of these important cells.
Topics: Dependovirus; Microglia; Capsid; Transgenes; Macrophages
PubMed: 37603759
DOI: 10.1073/pnas.2302997120 -
Molecular Therapy : the Journal of the... Feb 2016Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including...
Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector.
Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 10(5) vector genome containing particles (vg)/cell or greater than 1 × 10(14) vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 10(13) vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.
Topics: Batch Cell Culture Techniques; Bioreactors; Cell Adhesion; Culture Media, Serum-Free; Dependovirus; Genetic Vectors; HEK293 Cells; Humans; Vascular Endothelial Growth Factor Receptor-1
PubMed: 26437810
DOI: 10.1038/mt.2015.187 -
Blood Feb 2021In this issue of , Konkle et al report that 7 of 8 participants in a phase 1/2 trial of adeno-associated virus (AAV) vector (BAX335) for factor IX (FIX)-Padua gene...
In this issue of , Konkle et al report that 7 of 8 participants in a phase 1/2 trial of adeno-associated virus (AAV) vector (BAX335) for factor IX (FIX)-Padua gene transfer in patients with hemophilia B did not maintain expression despite steroid intervention, which the authors hypothesize is a result of the innate immune stimulatory effect of CpG motifs enriched within their vector cassette. Their study demonstrates that the cellular immune response to AAV vectors does not always respond to steroids and provides insight into mechanisms that may contribute to the AAV immune response with implications for the future design of AAV vectors.
Topics: Dependovirus; Gene Expression; Genetic Therapy; Hemophilia B; Humans; bcl-2-Associated X Protein
PubMed: 33570612
DOI: 10.1182/blood.2020009285 -
Quantification of adeno-associated virus particles and empty capsids by optical density measurement.Molecular Therapy : the Journal of the... Jan 2003We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid...
We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid proteins in solution. We determined the molar extinction coefficients of capsid protein to be 3.72 x 10(6) M(-1) cm(-1) at 260 nm and 6.61 x 10(6) M(-1) cm(-1) at 280 nm. For recombinant AAV vectors, extinction coefficients can be calculated by including the predicted absorbance of the vector DNA. Since the amount of empty capsids in purified vector preparations lowers the A(260)/A(280) ratio in a predictable manner, the vector genome (vg) and capsid particle (cp) titers in purified AAV vector preparations can be calculated from the absorbance at 260 nm and the A(260)/A(280) ratio. To validate this method, the vg and cp titers calculated by UV absorbance were compared with titers determined by quantitative (Q)-PCR and capsid ELISA. The vg titers determined by absorbance agreed well with titers determined by Q-PCR. The cp/vg ratio determined by the A(260)/A(280) method also correlated well with those determined by AAV capsid ELISA in conjunction with Q-PCR. This new method provides a simple and rapid means to determine AAV vg titers and the ratio of empty to full particles in purified virus preparations.
Topics: Capsid; Cell Line; Dependovirus; Genetic Vectors; Humans; Optics and Photonics; Polymerase Chain Reaction; Sensitivity and Specificity; Virion
PubMed: 12573625
DOI: 10.1016/s1525-0016(02)00019-9 -
Chemical Reviews Sep 2022Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery... (Review)
Review
Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery vector in emerging gene therapy treatments for inherited disorders, so its structure and molecular interactions with human hosts are of intense interest. A wide array of electron microscopic approaches have been used to visualize the virus and its complexes, depending on the scientific question, technology available, and amenability of the sample. Approaches range from subvolume tomographic analyses of complexes with large and flexible host proteins to detailed analysis of atomic interactions within the virus and with small ligands at resolutions as high as 1.6 Å. Analyses have led to the reclassification of glycan receptors as attachment factors, to structures with a new-found receptor protein, to identification of the epitopes of antibodies, and a new understanding of possible neutralization mechanisms. AAV is now well-enough characterized that it has also become a model system for EM methods development. Heralding a new era, cryo-EM is now also being deployed as an analytic tool in the process development and production quality control of high value pharmaceutical biologics, namely AAV vectors.
Topics: Cryoelectron Microscopy; Dependovirus; Epitopes; Genetic Therapy; Humans
PubMed: 35575684
DOI: 10.1021/acs.chemrev.1c00936 -
Molecular Therapy : the Journal of the... Dec 2023Recombinant adeno-associated viral (AAV) vectors are the current benchmark for systemic delivery of gene therapies to multiple organs in vivo. Despite clinical... (Review)
Review
Recombinant adeno-associated viral (AAV) vectors are the current benchmark for systemic delivery of gene therapies to multiple organs in vivo. Despite clinical successes, safe and effective gene delivery to extrahepatic tissues has proven challenging due to dose limiting toxicity arising from high liver uptake of AAV vectors. Deeper understanding of AAV structure, receptor biology, and pharmacology has enabled the design and engineering of liver-de-targeted capsids ushering in several new vector candidates. This next generation of AAVs offers significant promise for extrahepatic gene delivery to cardiovascular, musculoskeletal, and neurological tissues with improved safety profiles.
Topics: Dependovirus; Gene Transfer Techniques; Genetic Therapy; Capsid; Liver; Genetic Vectors
PubMed: 37805712
DOI: 10.1016/j.ymthe.2023.10.005