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Scientific Reports Dec 2023Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy...
Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.
Topics: Humans; Capsid; Retina; Capsid Proteins; Genetic Therapy; Bioengineering; Dependovirus; Genetic Vectors; Transduction, Genetic
PubMed: 38081924
DOI: 10.1038/s41598-023-49112-2 -
Theranostics 2023Despite recent advances in the use of adeno-associated viruses (AAVs) as potential vehicles for genetic intervention of central and peripheral nervous system-associated...
Despite recent advances in the use of adeno-associated viruses (AAVs) as potential vehicles for genetic intervention of central and peripheral nervous system-associated disorders, gene therapy for the treatment of neuropathology in adults has not been approved to date. The currently FDA-approved AAV-vector based gene therapies rely on naturally occurring serotypes, such as AAV2 or AAV9, which display limited or no transport across the blood-brain barrier (BBB) if systemically administered. Recently developed engineered AAV variants have shown broad brain transduction and reduced off-target liver toxicity in non-human primates (NHPs). However, these vectors lack spatial selectivity for targeted gene delivery, a potentially critical limitation for delivering therapeutic doses in defined areas of the brain. The use of microbubbles, in conjunction with focused ultrasound (FUS), can enhance regional brain AAV transduction, but methods to assess transduction in vivo are needed. In a murine model, we combined positron emission tomography (PET) and optical imaging of reporter gene payloads to non-invasively assess the spatial distribution and transduction efficiency of systemically administered AAV9 after FUS and microbubble treatment. Capsid and reporter probe accumulation are reported as percent injected dose per cubic centimeter (%ID/cc) for PET quantification, whereas results for assays are reported as percent injected dose per gram (%ID/g). In a study spanning accumulation and transduction, mean AAV9 accumulation within the brain was 0.29 %ID/cc without FUS, whereas in the insonified region of interest of FUS-treated mice, the spatial mean and maximum reached ~2.3 %ID/cc and 4.3 %ID/cc, respectively. Transgene expression assessed by PET reporter gene imaging employing the pyruvate kinase M2 (PKM2)/[F]DASA-10 reporter system increased up to 10-fold in the FUS-treated regions, as compared to mice receiving AAVs without FUS. Systemic injection of AAV9 packaging the EF1A-PKM2 transgene followed by FUS in one hemisphere resulted in 1) an average 102-fold increase in PKM2 mRNA concentration compared to mice treated with AAVs only and 2) a 12.5-fold increase in the insonified compared to the contralateral hemisphere of FUS-treated mice. Combining microbubbles with US-guided treatment facilitated a multi-hour BBB disruption and stable AAV transduction in targeted areas of the murine brain. This unique platform has the potential to provide insight and aid in the translation of AAV-based therapies for the treatment of neuropathologies.
Topics: Mice; Animals; Dependovirus; Tomography, X-Ray Computed; Brain; Blood-Brain Barrier; Positron-Emission Tomography; Genetic Vectors
PubMed: 37908737
DOI: 10.7150/thno.85549 -
Genome Biology Apr 2024Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA...
BACKGROUND
Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency.
RESULTS
Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol.
CONCLUSIONS
Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.
Topics: Gene Editing; Humans; Animals; Mice; CRISPR-Associated Protein 9; CRISPR-Cas Systems; HEK293 Cells; Dependovirus
PubMed: 38671524
DOI: 10.1186/s13059-024-03257-z -
CNS Neuroscience & Therapeutics Dec 2023Stroke has been associated with devastating clinical outcomes, with current treatment strategies proving largely ineffective. Therefore, there is a need to explore... (Review)
Review
Stroke has been associated with devastating clinical outcomes, with current treatment strategies proving largely ineffective. Therefore, there is a need to explore alternative treatment options for addressing post-stroke functional deficits. Gene therapy utilizing adeno-associated viruses (AAVs) as a critical gene vector delivering genes to the central nervous system (CNS) gene delivery has emerged as a promising approach for treating various CNS diseases. This review aims to provide an overview of the biological characteristics of AAV vectors and the therapeutic advancements observed in preclinical models of ischemic stroke. The study further investigates the potential of manipulating AAV vectors in preclinical applications, emphasizing the challenges and prospects in the selection of viral vectors, drug delivery strategies, immune reactions, and clinical translation.
Topics: Humans; Dependovirus; Ischemic Stroke; Genetic Therapy; Gene Transfer Techniques; Genetic Vectors; Stroke
PubMed: 37551863
DOI: 10.1111/cns.14392 -
Biotechnology Advances 2024Recombinant adeno-associated viruses (rAAVs) stand at the forefront of gene therapy applications, holding immense significance for their safe and efficient gene delivery... (Review)
Review
Recombinant adeno-associated viruses (rAAVs) stand at the forefront of gene therapy applications, holding immense significance for their safe and efficient gene delivery capabilities. The constantly increasing and unmet demand for rAAVs underscores the need for a more comprehensive understanding of AAV biology and its impact on rAAV production. In this literature review, we delved into AAV biology and rAAV manufacturing bioprocesses, unravelling the functions and essentiality of proteins involved in rAAV production. We discuss the interconnections between these proteins and how they affect the choice of rAAV production platform. By addressing existing inconsistencies, literature gaps and limitations, this review aims to define a minimal set of genes that are essential for rAAV production, providing the potential to advance rAAV biomanufacturing, with a focus on minimizing the genetic load within rAAV-producing cells.
Topics: Dependovirus; Animals; Genetic Vectors; Humans; Genetic Therapy
PubMed: 38692443
DOI: 10.1016/j.biotechadv.2024.108370 -
Biomolecules Sep 2023Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by progressive disabling heterotopic ossification (HO) at extra-skeletal...
Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by progressive disabling heterotopic ossification (HO) at extra-skeletal sites. Here, we developed adeno-associated virus (AAV)-based gene therapy that suppresses trauma-induced HO in FOP mice harboring a heterozygous allele of human () while limiting the expression in non-skeletal organs such as the brain, heart, lung, liver, and kidney. AAV gene therapy carrying the combination of codon-optimized human ACVR1 (ACVR1) and artificial miRNAs targeting Activin A and its receptor ACVR1 ablated the aberrant activation of BMP-Smad1/5 signaling and the osteogenic differentiation of skeletal progenitors. The local delivery of AAV gene therapy to HO-causing cells in the skeletal muscle resulted in a significant decrease in endochondral bone formation in mice. These mice showed little to no expression in a major AAV-targeted organ, the liver, due to liver-abundant miR-122-mediated repression. Thus, AAV gene therapy is a promising therapeutic strategy to explore in suppressing HO in FOP.
Topics: Animals; Humans; Mice; Activin Receptors, Type I; Activins; Dependovirus; Myositis Ossificans; Osteogenesis
PubMed: 37759764
DOI: 10.3390/biom13091364 -
Cell Jun 2024Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic...
Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.
Topics: Animals; Mice; Gene Regulatory Networks; Single-Cell Analysis; Forkhead Transcription Factors; Cerebral Cortex; Neurons; Female; Dependovirus; Humans; Nerve Tissue Proteins; CRISPR-Cas Systems; Genetic Vectors; Mice, Inbred C57BL; Transcriptome
PubMed: 38772369
DOI: 10.1016/j.cell.2024.04.050 -
Gene Therapy May 2024In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian...
In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 10, 1.4 × 10 and 1.4 × 10 genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.
Topics: Animals; Simian Immunodeficiency Virus; Macaca mulatta; Gene Editing; CRISPR-Cas Systems; Simian Acquired Immunodeficiency Syndrome; Tissue Distribution; Genetic Vectors; Genetic Therapy; Dependovirus
PubMed: 37587230
DOI: 10.1038/s41434-023-00410-4 -
Journal of Virology Jun 2023Unbiased selections of diverse capsid libraries can yield engineered capsids that overcome gene therapy delivery challenges like traversing the blood-brain barrier...
Unbiased selections of diverse capsid libraries can yield engineered capsids that overcome gene therapy delivery challenges like traversing the blood-brain barrier (BBB), but little is known about the parameters of capsid-receptor interactions that govern their improved activity. This hampers broader efforts in precision capsid engineering and is a practical impediment to ensuring the translatability of capsid properties between preclinical animal models and human clinical trials. In this work, we utilize the adeno-associated virus (AAV)-PHP.B-Ly6a model system to better understand the targeted delivery and BBB penetration properties of AAV vectors. This model offers a defined capsid-receptor pair that can be used to systematically define relationships between target receptor affinity and activity of engineered AAV vectors. Here, we report a high-throughput method for quantifying capsid-receptor affinity and demonstrate that direct binding assays can be used to organize a vector library into families with varied affinity for their target receptor. Our data indicate that efficient central nervous system transduction requires high levels of target receptor expression at the BBB, but it is not a requirement for receptor expression to be limited to the target tissue. We observed that enhanced receptor affinity leads to reduced transduction of off-target tissues but can negatively impact on-target cellular transduction and penetration of endothelial barriers. Together, this work provides a set of tools for defining vector-receptor affinities and demonstrates how receptor expression and affinity interact to impact the performance of engineered AAV vectors in targeting the central nervous system. Novel methods for measuring adeno-associated virus (AAV)-receptor affinities, especially in relation to vector performance , would be useful to capsid engineers as they develop AAV vectors for gene therapy applications and characterize their interactions with native or engineered receptors. Here, we use the AAV-PHP.B-Ly6a model system to assess the impact of receptor affinity on the systemic delivery and endothelial penetration properties of AAV-PHP.B vectors. We discuss how receptor affinity analysis can be used to isolate vectors with optimized properties, improve the interpretation of library selections, and ultimately translate vector activities between preclinical animal models and humans.
Topics: Humans; Antigens, Ly; Capsid; Capsid Proteins; Dependovirus; Gene Transfer Techniques; Genetic Vectors; Receptors, Virus; Protein Binding; Peptides; Peptide Library; Transgenes; Gene Expression; HEK293 Cells; Endothelium
PubMed: 37199615
DOI: 10.1128/jvi.00174-23 -
Molecular Therapy : the Journal of the... Mar 2024Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery...
Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood-brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuffled rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAV-BBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270-fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAV-PHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes in vivo. To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modifications based on rational design. Overall, our study highlights the potential of AAV1-like vectors for efficient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies.
Topics: Humans; Animals; Mice; Blood-Brain Barrier; Genetic Therapy; Capsid; Liver; Peptides; Dependovirus; Genetic Vectors; Transduction, Genetic
PubMed: 38297833
DOI: 10.1016/j.ymthe.2024.01.024