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Applied Microbiology and Biotechnology Jun 2024Recombinant adeno-associated virus (rAAV) is a major gene delivery vehicle. We have constructed a stable rAAV producer cell line by integrating essential rAAV genome,... (Comparative Study)
Comparative Study
Recombinant adeno-associated virus (rAAV) is a major gene delivery vehicle. We have constructed a stable rAAV producer cell line by integrating essential rAAV genome, viral and helper genes into the genome of HEK293 cell under the control of inducible promoters. Upon induction, the cell line produces transducing rAAV. To gain insight into enhancing rAAV productivity and vector quality, we performed a comparative transcriptomic and proteomic analysis of our synthetic cell line GX2 and two wild-type AAV (wtAAV) production systems, one by virus co-infection and the other by multi-plasmid transfection. The three systems had different kinetics in viral component synthesis but generated comparable copies of AAV genomes; however, the capsid titer of GX2 was an order of magnitude lower compared to those two wtAAV systems, indicating that its capsid production may be insufficient. The genome packaging efficiency was also lower in GX2 despite it produced higher levels of Rep52 proteins than either wtAAV systems, suggesting that Rep52 protein expression may not limit genome packaging. In the two wtAAV systems, VP were the most abundant AAV proteins and their levels continued to increase, while GX2 had high level of wasteful cargo gene expression. Furthermore, upregulated inflammation, innate immune responses, and MAPK signaling, as well as downregulated mitochondrial functions, were commonly observed in either rAAV or wtAAV systems. Overall, this comparative multi-omics study provided rich insights into host cell and viral factors that are potential targets for genetic and process intervention to enhance the productivity of synthetic rAAV producer cell lines. KEY POINTS: • wtAAV infection was more efficient in producing full viral particles than the synthetic cell GX2. • Capsid protein synthesis, genome replication, and packaging may limit rAAV production in GX2. • wtAAV infection and rAAV production in GX2 elicited similar host cell responses.
Topics: Dependovirus; Humans; HEK293 Cells; Proteomics; Transcriptome; Genetic Vectors; Kinetics; Genome, Viral; Gene Expression Profiling; Proteome
PubMed: 38896252
DOI: 10.1007/s00253-024-13203-5 -
Zoological Research Jul 2024Precise targeting of specific regions within the central nervous system (CNS) is crucial for both scientific research and gene therapy in the context of brain diseases....
Precise targeting of specific regions within the central nervous system (CNS) is crucial for both scientific research and gene therapy in the context of brain diseases. Adeno-associated virus 13 (AAV13) is known for its restricted diffusion range within the CNS, making it an ideal choice for precise labeling and administration within small brain regions. However, AAV13 mediates relatively low expression of target genes. Here, we introduced specifically engineered modifications to the AAV13 capsid protein to enhance its transduction efficiency. We first constructed AAV13-YF by mutating tyrosine to phenylalanine on the surface of the AAV13 capsid. We then inserted the 7m8 peptide, known to enhance cell transduction, into positions 587/588 and 585/586 of the AAV13 capsid, resulting in two distinct variants named AAV13-587-7m8 and AAV13-585-7m8, respectively. We found that AAV13-YF exhibited superior infectivity in HEK293T cells compared to AAV13, while AAV13-587-7m8 and AAV13-585-7m8 showed enhanced CNS infection capabilities in C57BL/6 mice, with AAV13-587-7m8 infection retaining a limited spread range. These modified AAV13 variants hold promising potential for applications in gene therapy and neuroscience research.
Topics: Dependovirus; Animals; Humans; Mice; HEK293 Cells; Mice, Inbred C57BL; Transduction, Genetic; Capsid Proteins
PubMed: 38894521
DOI: 10.24272/j.issn.2095-8137.2023.355 -
Translational Vision Science &... Jun 2024To report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a...
PURPOSE
To report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector.
DESIGN
Single-center, retrospective chart review.
METHODS
In this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated.
RESULTS
Three out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector.
CONCLUSIONS
We observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes.
TRANSLATIONAL RELEVANCE
Caution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.
Topics: Humans; Retrospective Studies; Genetic Vectors; Genetic Therapy; Male; Female; Child; Visual Acuity; Tomography, Optical Coherence; cis-trans-Isomerases; Dependovirus; Atrophy; Visual Fields
PubMed: 38888288
DOI: 10.1167/tvst.13.6.11 -
Protein Science : a Publication of the... Jul 2024Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of...
Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.
Topics: Dependovirus; Capsid Proteins; Genetic Vectors; Genetic Therapy; Capsid; Hydrogen Deuterium Exchange-Mass Spectrometry; Protein Stability; Humans; Protein Conformation; Models, Molecular
PubMed: 38888268
DOI: 10.1002/pro.5074 -
PLoS Pathogens Jun 2024Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of...
Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy.
Topics: Dependovirus; Humans; Animals; Evolution, Molecular; Capsid Proteins; Selection, Genetic; Genetic Variation; Genetic Therapy
PubMed: 38885242
DOI: 10.1371/journal.ppat.1012260 -
Nature Communications Jun 2024Individuals with autism spectrum disorder (ASD) have a higher prevalence of social memory impairment. A series of our previous studies revealed that hippocampal ventral...
Individuals with autism spectrum disorder (ASD) have a higher prevalence of social memory impairment. A series of our previous studies revealed that hippocampal ventral CA1 (vCA1) neurons possess social memory engram and that the neurophysiological representation of social memory in the vCA1 neurons is disrupted in ASD-associated Shank3 knockout mice. However, whether the dysfunction of Shank3 in vCA1 causes the social memory impairment observed in ASD remains unclear. In this study, we found that vCA1-specific Shank3 conditional knockout (cKO) by the adeno-associated virus (AAV)- or specialized extracellular vesicle (EV)- mediated in vivo gene editing was sufficient to recapitulate the social memory impairment in male mice. Furthermore, the utilization of EV-mediated Shank3-cKO allowed us to quantitatively examine the role of Shank3 in social memory. Our results suggested that there is a certain threshold for the proportion of Shank3-cKO neurons required for social memory disruption. Thus, our study provides insight into the population coding of social memory in vCA1, as well as the pathological mechanisms underlying social memory impairment in ASD.
Topics: Animals; Mice, Knockout; Male; Nerve Tissue Proteins; CA1 Region, Hippocampal; Autism Spectrum Disorder; Mice; Memory; Gene Editing; Social Behavior; Neurons; Dependovirus; Microfilament Proteins; Memory Disorders; Mice, Inbred C57BL
PubMed: 38866749
DOI: 10.1038/s41467-024-48430-x -
Journal of Cellular and Molecular... Jun 2024Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that...
Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that a long non-coding RNA, Neat1 as a primary regulator of matrix metalloproteinase (MMP) 3 and MMP13 activity, and its induction in the target joint has a deteriorating effect on articular cartilage. In the present study, we administered an Adeno-associated virus (AAV) 5 vector carrying an short hairpin (sh)RNA to Neat1 via intra-articular injection alone or in conjunction with systemic administration of a capsid-modified AAV8 (K31Q) vector carrying F8 gene (F8-BDD-V3) to study its impact on HA. AAV8K31Q-F8 vector administration at low dose, led to an increase in FVIII activity (16%-28%) in treated mice. We further observed a significant knockdown of Neat1 (~40 fold vs. untreated injured joint, p = 0.005) in joint tissue of treated mice and a downregulation of chondrodegenerative enzymes, MMP3, MMP13 and the inflammatory mediator- cPLA2, in mice receiving combination therapy. These data demonstrate that AAV mediated Neat1 knockdown in combination with F8 gene augmentation can potentially impact mediators of haemophilic joint disease.
Topics: Animals; Hemophilia A; Dependovirus; RNA, Long Noncoding; Matrix Metalloproteinase 13; Mice; Matrix Metalloproteinase 3; Genetic Vectors; Factor VIII; Joint Diseases; Humans; Genetic Therapy; Mice, Inbred C57BL; Cartilage, Articular; Disease Models, Animal; Male
PubMed: 38864710
DOI: 10.1111/jcmm.18460 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... May 2024To investigate the effects of an adeno-associated virus (AAV2) vector expressing secretory transforming growth factor-β (TGF-β) type Ⅱ receptor (sTβRⅡ)...
[A recombinant adeno-associated virus expressing secretory TGF-β type Ⅱ receptor inhibits triple-negative murine breast cancer 4T1 cell proliferation and lung metastasis in mice].
OBJECTIVE
To investigate the effects of an adeno-associated virus (AAV2) vector expressing secretory transforming growth factor-β (TGF-β) type Ⅱ receptor (sTβRⅡ) extracellular domain-IgG2a Fc fusion protein (sTβRⅡ-Fc) on proliferation and migration of triple-negative murine breast cancer 4T1 cells in mice.
METHODS
The pAAV-sTβRⅡ-Fc vector expressing sTβRⅡ-Fc fusion protein constructed by molecular cloning, the capsid protein-expressing vector pAAV2 and the helper vector were co-transfected into HEK 293T cells to prepare the recombinant AAV2-sTβRⅡ virus, which was purified by density gradient centrifugation with iodixanol. Western blotting was used to examine the effects of AAV-sTβRⅡ virus on Smad2/3 phosphorylation in 4T1 cells and on expression levels of E-cadherin, vimentin and p-Smad2/3 in 4T1 cell xenografts in mice. BALB/c mice bearing subcutaneous xenografts of luciferase-expressing 4T1 cells received intravenous injections of AAV-sTβRⅡ virus, AAV-GFP virus or PBS (=6) through the tail vein, and the proliferation and migration of 4T1 cells were analyzed with in vivo imaging. Ki67 expression in the tumor tissues and sTβRⅡ protein expressions in mouse livers were detected with immunohistochemistry and immunofluorescence staining, and tumor metastases in the vital organs were examined with HE staining.
RESULTS
The recombinant pAAV-sTβRⅡ-Fc vector successfully expressed sTβRⅡ in HEK 293T cells. Infection with AAV2-sTβRⅡ virus significantly reduced TGF-β1-induced Smad2/3 phosphorylation in 4T1 cells and effectively inhibited proliferation and lung metastasis of 4T1 xenografts in mice (<0.05). In the tumor-bearing mice, intravenous injection of AAV-sTβRⅡ virus significantly increased E-cadherin expression, reduced vimentin and Ki67 protein expressions and Smad2/3 phosphorylation level in the tumor tissues (<0.05 or 0.01), and induced liver-specific sTβRⅡ expression without causing body weight loss or heart, liver, spleen or kidney pathologies.
CONCLUSION
The recombinant AVV2 vector encoding sTβRⅡ extracellular domain is capable of blocking the TGF-β signaling pathway to inhibit the proliferation and lung metastasis of 4T1 cells in mice.
Topics: Animals; Mice; Dependovirus; Cell Proliferation; Mice, Inbred BALB C; Humans; HEK293 Cells; Genetic Vectors; Lung Neoplasms; Female; Receptor, Transforming Growth Factor-beta Type II; Cell Line, Tumor; Triple Negative Breast Neoplasms; Cadherins; Smad3 Protein; Cell Movement; Smad2 Protein
PubMed: 38862439
DOI: 10.12122/j.issn.1673-4254.2024.05.03 -
Nature Communications Jun 2024Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific...
Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific delivery of common gene vectors such as adeno-associated viruses (AAVs) is typically performed via invasive injections, which limit its applicable scope of research and clinical applications. Alternatively, focused ultrasound blood-brain-barrier opening (FUS-BBBO), performed noninvasively, enables the site-specific entry of AAVs into the brain from systemic circulation. However, when used in conjunction with natural AAV serotypes, this approach has limited transduction efficiency and results in substantial undesirable transduction of peripheral organs. Here, we use high throughput in vivo selection to engineer new AAV vectors specifically designed for local neuronal transduction at the site of FUS-BBBO. The resulting vectors substantially enhance ultrasound-targeted gene delivery and neuronal tropism while reducing peripheral transduction, providing a more than ten-fold improvement in targeting specificity in two tested mouse strains. In addition to enhancing the only known approach to noninvasively target gene delivery to specific brain regions, these results establish the ability of AAV vectors to be evolved for specific physical delivery mechanisms.
Topics: Animals; Genetic Vectors; Dependovirus; Gene Transfer Techniques; Mice; Blood-Brain Barrier; Brain; Humans; Neurons; Transduction, Genetic; Mice, Inbred C57BL; Genetic Engineering; Female; Male; HEK293 Cells
PubMed: 38858354
DOI: 10.1038/s41467-024-48974-y -
STAR Protocols Jun 2024Studying synapses in vivo presents challenges due to the complexity of accurately targeting and visualizing specific synaptic proteins within the brain. Here, we...
Studying synapses in vivo presents challenges due to the complexity of accurately targeting and visualizing specific synaptic proteins within the brain. Here, we present a protocol for in vivo analysis of pre- and post-synaptic protein function in mice. We describe steps for combining adeno-associated virus (AAV)-mediated gene transfer to manipulate specific neuron subtypes. We also describe immunofluorescence on artificial cerebrospinal fluid (ACSF)-perfused brain sections to enhance the visualization of synaptic proteins. For complete details on the use and execution of this protocol, please refer to Cramer et al..
Topics: Animals; Mice; Synapses; Dependovirus; Brain; Neurons; Gene Transfer Techniques; Nerve Tissue Proteins
PubMed: 38857153
DOI: 10.1016/j.xpro.2024.103117