No CpGs for AAVs?
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ABSTRACT:
SUBMITTER: George LA
PROVIDER: S-EPMC7885819 | biostudies-literature | 2021 Feb
REPOSITORIES: biostudies-literature
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Project description:Adeno-associated viruses (AAVs) are popular gene therapy delivery vectors, but their application can be limited by anti-vector immunity. Both preexisting neutralizing antibodies (NAbs) and post-administration NAbs can limit transgene expression and reduce the clinical utility of AAVs. The development of novel AAVs will advance our understanding of AAV immunity and may also have practical applications. In this study, we identified five novel AAV capsids from rhesus macaques. RhAAV4282 exhibited 91.4% capsid sequence similarity with AAV7 and showed similar tissue tropism with slightly diminished overall signal. Despite this sequence homology, RhAAV4282 and AAV7 showed limited cross-neutralization. We determined a cryo-EM structure of the RhAAV4282 capsid at 2.57 Å resolution and identified a small segment within the hypervariable region IV, involving seven amino acids that formed a shortened external loop in RhAAV4282 compared with AAV7. We generated RhAAV4282 and AAV7 mutants that involved swaps of this region and showed that this region partially determined neutralization phenotype. We termed this region the hypervariable region IV neutralizing epitope (HRNE). Our data suggests that modification of the HRNE can lead to AAVs with altered neutralization profiles.
| S-EPMC11513466 | biostudies-literature
Project description:Neural signals for locomotion are influenced both by the neural network architecture and sensory inputs coordinating and adapting the gait to the environment. Adaptation relies on the ability to change amplitude, frequency, and phase of the signals within the sensorimotor loop in response to external stimuli. However, in order to experiment with closed-loop control, we first need a better understanding of the dynamics of the system and how adaptation works. Based on insights from biology, we developed a spiking neural network capable of continuously changing amplitude, frequency, and phase online. The resulting network is deployed on a hexapod robot in order to observe the walking behavior. The morphology and parameters of the network results in a tripod gait, demonstrating that a design without afferent feedback is sufficient to maintain a stable gait. This is comparable to results from biology showing that deafferented samples exhibit a tripod-like gait and adds to the evidence for a meaningful role of network topology in locomotion. Further, this work enables research into the role of sensory feedback and high-level control signals in the adaptation of gait types. A better understanding of the neural control of locomotion relates back to biology where it can provide evidence for theories that are currently not testable on live insects.
| S-EPMC7333644 | biostudies-literature
Project description:Hereditary diseases are caused by mutations in genes, and more than 7,000 rare diseases affect over 30 million Americans. For more than 30 years, hundreds of researchers have maintained that genetic modifications would provide effective treatments for many inherited human diseases, offering durable and possibly curative clinical benefit with a single treatment. This review is limited to gene therapy using adeno-associated virus (AAV) because the gene delivered by this vector does not integrate into the patient genome and has a low immunogenicity. There are now five treatments approved for commercialization and currently available, i.e., Luxturna, Zolgensma, the two chimeric antigen receptor T cell (CAR-T) therapies (Yescarta and Kymriah), and Strimvelis (the gammaretrovirus approved for adenosine deaminase-severe combined immunodeficiency [ADA-SCID] in Europe). Dozens of other treatments are under clinical trials. The review article presents a broad overview of the field of therapy by in vivo gene transfer. We review gene therapy for neuromuscular disorders (spinal muscular atrophy [SMA]; Duchenne muscular dystrophy [DMD]; X-linked myotubular myopathy [XLMTM]; and diseases of the central nervous system, including Alzheimer's disease, Parkinson's disease, Canavan disease, aromatic l-amino acid decarboxylase [AADC] deficiency, and giant axonal neuropathy), ocular disorders (Leber congenital amaurosis, age-related macular degeneration [AMD], choroideremia, achromatopsia, retinitis pigmentosa, and X-linked retinoschisis), the bleeding disorder hemophilia, and lysosomal storage disorders.
| S-EPMC7854298 | biostudies-literature
Project description:Brain endothelial cells are critical for homeostasis of the central nervous system. Novel adeno-associated viruses (AAV) with brain endothelial cell tropism have been developed and are beginning to be employed in mechanistic and therapeutic research. Studies using AAVs can be involved in terms of cost, time and personnel, and many groups, including our own, are not experts on the technology. Therefore, it is important to report data using AAVs with the research community as a guide for ongoing and future studies. Here, we detail our initial experience with the two most prevalent AAVs with tropism for brain endothelial cells, AAV-BR1 and AAV-BI30. One of our long-term goals is to express key proteins in brain endothelial cells and determine the impact on brain function. For method development, we administered AAV-BR1 and AAV-BI30 with a CMV-driven fluorescent reporter (CMV-P2A-mCherry) to wild-type mice intravenously (retro-orbital) and measured expression in brain and peripheral tissues by RT-PCR and immunostaining. We found that AAV-BR1 transduces neurons and endothelial cells in the brain, and the lung and liver, whereas AAV-BI30 transduces brain endothelial cells and peripheral tissue. Our data highlights the importance of using the AAV best suited to the scientific question.
| S-EPMC11792159 | biostudies-literature
Project description:BackgroundThe human chromosome 14q32.2 imprinted region harbors the primary MEG3/DLK1:IG-differentially methylated region (DMR) and secondary MEG3:TSS-DMR. The MEG3:TSS-DMR can remain unmethylated only in the presence of unmethylated MEG3/DLK1:IG-DMR in somatic tissues, but not in the placenta, because of a hierarchical regulation of the methylation pattern between the two DMRs.MethodsWe performed molecular studies in a 4-year-old Japanese girl with Temple syndrome (TS14).ResultsPyrosequencing analysis showed extremely low methylation levels of five CpGs at the MEG3:TSS-DMR and grossly normal methylation levels of four CpGs at the MEG3/DLK1:IG-DMR in leukocytes. HumanMethylation450 BeadChip confirmed marked hypomethylation of the MEG3:TSS-DMR and revealed multilocus imprinting disturbance (MLID) including mild hypomethylation of the H19/IGF2:IG-DMR and mild hypermethylation of the GNAS A/B:TSS-DMR in leukocytes. Bisulfite sequencing showed markedly hypomethylated CpGs at the MEG3:TSS-DMR and irregularly and non-differentially methylated CpGs at the MEG3/DLK1:IG-DMR in leukocytes and apparently normal methylation patterns of the two DMRs in the placenta. Maternal uniparental disomy 14 and a deletion involving this imprinted region were excluded.ConclusionsSuch a methylation pattern of the MEG3/DLK1:IG-DMR has not been reported in patients with TS14. It may be possible that a certain degree of irregular hypomethylation at the MEG3/DLK1:IG-DMR has prevented methylation of the MEG3:TSS-DMR in somatic tissues and that a hypermethylation type MLID has occurred at the MEG3/DLK1:IG-DMR to yield the apparently normal methylation pattern in the placenta.
| S-EPMC6407230 | biostudies-literature
Project description:Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV), makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs), critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg) than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.
| S-EPMC3525534 | biostudies-literature
Project description:Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how the development, physiology, and functions of CINs influence cortical circuit activity and behavior in model organisms such as mice and primates. While transgenic driver lines are powerful tools for studying CINs in mice, this technology is limited in other species. An alternative approach is to use viral vectors such as AAV, which can be used in multiple species including primates and also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell types. The present study describes the systematic genome-wide identification of putative REs (pREs) that are preferentially active in immature CINs by histone modification chromatin immunoprecipitation and sequencing (ChIP-seq). We evaluated two novel pREs in AAV vectors, alongside the well-established Dlx I12b enhancer, and found that they drove CIN-specific reporter expression in adult mice. We also showed that the identified Arl4d pRE could drive sufficient expression of channelrhodopsin for optogenetic rescue of behavioral deficits in the Dlx5/6+/- mouse model of fast-spiking CIN dysfunction.
| S-EPMC7768279 | biostudies-literature
Project description:High levels of anti-citrullinated protein antibodies (ACPA) are often observed prior to a diagnosis of rheumatoid arthritis (RA). We undertook a replication study to confirm CpG sites showing evidence of differential methylation in subjects positive vs. negative for ACPA, in a new subset of 112 individuals sampled from the population cohort and biobank CARTaGENE in Quebec, Canada. Targeted custom capture bisulfite sequencing was conducted at approximately 5.3 million CpGs located in regulatory or hypomethylated regions from whole blood; library and protocol improvements had been instituted between the original and this replication study, enabling better coverage and additional identification of differentially methylated regions (DMRs). Using binomial regression models, we identified 19,472 ACPA-associated differentially methylated cytosines (DMCs), of which 430 overlapped with the 1909 DMCs reported by the original study; 814 DMRs of relevance were clustered by grouping adjacent DMCs into regions. Furthermore, we performed an additional integrative analysis by looking at the DMRs that overlap with RA related loci published in the GWAS Catalog, and protein-coding genes associated with these DMRs were enriched in the biological process of cell adhesion and involved in immune-related pathways.
| S-EPMC8472734 | biostudies-literature
Project description:Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%). We apply these systems to install putative protective mutations in vivo for Alzheimer's disease in astrocytes and for coronary artery disease in hepatocytes. In vivo prime editing with v3em PE-AAV caused no detectable off-target effects or significant changes in liver enzymes or histology. Optimized PE-AAV systems support the highest unenriched levels of in vivo prime editing reported to date, facilitating the study and potential treatment of diseases with a genetic component.
| S-EPMC10869272 | biostudies-literature
Project description:BackgroundWhole-genome bisulfite sequencing (WGBS) is the gold standard for studying landscape DNA methylation. Current computational methods for WGBS are mainly designed for gene regulatory regions with multiple under-methylated CpGs (UMCs), such as promoters and enhancers.ResultsTo reliably predict the functional importance of single isolated UMCs across the genome, which is usually not achievable using traditional methods, we develop a multi-sample-based method. We identified 9421 sparse conserved under-methylated CpGs (scUMCs) from 31 high-quality methylomes, which are enriched in distal interacting anchor regions co-occupied by multiple chromatin-loop factors and are flanked by highly methylated CpGs. Moreover, cell lineage-specific scUMCs are associated with essential developmental genes, regulators of cell differentiation, and chromatin remodeling enzymes. Dynamic methylation levels of scUMCs correlate with the intensity of chromatin interactions and binding of looping factors as well as patterns of gene expression.ConclusionsWe introduce an innovative computational method for the identification of scUMCs, which are novel epigenetic features associated with high-order chromatin structure, opening new directions in the study of the inter-relationships between DNA methylation and chromatin structure.
| S-EPMC5580327 | biostudies-literature