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Current Protocols Jun 2024Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term...
Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of AAV9 vectors in 293EB cells Basic Protocol 2: Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration Basic Protocol 3: Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography Basic Protocol 4: Analysis of the purified AAV9 vectors.
Topics: Dependovirus; Genetic Vectors; Humans; Ceramics; Serogroup; Durapatite; Chromatography; HEK293 Cells
PubMed: 38837274
DOI: 10.1002/cpz1.1068 -
ACS Nano Jun 2024Using viral vectors as gene delivery vehicles for gene therapy necessitates their quality control. Here, we report on nanopore sensing for nondestructively inspecting...
Using viral vectors as gene delivery vehicles for gene therapy necessitates their quality control. Here, we report on nanopore sensing for nondestructively inspecting genomes inside the nanoscale cargoes at the single-molecule level. Using ionic current measurements, we motion-tracked the adeno-associated virus (AAV) vectors as they translocated through a solid-state nanopore. Considering the varying contributions of the electrophoretic forces from the negatively charged internal polynucleotides of different lengths, the nanocargoes carrying longer DNA moved more slowly in the nanochannel. Moreover, ion blockage characteristics revealed their larger volume by up to approximately 3600 nm in proportion to the length of single-stranded DNA packaged inside, thereby allowing electrical discriminations of AAV vectors by the gene-derived physical features. The present findings can be a promising tool for the enhanced quality control of AAV products by enabling the screening of empty and intermediate vectors at the single-particle level.
Topics: Nanopores; Dependovirus; Genetic Vectors; DNA, Single-Stranded; Humans
PubMed: 38836590
DOI: 10.1021/acsnano.4c01888 -
Generation of nanobodies from transgenic 'LamaMice' lacking an endogenous immunoglobulin repertoire.Nature Communications Jun 2024Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in...
Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.
Topics: Animals; Single-Domain Antibodies; Camelids, New World; Immunoglobulin Heavy Chains; Mice, Transgenic; Mice; Spike Glycoprotein, Coronavirus; Lectins, C-Type; SARS-CoV-2; Immunoglobulin E; Humans; Dependovirus; Immunoglobulin G; COVID-19; B-Lymphocytes
PubMed: 38830864
DOI: 10.1038/s41467-024-48735-x -
Mutation Research. Genetic Toxicology... 2024Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application... (Review)
Review
Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application requires the development of appropriate safety testing strategies. Several guidelines have been provided by health authorities to address these concerns. These guidelines state that non-clinical testing should be carried out on a case-by-case basis depending on the modality. This review focuses on the genome safety assessment of frequently used gene therapy modalities, namely Adeno Associated Viruses (AAVs), Lentiviruses, designer nucleases and mRNAs. Important safety considerations for these modalities, amongst others, are vector integrations into the patient genome (insertional mutagenesis) and off-target editing. Taking into account the constraints of in vivo studies, health authorities endorse the development of novel approach methodologies (NAMs), which are innovative in vitro strategies for genotoxicity testing. This review provides an overview of NAMs applied to viral and CRISPR/Cas9 safety, including next generation sequencing-based methods for integration site analysis and off-target editing. Additionally, NAMs to evaluate the oncogenicity risk arising from unwanted genomic modifications are discussed. Thus, a range of promising techniques are available to support the safe development of gene therapies. Thorough validation, comparisons and correlations with clinical outcomes are essential to identify the most reliable safety testing strategies. By providing a comprehensive overview of these NAMs, this review aims to contribute to a better understanding of the genome safety perspectives of gene therapies.
Topics: Genetic Therapy; Humans; Gene Editing; Animals; Dependovirus; Genetic Vectors; CRISPR-Cas Systems; Lentivirus; Endonucleases; Mutagenicity Tests; Nucleotides
PubMed: 38821669
DOI: 10.1016/j.mrgentox.2024.503767 -
The Science of the Total Environment Aug 2024No single microbial source tracking (MST) marker can be applied to determine the sources of fecal pollution in all water types. This study aimed to validate a...
No single microbial source tracking (MST) marker can be applied to determine the sources of fecal pollution in all water types. This study aimed to validate a high-throughput quantitative polymerase chain reaction (HT-qPCR) method for the simultaneous detection of multiple MST markers. A total of 26 fecal-source samples that had been previously collected from human sewage (n = 6) and ruminant (n = 3), dog (n = 6), pig (n = 6), chicken (n = 3), and duck (n = 2) feces in the Kathmandu Valley, Nepal, were used to validate 10 host-specific MST markers, i.e., Bacteroidales (BacHum, gyrB, BacR, and Pig2Bac), mitochondrial DNA (mtDNA) (swine, bovine, and Dog-mtDNA), and viral (human adenovirus, porcine adenovirus, and chicken/turkey parvovirus) markers, via HT-qPCR. Only Dog-mtDNA showed 100 % accuracy. All the tested bacterial markers showed a sensitivity of 100 %. Nine of the 10 markers were further used to identify fecal contamination in groundwater sources (n = 54), tanker filling stations (n = 14), drinking water treatment plants (n = 5), and river water samples (n = 6). The human-specific Bacteroidales marker BacHum and ruminant-specific Bacteroidales marker BacR was detected at a high ratio in river water samples (83 % and 100 %, respectively). The results of HT-qPCR were in agreement with the standard qPCR. The comparable performances of HT-qPCR and standard qPCR as well as the successful detection of MST markers in the fecal-source and water samples demonstrated the potential applicability of these markers for detecting fecal contamination sources via HT-qPCR.
Topics: Environmental Monitoring; Feces; Water Microbiology; Animals; Nepal; Real-Time Polymerase Chain Reaction; Humans; Sewage; Water Pollution
PubMed: 38821279
DOI: 10.1016/j.scitotenv.2024.173604 -
Medecine Sciences : M/S May 2024
Topics: Animals; Mice; Dependovirus; Deafness; Membrane Proteins; Genetic Vectors; Humans; Serine Endopeptidases; Genetic Therapy; Aging; Hearing; Disease Progression; Mice, Mutant Strains; Mutation; Neoplasm Proteins
PubMed: 38819271
DOI: 10.1051/medsci/2024042 -
Virusdisease Mar 2024Parvoviruses are ubiquitous pathogens that cause fatal disease in cats. Feline panleukopenia virus (FPV) is a primitive virus reported first and canine parvovirus (CPV)...
UNLABELLED
Parvoviruses are ubiquitous pathogens that cause fatal disease in cats. Feline panleukopenia virus (FPV) is a primitive virus reported first and canine parvovirus (CPV) evolved from FPV and was reported later. Both induce disease in cats and dogs with correlative signs. FPV in domestic cats is genetically diverse and some strains may differ from those used for vaccination. In this study, a virus of FPV strain, ABT/MVC/2022/FPV/001, was identified from a fecal sample of the suspected cat with severe haemorrhagic gastroenteritis. The phylogenetic analysis and complete genome sequence of the strain share 99.75% nucleotide identity with FPV variant MH559110 belonging to Tamil Nadu, India. The results also reveal similarities to strains isolated from Italy, Belgium, and China. The deduced amino acid sequence of isolated strain revealed specific amino acid substitution (Pro5Ala, Phe6Val, His7Gln, Asn9Asp, Lys16Arg, Lys19Arg, Asn52Lys, Gly58Trp, Thr66Ser, Lys67Arg, Leu70His, Asn373Asp and Ala390Thr) which differed from MH559110 and other strains. The complete genomic analysis revealed that the FPV strain circulating in India is evolving rapidly with unique antigenic variations between field FPV, CPV and vaccine strains which may be the major cause for vaccine failure in vaccinated cats.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13337-023-00854-7.
PubMed: 38817404
DOI: 10.1007/s13337-023-00854-7 -
Comparative Immunology, Microbiology... Jul 2024Canine parvovirus type 2 (CPV-2) is a major cause of fatal gastroenteritis and myocarditis in puppies of domestic and wild carnivores. CPV-2 has accumulated changes over...
Canine parvovirus type 2 (CPV-2) is a major cause of fatal gastroenteritis and myocarditis in puppies of domestic and wild carnivores. CPV-2 has accumulated changes over time lead to the emergence of three antigenic variants CPV-2a, CPV-2b, and CPV-2c. VP2 is the major capsid protein that determines virus antigenicity, and host range. Although the three CPV-2 variants were previously identified in Egypt, most reports covered a restricted geographic region and/or time period, and only analyzed partial fragments of VP2 gene. Therefore, this study was designed to test 100 rectal swabs collected from 7 Egyptian governorates between 2019 and 2021 for CPV-2 using PCR. A total of 65 positive samples were identified, mostly in pure dog breeds of young age. The three variants co-circulated in 2019, while CPV-2b was not detected in 2020 and 2021. The frequency of CPV-2b and CPV-2c was higher in 2019 and 2021, respectively. Analysis of CPV-2 full-length VP2 gene sequence from 19/65 positive samples has identified four common amino acid substitutions F267Y, S297A, A300G, Y324I, which are characteristic for the new CPV-2 variants currently circulating worldwide. Unique substitutions including A5G, G36R, V38E, Q370R, and G392V were recognized in certain samples, and appears to have distinct effect on receptor binding, nuclear translocation, and inter-species transmission. Phylogenetic analysis showed separation of CPV-2 strains into two clades. All strains of this study were classified in clade I with Asian strains. In conclusion, this study provides updated comprehensive molecular analysis of CPV-2 variants in Egypt.
Topics: Animals; Egypt; Dogs; Parvovirus, Canine; Capsid Proteins; Parvoviridae Infections; Phylogeny; Dog Diseases; Amino Acid Substitution
PubMed: 38815398
DOI: 10.1016/j.cimid.2024.102190 -
PLoS Biology May 2024The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the...
The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
Topics: Dependovirus; Humans; Gene Editing; Genetic Vectors; CRISPR-Cas Systems; Animals; HEK293 Cells; Genetic Therapy; CRISPR-Associated Proteins; Mice; Endodeoxyribonucleases; Bacterial Proteins
PubMed: 38814985
DOI: 10.1371/journal.pbio.3002619