-
Biomaterials Dec 2023Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types...
Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy.
Topics: Mice; Humans; Animals; Designed Ankyrin Repeat Proteins; Transduction, Genetic; Dependovirus; Genetic Vectors; Genetic Therapy; HIV Infections
PubMed: 37992599
DOI: 10.1016/j.biomaterials.2023.122399 -
Analytical Biochemistry Mar 2024Development of clinically desirable adeno-associated virus (AAV) vectors with optimal genome design requires rapid and accurate analytical methods to assess AAV quality....
Development of clinically desirable adeno-associated virus (AAV) vectors with optimal genome design requires rapid and accurate analytical methods to assess AAV quality. Anion-exchange (AEX) chromatography provides a powerful analytical method for full/empty AAV capsid ratio determination. However, the current AEX methodology for separation of empty and full AAV capsids largely relies on the use of the highly toxic tetramethylammonium chloride (TMAC). Here, we describe a novel analytical AEX method for separation of empty and full AAV capsids that uses only non-toxic, choline-type compounds that contain structural similarity to the quaternary ammonium ligand present on the surface of AEX resin. Choline-Cl gradient, combined with sensitive fluorescence detection, allowed a safe and effective separation of empty and full AAV capsids with reproducible empty/full ratio determination. The choline-based assay was suitable for commonly used serotypes, AAV2, AAV5, AAV6, and AAV8. The limit of detection was ∼3.9 × 10 virus particles in the assay. A gradient-hold step-gradient elution with choline-Cl resulted in enhanced baseline separation of empty and full AAV8 capsids. In summary, the use of choline-Cl in the AEX assay is recommended for empty/full capsid ratio determination and other applications in AAV production, and it eliminates the necessity of using toxic TMAC.
Topics: Capsid; Dependovirus; Salts; Choline; Genetic Vectors; Capsid Proteins; Chromatography
PubMed: 38061416
DOI: 10.1016/j.ab.2023.115421 -
Molecular Therapy : the Journal of the... Oct 2023Hepatotoxicity associated with intravenous/intrathecal adeno-associated virus (AAV) gene therapy has been observed in preclinical species and patients. In nonhuman...
Hepatotoxicity associated with intravenous/intrathecal adeno-associated virus (AAV) gene therapy has been observed in preclinical species and patients. In nonhuman primates, hepatotoxicity following self-complementary AAV9 administration varies from asymptomatic transaminase elevation with minimal to mild microscopic changes to symptomatic elevations of liver function and thromboinflammatory markers with microscopic changes consistent with marked hepatocellular necrosis and deteriorating clinical condition. These transient acute liver injury marker elevations occur from 3-4 days post intravenous administration to ∼2 weeks post intrathecal administration. No transaminase elevation or microscopic changes were observed with intrathecal administration of empty capsids or a "promoterless genome" vector, suggesting that liver injury after cerebrospinal fluid dosing in nonhuman primates is driven by viral transduction and transgene expression. Co-administration of prednisolone after intravenous or intrathecal dosing did not prevent liver enzyme or microscopic changes despite a reduction of T lymphocyte infiltration in liver tissue. Similarly, co-administration of rituximab/everolimus with intrathecal dosing failed to block AAV-driven hepatotoxicity. Self-complementary AAV-induced acute liver injury appears to correlate with high hepatocellular vector load, macrophage activation, and type 1 interferon innate virus-sensing pathway responses. The current work characterizes key aspects pertaining to early AAV-driven hepatotoxicity in cynomolgus macaques, highlighting the usefulness of this nonclinical species in that context.
Topics: Animals; Humans; Macaca fascicularis; Genetic Therapy; Administration, Intravenous; Chemical and Drug Induced Liver Injury; Dependovirus; Genetic Vectors
PubMed: 37515322
DOI: 10.1016/j.ymthe.2023.07.020 -
Frontiers in Cellular and Infection... 2023Gene therapy involves introducing and editing foreign genes in the body to treat and prevent genetic diseases. Adeno-associated virus (AAV) vector has become a widely...
BACKGROUND
Gene therapy involves introducing and editing foreign genes in the body to treat and prevent genetic diseases. Adeno-associated virus (AAV) vector has become a widely used tool in gene therapy due to its high safety and transfection efficiency.
METHODS
This study employs bibliometric analysis to explore the foundation and current state of AAV vector application in gene therapy research. A total of 6,069 publications from 1991 to 2022 were analyzed, retrieved from the Science Citation Index Expanded (SCI-E) within the Web of Science Core Collection (WoSCC) of Clarivate Analytics. Institutions, authors, journals, references, and keywords were analyzed and visualized by using VOSviewer and CiteSpace. The R language and Microsoft Excel 365 were used for statistical analyses.
RESULTS
The global literature on AAV vector and gene therapy exhibited consistent growth, with the United States leading in productivity, contributing 3,868 papers and obtaining the highest H-index. Noteworthy authors like Wilson JM, Samulski RJ, Hauswirth WW, and Mingozzi F were among the top 10 most productive and co-cited authors. The journal "Human Gene Therapy" published the most papers (n = 485) on AAV vector and gene therapy. Current research focuses on "gene editing," "gene structure," "CRISPR," and "AAV gene therapy for specific hereditary diseases."
CONCLUSION
The application of AAV vector in gene therapy has shown continuous growth, fostering international cooperation among countries and institutions. The intersection of gene editing, gene structure, CRISPR, and AAV gene therapy for specific hereditary diseases and AAV vector represents a prominent and prioritized focus in contemporary gene therapy research. This study provides valuable insights into the trends and characteristics of AAV gene therapy research, facilitating further advancements in the field.
Topics: Humans; Dependovirus; Bibliometrics; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; Genetic Therapy
PubMed: 38145048
DOI: 10.3389/fcimb.2023.1301915 -
Molecular Therapy : the Journal of the... Nov 2023The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines...
The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.
Topics: Animals; Humans; Mice; Angiotensin-Converting Enzyme 2; Antibodies, Monoclonal; Antibodies, Viral; COVID-19; Dependovirus; RNA, Viral; SARS-CoV-2; Broadly Neutralizing Antibodies
PubMed: 37689971
DOI: 10.1016/j.ymthe.2023.09.002 -
Cell Reports Nov 2023Promoters are essential tools for basic and translational neuroscience research. An ideal promoter should possess the shortest possible DNA sequence with cell-type...
Promoters are essential tools for basic and translational neuroscience research. An ideal promoter should possess the shortest possible DNA sequence with cell-type selectivity. However, whether ultra-compact promoters can offer neuron-specific expression is unclear. Here, we report the development of an extremely short promoter that enables selective gene expression in neurons, but not glial cells, in the brain. The promoter sequence originates from the human CALM1 gene and is only 120 bp in size. The CALM1 promoter (pCALM1) embedded in an adeno-associated virus (AAV) genome directed broad reporter expression in excitatory and inhibitory neurons in mouse and monkey brains. Moreover, pCALM1, when inserted into an all-in-one AAV vector expressing SpCas9 and sgRNA, drives constitutive and conditional in vivo gene editing in neurons and elicits functional alterations. These data demonstrate the ability of pCALM1 to conduct restricted neuronal gene expression, illustrating the feasibility of ultra-miniature promoters for targeting brain-cell subtypes.
Topics: Mice; Animals; Humans; RNA, Guide, CRISPR-Cas Systems; Neurons; Brain; Neuroglia; Genetic Therapy; Genetic Vectors; Dependovirus
PubMed: 37910509
DOI: 10.1016/j.celrep.2023.113348 -
International Journal of Molecular... Jul 2023Adeno-associated viruses (AAV) are one of the most commonly used vehicles in gene therapies for the treatment of rare diseases. During the AAV manufacturing process,...
Adeno-associated viruses (AAV) are one of the most commonly used vehicles in gene therapies for the treatment of rare diseases. During the AAV manufacturing process, particles with little or no genetic material are co-produced alongside the desired AAV capsid containing the transgene of interest. Because of the potential adverse health effects of these byproducts, they are considered impurities and need to be monitored carefully. To date, analytical ultracentrifugation (AUC), transmission electron microscopy (TEM) and charge-detection mass spectrometry (CDMS) are used to quantify these subspecies. However, they are associated with long turnaround times, low sample throughput and complex data analysis. Mass photometry (MP) is a fast and label-free orthogonal technique which is applicable to multiple serotypes without the adaption of method parameters. Furthermore, it can be operated with capsid titers as low as 8 × 10 cp mL with a CV < 5% using just 10 µL total sample volume. Here we demonstrate that mass photometry can be used as an orthogonal method to AUC to accurately quantify the proportions of empty, partially filled, full and overfull particles in AAV samples, especially in cases where ion-exchange chromatography yields no separation of the populations. In addition, it can be used to confirm the molar mass of the packaged genomic material in filled AAV particles.
Topics: Dependovirus; Genetic Vectors; Capsid; Capsid Proteins; Microscopy, Electron, Transmission
PubMed: 37446211
DOI: 10.3390/ijms241311033 -
Nature Communications Nov 2023Astrocytes, one of the most prevalent cell types in the central nervous system (CNS), are critically involved in neural function. Genetically manipulating astrocytes is...
Astrocytes, one of the most prevalent cell types in the central nervous system (CNS), are critically involved in neural function. Genetically manipulating astrocytes is an essential tool in understanding and affecting their roles. Adeno-associated viruses (AAVs) enable rapid genetic manipulation; however, astrocyte specificity of AAVs can be limited, with high off-target expression in neurons and sparsely in endothelial cells. Here, we report the development of a cassette of four copies of six miRNA targeting sequences (4x6T) which triggers transgene degradation specifically in neurons and endothelial cells. In combination with the GfaABC1D promoter, 4x6T increases astrocytic specificity of Cre with a viral reporter from <50% to >99% in multiple serotypes in mice, and confers astrocyte specificity in multiple recombinases and reporters. We also present empty vectors to add 4x6T to other cargo, independently and in Cre/Dre-dependent forms. This toolbox of AAVs allows rapid manipulation of astrocytes throughout the CNS, is compatible with different AAV serotypes, and demonstrates the efficacy of using multiplexed miRNA targeting sequences to decrease expression in multiple off-target cell populations simultaneously.
Topics: Mice; Animals; Astrocytes; MicroRNAs; Serogroup; Endothelial Cells; Genetic Vectors; Dependovirus
PubMed: 37973910
DOI: 10.1038/s41467-023-42746-w -
Journal of Virology Nov 2023The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally...
The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.
Topics: Humans; Capsid; Capsid Proteins; Dependovirus; Genetic Therapy; Genetic Vectors; Phylogeny; Tissue Distribution
PubMed: 37843374
DOI: 10.1128/jvi.01163-23 -
Journal of Virology Jul 2023Parvoviruses are among the smallest and superficially simplest animal viruses, infecting a broad range of hosts, including humans, and causing some deadly infections. In... (Review)
Review
The Structures and Functions of Parvovirus Capsids and Missing Pieces: the Viral DNA and Its Packaging, Asymmetrical Features, Nonprotein Components, and Receptor or Antibody Binding and Interactions.
Parvoviruses are among the smallest and superficially simplest animal viruses, infecting a broad range of hosts, including humans, and causing some deadly infections. In 1990, the first atomic structure of the canine parvovirus (CPV) capsid revealed a 26-nm-diameter T=1 particle made up of two or three versions of a single protein, and packaging about 5,100 nucleotides of single-stranded DNA. Our structural and functional understanding of parvovirus capsids and their ligands has increased as imaging and molecular techniques have advanced, and capsid structures for most groups within the family have now been determined. Despite those advances, significant questions remain unanswered about the functioning of those viral capsids and their roles in release, transmission, or cellular infection. In addition, the interactions of capsids with host receptors, antibodies, or other biological components are also still incompletely understood. The parvovirus capsid's apparent simplicity likely conceals important functions carried out by small, transient, or asymmetric structures. Here, we highlight some remaining open questions that may need to be answered to provide a more thorough understanding of how these viruses carry out their various functions. The many different members of the family share a capsid architecture, and while many functions are likely similar, others may differ in detail. Many of those parvoviruses have not been experimentally examined in detail (or at all in some cases), so we, therefore, focus this minireview on the widely studied protoparvoviruses, as well as the most thoroughly investigated examples of adeno-associated viruses.
Topics: Animals; Humans; Capsid; Capsid Proteins; DNA, Viral; Parvoviridae; Parvoviridae Infections; Dependovirus
PubMed: 37367301
DOI: 10.1128/jvi.00161-23