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Journal of Controlled Release :... Jan 2022To meet the present and future challenges in achieving therapeutic in vivo gene delivery using adeno-associated virus (AAV), new innovations are required that integrate... (Review)
Review
To meet the present and future challenges in achieving therapeutic in vivo gene delivery using adeno-associated virus (AAV), new innovations are required that integrate knowledge from disciplines ranging from biomaterials science, drug delivery, immunobiology, to tissue engineering. One of the foremost challenges remaining is in addressing pre-existing and therapy induced immune responses to AAV which significantly limit its therapeutic effect. In addition, functional correction of diseased tissues will depend on the ability of AAVs to retain activity after local or systemic administration and broadly distribute in target tissues. In this contribution to the Orations - New Horizons of the Journal of Controlled Release, I will introduce new concepts and potential strategies pursued by our lab and others to better understand and overcome these hurdles to effective AAV gene therapy. These multi-disciplinary approaches may open the door to the creation of precision gene therapies to treat heavily burdensome and often deadly diseases.
Topics: Dependovirus; Gene Transfer Techniques; Genes, Viral; Genetic Therapy; Genetic Vectors
PubMed: 34785314
DOI: 10.1016/j.jconrel.2021.11.013 -
Molecular Therapy : the Journal of the... Oct 2021Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal...
Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal vector, its single-stranded DNA genome is prone to intra- and inter-molecular recombination leading to rearrangements and integration into the host cell genome. Here, we ascertained the integration frequency of rAAV in human hepatocytes transduced either ex vivo or in vivo and subsequently expanded in a mouse model of xenogeneic liver regeneration. Chromosomal rAAV integration events and vector integrity were determined using the capture-PacBio sequencing approach, a long-read next-generation sequencing method that has not previously been used for this purpose. Chromosomal integrations were found at a surprisingly high frequency of 1%-3% both in vitro and in vivo. Importantly, most of the inserted rAAV sequences were heavily rearranged and were accompanied by deletions of the host genomic sequence at the integration site.
Topics: Animals; Cells, Cultured; Chromosomes; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Hepatocytes; Humans; Liver Regeneration; Mice; Transduction, Genetic; Virus Integration
PubMed: 34461297
DOI: 10.1016/j.ymthe.2021.08.031 -
Current Opinion in Pharmacology Oct 2015Gene transfer vectors based on adeno-associated virus (AAV) are showing exciting therapeutic promise in early phase clinical trials. The ability to cross-package the... (Review)
Review
Gene transfer vectors based on adeno-associated virus (AAV) are showing exciting therapeutic promise in early phase clinical trials. The ability to cross-package the prototypic AAV2 vector genome into different capsids is a powerful way of conferring novel tropism and biology, with evolving capsid engineering technologies and directed evolution approaches further enhancing the utility and flexibility of these vectors. Novel properties of specific capsids show unpredictable species and cell-type specificity. Therefore, full realisation of the therapeutic potential of AAV vectors requires the development of more therapeutically predictive preclinical methods for evaluating capsid performance. This will strongly complement an iterative approach to the evaluation of capsid variants in the clinic and, should wherever possible, include the determination of gene transfer efficiencies.
Topics: Animals; Capsid; Dependovirus; Genetic Therapy; Genetic Vectors; Humans; Serogroup
PubMed: 26291407
DOI: 10.1016/j.coph.2015.07.006 -
Current Opinion in Virology Oct 2014Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue... (Review)
Review
Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue tropism, and limited viral host response has made it an attractive vector system for gene therapy. The viral protein capsid, the primary interface with the host, is the main determinant for these phenotypes, is highly variable, and is most subject to pressures during replication. Here, we explore the evolutionary path of AAV and other parvoviruses in respect to these phenotypes, as well as directed evolution and engineering strategies that have exploited the lessons learned from natural selection in order to address remaining limitations of AAV as a therapeutic gene transfer platform.
Topics: Biological Evolution; Dependovirus; Directed Molecular Evolution; Genetic Therapy; Genetic Vectors; Humans; Selection, Genetic; Viral Tropism
PubMed: 25128609
DOI: 10.1016/j.coviro.2014.07.008 -
Current Opinion in Biotechnology Apr 2022Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical... (Review)
Review
Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical breakthroughs. As of April 2021, there are two rAAV-based gene therapies approved and more than two-hundred active clinical trials (approximately thirty in Phase III). However, the expected increase in demand for rAAV vectors still poses several challenges. Discussed herein are key aspects related to R&D needs and Chemistry, Manufacturing and Control (CMC) efforts required to attend this growing demand. Authors provide their perspective on strategic topics for rAAV-based therapies success: scalability and productivity; improved safety; increased process understanding combined with development of orthogonal bioanalytics that are able to identify, monitor and control Critical Quality Attributes (CQAs) during bioprocessing.
Topics: Dependovirus; Genetic Therapy; Genetic Vectors
PubMed: 35007989
DOI: 10.1016/j.copbio.2021.12.009 -
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 -
Bioanalysis Jan 2021
Topics: Dependovirus; Genetic Therapy; Humans; Neoplasms; Rare Diseases
PubMed: 33496630
DOI: 10.4155/bio-2020-0295 -
Toxicologic Pathology Oct 2023This article reviews the presentation given at the 2023 annual meeting of the Society of Toxicologic Pathology (STP) on liver toxicity observed with adeno-associated... (Review)
Review
This article reviews the presentation given at the 2023 annual meeting of the Society of Toxicologic Pathology (STP) on liver toxicity observed with adeno-associated viral vector (AAV) gene therapy. After decades as a therapeutic modality largely confined to the academic research environment, gene therapy has emerged in recent years as a rapidly expanding therapeutic approach in the biopharmaceutical industry with AAV as the most commonly used viral vector for gene delivery. This interest in the field of gene therapy by industry has been enhanced by the recent success of approved therapies for curing genetic diseases such as ZOLGENSMA for spinal muscular atrophy and LUXTURNA for Leber congenital amaurosis. However, recently reported clinical and nonclinical toxicities highlight the challenges in safely developing AAV gene therapies that require high dose systemic administration. The presentation reviewed general attributes of AAV as a gene therapy vector, clinical and nonclinical liver toxicity associated with AAV gene therapy and the potential for a multimodal immune suppression strategy that may mitigate toxicities.
Topics: Dependovirus; Genetic Therapy; Gene Transfer Techniques; Genetic Vectors; Liver
PubMed: 37772805
DOI: 10.1177/01926233231201408 -
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 -
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