A novel approach to detarget liver transduction is developed by transiently downregulating the expression of key entry factors in this tissue using GalNac-siRNAs prior to AAV9 administration, which blunted hepatic transduction but also redirected the vector to other transduction-permissive tissues.
Abstract
Clinical successes in gene therapy using adeno-associated vectors (AAV) are offering a hopeful path toward the correction of several monogenic disorders. At its core, AAV gene delivery relies on multiple interactions between the capsid or vector genome and numerous host cell factors, a complex process that remains incompletely understood. Whether different serotypes similarly hijack the intracellular machinery in a cell, and whether these processes are necessarily conserved across species, are not known. To identify host factors enabling or preventing transduction across species or serotypes, CRISPR/Cas9 whole-genome knockdown screens were conducted in mouse (AML12) and human (HuH-7) cell lines before transduction with AAV2 or AAV9. Key common entry factors (AAVR, GPR108) were confirmed, with many other serotype- and/or species-specific host cell modulators identified. Interestingly, while serotype-specific differences were observed for successful transduction of a specific cell line, species-specific divergences were even more striking when comparing results across murine and human cells for the same serotype. Leveraging these data, we developed a novel approach to detarget liver transduction by transiently downregulating the expression of key entry factors in this tissue using GalNac-siRNAs prior to AAV9 administration. Such manipulation not only blunted hepatic transduction but also redirected the vector to other transduction-permissive tissues.
The data support the genomic safety of rAAV6 and its applicability to hematological gene therapy and functional enrichment analysis indicated associations with general cellular and structural processes, without enrichment in oncogenic pathways.
H. Lee, Nayoung Park, In-Byung Park et al.· International Journal of Ste...· 0 citations
Lentiviral vectors have revolutionized gene therapy by efficient and stable transduction of dividing and non-dividing cells, their large packaging capacity, and their compatibility with pseudotyping to alter viral tropism. The vesicular stomatitis virus glycoprotein (VSV-G) is widely used as a viral envelope protein of choice to pseudotype lentiviral vector particles as it confers exceptional particle stability and a broad tropism, due to the ubiquitous nature of the low-density lipoprotein receptor (LDLR). While this broad tropism facilitates transduction of diverse cell types, it precludes accurate in vivo targeting of specific cell populations. Structural insights into VSV-G have made receptor-blinding possible and revealed sites amenable to mutation while preserving fusion capacity. Coupled with targeting moieties, VSV-G pseudotyped lentiviral particles are redirected towards cells expressing target antigens. Such targeted vectors open new possibilities for in vivo gene therapy across oncology, infectious diseases, transplantation medicine, and other diseases. Use of targeted vectors will make in vivo gene therapy more accessible than cost-intensive ex vivo gene therapies. Since targeted vectors will be available as 'off-the-shelf' drugs, they will also drastically reduce time-to-treatment. This review highlights advances in bioengineering to exploit the versatility of VSV-G-pseudotyped lentiviral vectors and explores their vast potential for targeted gene delivery.
Anjali Shrivastava, Felix L. Warnecke, J. Schott et al.· Molecular Therapy· 0 citations
This study generated a novel recombinant AAV vector rAAV.hu.hu.S17, derived from the human spleen isolate AAV.hu.S17, and systematically evaluated its capsid features, in vitro transduction, and in vivo tissue tropism.
Wenyan Guo, Jiawen Sun, Fei Wang et al.· Journal of Genetic Engineeri...· 0 citations
The results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.
Carmen Unzu, Amanda X. Chen, Liliana Mancio-Silva et al.· bioRxiv· 0 citations
Tumor-specific targeting remains a major obstacle for the development of precision cancer therapies. For several tumors and specifically glioblastoma (GBM), an aggressive brain tumor with poor prognosis, more effective treatments are urgently needed. Recombinant adeno-associated viruses (rAAVs), with their established clinical utility and mutational capsid plasticity, offer a promising platform for targeted delivery. We engineered and evaluated modified AAV capsids derived from the established AAV2 with HSPG-tropism knockdown and two less-characterized, tropism-reduced AAV9 variants. The epidermal growth factor receptor (EGFR), a tumor marker often overexpressed in GBM, was targeted by inserting an affibody (ZEGFR:1907). Also chlorotoxin (CLTX) was inserted, a peptide from scorpion venom reported to bind GBM. Transduction efficiencies were initially assessed with established cell lines (A431, HeLa, U251MG, MCF7). Affibody-displaying capsids exhibited EGFR-dependent transduction, with AAV9-affibody variants surpassing AAV9 wild-type. Several affibody-displaying capsids also transduced patient-derived GBM explants, as confirmed by fluorescence microscopy. These findings highlight the potential of retargeting AAV9 variants and the use of human surgical tissue samples for the initial evaluation of newly designed AAV capsids.
Claire Rothschild‐Gronau, Philipp Borchert, M. Radukic et al.· Scientific Reports· 0 citations
