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.
Abstract
Background&Aims Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods Two human hepatocyte models were tested: Fah−/−/Rag2−/−/Il2rg−/− (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions Our 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.
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
Background: Adeno-associated virus (AAV) vector-mediated gene transfer is an emerging treatment strategy for severe cardiac disorders with genetic etiology. We refined the AAV toolkit to achieve efficient and selective expression of the therapeutic transgene in mouse hearts. Methods: Using vectors with a reporter transgene, we evaluated AAV administration routes, AAV serotype tropism to the myocardium, and cardiospecific promoters. Results: We showed that systemic AAV administration provides potent delivery and uniform transduction of cardiac tissue, outperforming localized injection techniques. The MyoAAV 2A capsid variant enabled an improved heart-to-liver transduction ratio compared to the parental AAV9 serotype. Screening a panel of cardiac and pan-muscular promoters in vitro and in vivo verified the superiority of the cardiac troponin T (cTnT) promoter for robust heart-specific transgene expression. Finally, we demonstrated that the cumulative properties of systemic AAV delivery, the MyoAAV 2A serotype, and the cTnT promoter allowed for efficient cardiac synthesis of the therapeutic transgene—an artificial miRNA designed for the gene suppression strategy of FLNC-related cardiomyopathy. Conclusions: Our findings establish an effective AAV approach for transgene transfer into the mouse heart and promote the development of gene therapy for cardiac disorders.
I. Galkin, V. Skopenkova, Maria Y. Shubina et al.· BioTech· 0 citations
RAAV integration patterns in primary human hepatocytes xenografted into FRG mouse livers and in hepatocytes from cynomolgus macaques following systemic rAAV administration support a low oncogenic risk profile for the evaluated vector while reinforcing the value of direct human liver integration analyses to refine risk assessment and guide the development of safer gene therapy platforms.
S. Scott, C. Hallwirth, Natsuki Sasaki et al.· Molecular Therapy· 0 citations
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.
Katie Kubek-Luck, J. Velazquez, Xiao-Rui Yao et al.· Molecular Therapy· 0 citations
Adeno-associated virus (AAV) gene therapy is frequently hindered by dose-limiting toxicities and preexisting neutralizing antibodies (nAbs). Here, we evaluate enhancer polymer (ePL), a cargo-less poly(L-lactic-co-glycolic acid) nanoparticle, the systemic administration of which demonstrated biocompatibility in nonhuman primates (NHPs) and generated a protransduction serum secretome that significantly enhanced AAV uptake in human iPSC-derived cardiomyocytes. Proteomic analysis of human peripheral blood mononuclear cell-derived secretomes identified a transient upregulation of AAV-entry factors, including ITGB3, alongside a downregulation of inflammatory cytokine pathways. Mechanistically, ePL-stimulated macrophages release a paracrine secretome that triggers the rapid, transient degradation of hepatic insulin receptor substrate 2 and subsequent AKT inhibition in liver and isolated primary mouse hepatocytes. Additionally, ePL attenuated the levels of NHP-derived nAbs in vitro and in a mouse-NHP xenotransfusion model without depleting total systemic immunoglobulins. Finally, ePL reduced Immunoglobulin G (IgG) secretory capacity of splenic B cells, suggesting a mechanistic link by which levels of nAbs are likely attenuated. Together, these findings highlight ePL as a promising supporting agent to improve AAV gene transfer, warranting its further optimization for clinical translation.
Lauren Switala, A. Khandaker, Sumita Dutta et al.· Human Gene Therapy· 0 citations