Loss of transgene expression limits liver gene therapy in primates

Abstract

Efforts to improve liver gene therapy have focused on next-generation adeno-associated virus (AAV) vector capsids, transgene delivery, and immunomodulating drugs, such as corticosteroids, to avoid destructive T-cell responses. We conducted a detailed characterization of AAV transduction in nonhuman primate liver across multiple capsids and transgenes to better define interactions that may limit stable and efficient transgene expression. We show that the initial transduction of hepatocytes is high, but the transduction rapidly diminishes to a lower stable baseline of <1% of cells, even though ~10% of the cells retain vector DNA that is localized within the nucleus. Further characterization showed genomic vector integration at frequencies of 1/100 to 1/1,000 genomes, suggesting that one mechanism for stable expression may occur via genome integration. These studies suggest that strategies to enhance durable transgene expression by activating retained nuclear episomes or by increasing the frequency of vector integrations may improve liver directed AAV gene therapy.

Base editing rescue of spinal muscular atrophy in cells and in mice

“…Thus, off-target editing outcomes observed in cell culture experiments were consistent with those observed in vivo over 18 weeks (79). The ABE strategy did not induce any detected coding mutations in either human or mouse genomes, and off-target editing in vivo was lower than in cell culture (reduced by 50% at Muc16 intron 54), likely because of lower copy number and expression levels in transduced cells in vivo or in vivo gene silencing over time (33,36,37).…”

“…In the spinal cord, Zolgensma up-regulates SMN transcript levels by ~25% (35), while in other tissues such as the liver and dorsal root ganglia, gene complementation may cause SMN overexpression that under some circumstances can cause long-term toxicity (21). We do not yet know whether SMN overexpression induces toxicity in patients treated with Zolgensma or how long AAVmediated expression will persist in motor neurons in patients (36,37). As such, a therapeutic modality that restores endogenous gene expression and preserves native SMN regulation by a one-time permanent treatment may address remaining limitations of existing SMA therapies.…”

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates

“…Another challenge remaining for AAV gene delivery is successful re-administration, which may be needed to maximize therapeutic effect, particularly given the loss of transgene expression over time observed with AAV gene delivery (Greig et al 2022). While neutralizing antibodies induced by initial AAV administration can prevent sequential administration of the same AAV (Hamilton and Wright 2021), switching to another AAV serotype with similar or complementary features is a potential solution (Bočkor et al 2017; Colella, Ronzitti, and Mingozzi 2018) that remains underexplored.…”

Genome-scale analysis of cellular restriction factors that inhibit transgene expression from adeno-associated virus vectors

“…(1) Large-scale manufacturing of rAAV for high dose administration (>10 14 viral particles per patient), is expensive and difficult; (2) immunological barriers such as the presence of pre-existing neutralizing antibodies against AAV or the triggering of a robust humoral immune response upon treatment prevent (repeated) administration; (3) hepatic and neuronal genotoxicity has been observed in a number of high-dose administrations; (4) transgene expression can be lost over time due to the clearance of transduced cells through a cytotoxic T cell response and/or potential silencing of transgene expression [4][5][6] .…”

“…Despite the recent clinical successes, rAAV-based gene therapies face several challenges: (i) Large-scale manufacturing of rAAV for high dose administration (>10 14 viral particles per patient), is expensive and difficult; (ii) immunological barriers such as the presence of pre-existing neutralizing antibodies against AAV or the triggering of a robust humoral immune response upon treatment prevent (repeated) administration; (iii) hepatic and neuronal genotoxicity has been observed in a number of high-dose administrations; (iv) transgene expression can be lost over time due to the clearance of transduced cells through a cytotoxic T cell response and/or potential silencing of transgene expression (4–6). Increased efficiency in rAAV transgene expression could therefore allow for a reduction in the therapeutic doses needed, which consequently could decrease costs, elicit fewer neutralizing antibodies, reduce the risk of genotoxicity, and lower AAV capsid-directed T lymphocyte-mediated cytotoxicity.…”