Enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vectorinduced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of Im-mTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

ImmTOR biodegradable nanoparticles encapsulating rapamycin have been shown to induce a durable tolerogenic immune response to co-administered biologics and gene therapy vectors. Prior mechanism of action studies have demonstrated selective biodistribution of ImmTOR to the spleen and liver following intravenous (IV) administration. In the spleen, ImmTOR has been shown to induce tolerogenic dendritic cells and antigen-specific regulatory T cells and inhibit antigen-specific B cell activation. Splenectomy of mice resulted in partial but incomplete abrogation of the tolerogenic immune response induced by ImmTOR. Here we investigated the ability of ImmTOR to enhance the tolerogenic environment in the liver. All the major resident populations of liver cells, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KC), stellate cells (SC), and hepatocytes, actively took up fluorescent-labeled ImmTOR particles, which resulted in downregulation of MHC class II and co-stimulatory molecules and upregulation of the PD-L1 checkpoint molecule. The LSEC, known to play an important role in hepatic tolerance induction, emerged as a key target cell for ImmTOR. LSEC isolated from ImmTOR treated mice inhibited antigen-specific activation of ovalbumin-specific OT-II T cells. The tolerogenic environment led to a multi-pronged modulation of hepatic T cell populations, resulting in an increase in T cells with a regulatory phenotype, upregulation of PD-1 on CD4+ and CD8+ T cells, and the emergence of a large population of CD4–CD8– (double negative) T cells. ImmTOR treatment protected mice in a concanavalin A-induced model of acute hepatitis, as evidenced by reduced production of inflammatory cytokines, infiltrate of activated leukocytes, and tissue necrosis. Modulation of T cell phenotype was seen to a lesser extent after administration by empty nanoparticles, but not free rapamycin. The upregulation of PD-1, but not the appearance of double negative T cells, was inhibited by antibodies against PD-L1 or CTLA-4. These results suggest that the liver may contribute to the tolerogenic properties of ImmTOR treatment.

Background Intravenous administration of adeno‐associated virus (AAV) vectors is a promising gene therapy approach for monogenic diseases. However, re‐administration of the same AAV serotype is impossible because of the induction of anti‐AAV neutralizing antibodies (NAbs). Here, we examined the feasibility of re‐administrating AAV vector serotypes different from the initial AAV vector serotype. Methods Liver‐targeting AAV3B, AAV5, and AAV8 vectors were intravenously injected in C57BL/6 mice, and the emergence of NAbs and the transduction efficacy following re‐administration were evaluated. Results For all serotypes, re‐administration of the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5, anti‐AAV5 NAbs did not react with other serotypes, resulting in successful re‐administration with the other serotypes. AAV5 re‐administration was also successful in all mice treated with AAV3B and AAV8. Effective secondary administration of AAV3B and AAV8 was observed in most mice initially administrated AAV8 and AAV3B, respectively. However, few mice developed NAbs cross‐reacting with the other serotypes, especially those with close sequence homology. Conclusions In summary, AAV vector administration induced NAbs relatively specific to the administrated serotype. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice.