Biodistribution and safety of a single rAAV3B-AAT vector for silencing and replacement of alpha-1 antitrypsin in Cynomolgus macaques

Abstract

Alpha-1 antitrypsin deficiency (AATD) is characterized by both chronic lung disease due to loss of wild-type AAT (M-AAT) antiprotease function and liver disease due to toxicity from delayed secretion, polymerization, and aggregation of misfolded mutant AAT (Z-AAT). The ideal gene therapy for AATD should therefore comprise both endogenous Z-AAT suppression and M-AAT overexpression. We designed a dual-function rAAV3B (df-rAAV3B) construct, which was effective at transducing hepatocytes, resulting in a considerable decrease of Z-AAT levels and safe M-AAT augmentation in mice. We optimized df-rAAV3B and created two variants, AAV3B-E12 and AAV3B-G3, to simultaneously enhance the concentration of M-AAT in the bloodstream to therapeutic levels and silence endogenous AAT liver expression in cynomolgus monkeys. Our results demonstrate that AAV3b-WT, AAV3B-E12, and AAV3B-G3 were able to transduce the monkey livers and achieve high M-AAT serum levels efficiently and safely. In this nondeficient model, we did not find downregulation of endogenous AAT. However, the dual-function vector did serve as a potentially "liver-sparing" alternative for high-dose liver-mediated AAT gene replacement in the context of underlying liver disease.

Keywords: AAV gene therapy; AAV3B; alpha-1 antitrypsin deficiency; biodistribution; gene silencing; miRNA; preclinical.

Graphical abstract

Figure 1

Hepatic toxicity from rAAV-AAT is mitigated by co-expression of synthetic miRNA (A) Serum ALT levels (a marker of liver injury; mean ± standard error of the mean) are elevated 8 weeks after dosing mice with the standard AAT transgene, but not with the dual-function construct. (B and C) Histopathologic examination of the liver shows more necrosis and large inclusions in the mice treated with the standard transgene (B) than the dual-function vector (C).

Figure 2

Design of the dual-function vector containing a CBA promoter, a syn-miR sequence complementary to regions of the AAT gene-coding sequence, an AAT gene containing silent mutations to induce mismatches with the miRNA, and a c-MYC tag

Figure 3

The AAV3B-G3 capsid showed higher concentration in the heart and lung than the WT and E12 capsids, but no difference in the liver The lines and error bars indicate mean ± standard deviation; ∗p < 0.05, ∗∗p < 0.01 determined by 1-way ANOVA, followed by Tukey post hoc test.

Figure 4

Circulating AST and ALT levels, as well as platelet count, white blood cell (WBC) count, and blood hemoglobin (HGB) concentration were within normal ranges and showed no significant differences among the groups during the study. Lines indicate means.

Figure 5

c-MYC staining indicates that the transgene was successfully expressed in the liver in all 3 groups, and no abnormalities were found in the liver after histopathological examination Green: c-MYC; blue: DAPI.

Figure 6

Concentration of c-MYC-tagged AAT in the serum increased in all groups and was maintained through the course of the study at nearly 100 μg/mL These levels are ∼20% of the therapeutic threshold (570,000 ng/mL). Data are mean ± standard error of the mean.

Figure 7

Endogenous AAT levels were not reduced in the serum after dosing with the dual-function vector in any of the groups, indicating that the expressed miRNA was not able to silence expression of the gene. Lines indicate median values.

Figure 8

Expression of the miRNA in the liver was validated by qPCR, and was found to be expressed in all 3 groups. Lines indicate median values.