Preclinical Evaluation of the Systemic Safety, Efficacy, and Biodistribution of a Recombinant AAV8 Vector Expressing FIX-TripleL in Hemophilia B Mice: Implications for Human Gene Therapy

Abstract

Gene therapy for hemophilia B offers the advantage of a single administration with sustained therapeutic effects. This study evaluated the systemic safety, efficacy, biodistribution, and immunogenicity of AAV8-FIX-TripleL, a recombinant adeno-associated virus type 8 (AAV8) vector encoding a modified factor IX (FIX) variant with increased activity. In this good laboratory practice (GLP)-compliant study, 180 male FIX-knockout hemophilia B mice were randomized into 12 groups (n = 15) and received intravenous AAV8-FIX-TripleL at therapeutic (5 × 10¹¹ VG/kg) or supraphysiological (5 × 10¹² VG/kg) doses on Day 1. The mice were sacrificed on Days 2, 15, 28, and 91 for comprehensive evaluations, including hematological and biochemical assessments, histopathological examination, FIX protein/activity analysis, immunogenicity assessment, and vector biodistribution via quantitative polymerase chain reaction (qPCR) in major organs. AAV8-FIX-TripleL demonstrated dose-dependent increases in FIX activity and protein levels, with FIX activity exceeding physiological levels and the maintenance of a favorable safety profile. Biodistribution analysis confirmed predominant hepatic accumulation and vector persistence up to 91 days post-injection, with minimal off-target distribution. These findings indicate that AAV8-FIX-TripleL is a promising gene therapy candidate for hemophilia B, as it has robust expression, sustained efficacy, and a favorable safety profile, and that further translational studies are warranted.

Keywords: AAV8; FIX; FIX-TripleL; gene therapy; hemophilia B.

Figure 1

Expression of hFIX following intravenous injection of AAV8-FIX-TripleL in hemophilia B mice. Hemophilia B mice were injected with AAV8-FIX-TripleL at doses of 5 × 10¹¹ VG/kg or 5 × 10¹² VG/kg and sacrificed on Days 2, 15, 29, and 91. Plasma samples were collected to assess FIX activity, protein levels, and specific activity. The data are presented as the means ± SDs (n = 15 per group). (A) Circulating FIX activity measured using a one-stage clotting assay (FIX-specific activated partial thromboplastin time). (B) Total hFIX protein levels detected by ELISA. On Day 2, FIX protein was undetectable in all groups. (C) Specific activity of circulating FIX, calculated by dividing FIX activity (U/mL) by the FIX protein concentration (mg/mL). Since FIX protein was undetectable on Day 2, no specific activity data are available for this time point. hFIX, human factor IX; FIX, factor IX; AAV, adeno-associated virus; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; VG, viral genome. The limit of detection (LOD) for the FIX ELISA was 312.5 ng/mL, and the LOD for FIX activity was 0.625%.

Figure 2

Biodistribution of AAV8-FIX-TripleL following intravenous injection of AAV8-FIX-TripleL in hemophilia B mice. Gene copy numbers of AAV8-FIX-TripleL DNA were assessed in various tissues, including the heart, liver, spleen, lung, kidney, brain, and testis, on Day 2 (A), Day 15 (B), Day 29 (C), and Day 91 (D) following intravenous administration at doses of 5 × 10¹¹ VG/kg or 5 × 10¹² VG/kg. AAV8-FIX-TripleL copy numbers are expressed as copies per 10 ng of total DNA. The data are presented as the means ± SDs (n = 15 per group). Copy numbers in the control group were undetectable and therefore are not displayed. FIX, factor IX; AAV, adeno-associated virus; SD, standard deviation; VG, viral genome.

Figure 3

Liver enzyme profiles in the serum of hemophilia B mice following AAV8-FIX-TripleL administration. Serum levels of AST (A), ALT (B), ALP (C), and LDH (D) were measured at Days 2, 15, 29, and 91 following intravenous administration of AAV8-FIX-TripleL at doses of 5 × 10¹¹ VG/kg or 5 × 10¹² VG/kg. The data are presented as the means ± SDs (n = 15 per group). No statistically significant differences were observed between the dose groups and the control group at any time point. FIX, factor IX; AAV, adeno-associated virus; SD, standard deviation; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; VG, viral genome.

Figure 4

Coagulation activation markers, including thrombin–antithrombin complexes and D-dimers, at various time points following AAV8-FIX-TripleL administration. Plasma levels of TAT (A) and D-dimers (B) were measured at Days 2, 15, 29, and 91 following intravenous administration of AAV8-FIX-TripleL at doses of 5 × 10¹¹ VG/kg or 5 × 10¹² VG/kg. The data are presented as the means ± SDs (n = 15 per group). No statistically significant differences were observed between the dose groups and the control group at any time point. FIX, factor IX; AAV, adeno-associated virus; SD, standard deviation; TAT, thrombin–antithrombin; VG, viral genome.