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
1. Introduction
2. Results
2.1. Evaluation of FIX Activity and Protein Expression Following AAV8-FIX-TripleL Administration in Hemophilia B Mice
2.2. Biodistribution of AAV8-FIX-TripleL in Hemophilia B Mice
2.3. Safety Evaluation of AAV8-FIX-TripleL in Hemophilia B Mice
2.4. AAV8-FIX-TripleL Does Not Increase the Levels of the Prothrombotic Markers Thrombin-Antithrombin Complex and D-Dimers
2.5. Immunogenicity Evaluation of AAV8-FIX-TripleL in Hemophilia B Mice
3. Discussion
4. Materials and Methods
4.1. Recombinant AAV Vectors
4.2. Animal Experiments
4.3. ELISA for Quantitating hFIX in Plasma
4.4. FIX Activity Assay
4.5. Detection of hFIX DNA in Organs
4.6. Measurements of TAT and D-Dimer Concentrations
4.7. Detection of the Anti-AAV8 Capsid IgG Antibody
4.8. Detection of Anti-FIX IgG Antibodies
4.9. FIX-Neutralizing Antibody Detection (Bethesda Inhibitor)
4.10. Safety Assessments
4.11. Histopathological Examination
4.12. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Group | Vector Treatment | Dose (VG/kg) | Harvest Day | Animal Number/Sex |
---|---|---|---|---|
1 | PBS | - | Day 2 | 15/Male |
2 | AAV8-FIX-TripleL | 5 × 1011 | Day 2 | 15/Male |
3 | AAV8-FIX-TripleL | 5 × 1012 | Day 2 | 15/Male |
4 | PBS | - | Day 15 | 15/Male |
5 | AAV8-FIX-TripleL | 5 × 1011 | Day 15 | 15/Male |
6 | AAV8-FIX-TripleL | 5 × 1012 | Day 15 | 15/Male |
7 | PBS | - | Day 29 | 15/Male |
8 | AAV8-FIX-TripleL | 5 × 1011 | Day 29 | 15/Male |
9 | AAV8-FIX-TripleL | 5 × 1012 | Day 29 | 15/Male |
10 | PBS | - | Day 91 | 15/Male |
11 | AAV8-FIX-TripleL | 5 × 1011 | Day 91 | 15/Male |
12 | AAV8-FIX-TripleL | 5 × 1012 | Day 91 | 15/Male |
Days | Day 2 | Day 15 | Day 29 | Day 91 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Dose (VG/kg) | - | 5 × 1011 | 5 × 1012 | - | 5 × 1011 | 5 × 1012 | - | 5 × 1011 | 5 × 1012 | - | 5 × 1011 | 5 × 1012 | |
Lung | Osseous metaplasia, alveoli, focal, minimal | 1/15 | - | - | - | - | - | - | - | - | - | - | - |
Score value | 0.07 ± 0.26 | - | - | - | - | - | - | - | - | - | - | - | |
Infiltration, mononuclear cell, focal, minimal | - | - | 1/15 | - | - | 1/15 | - | - | 1/15 | 1/15 | - | 1/15 | |
Score value | - | - | 0.07 ± 0.26 | - | - | 0.07 ± 0.26 | - | - | 0.07 ± 0.26 | 0.07 ± 0.26 | - | 0.07 ± 0.26 | |
Heart | Infiltration, mononuclear cell, focal, minimal | 1/15 | - | - | - | - | - | 1/15 | - | - | - | - | - |
Score value | 0.07 ± 0.26 | - | - | - | - | - | 0.07 ± 0.26 | - | - | - | - | - | |
Mineralization, focal, minimal | - | - | - | 1/15 | - | - | - | - | - | - | - | - | |
Score value | - | - | - | 0.07 ± 0.26 | - | - | - | - | - | - | - | - | |
Cardiomyopathy, focal, minimal | - | - | - | - | - | - | - | - | 1/15 | - | - | - | |
Score value | - | - | - | - | - | - | - | - | 0.07 ± 0.26 | - | - | - | |
Spleen | Pigment, focal, minimal | 1/15 | 1/15 | - | - | - | - | - | - | - | - | - | - |
Score value | 0.07 ± 0.26 | 0.07 ± 0.26 | - | - | - | - | - | - | - | - | - | - | |
Pancreas | Vacuolation, focal, minimal | 1/15 | - | - | - | - | - | - | - | - | - | - | - |
Score value | 0.07 ± 0.26 | - | - | - | - | - | - | - | - | - | - | - | |
Liver | Infiltration, mononuclear cell, focal, minimal | 8/15 | 5/15 | 7/15 | 9/15 | 9/15 | 6/15 | 5/15 | 9/15 | 8/15 | 8/15 | 7/15 | 2/15 |
Score value | 0.53 ± 0.52 | 0.33 ± 0.49 | 0.47 ± 0.52 | 0.60 ± 0.51 | 0.60 ± 0.51 | 0.40 ± 0.51 | 0.33 ± 0.49 | 0.60 ± 0.51 | 0.60 ± 0.63 | 0.53 ± 0.52 | 0.47 ± 0.52 | 0.13 ± 0.35 * | |
Necrosis, focal, minimal | - | - | - | 2/15 | - | - | - | - | - | - | - | - | |
Score value | - | - | - | 0.13 ± 0.35 | - | - | - | - | - | - | - | - | |
Fibrosis, focal, mild | - | - | - | - | - | - | - | - | 1/15 | - | - | - | |
Score value | - | - | - | - | - | - | - | - | 0.13 ± 0.52 | - | - | - | |
Kidneys | Infiltration, mononuclear cell, cortex, focal, minimal | 1/15 | 3/15 | 1/15 | 3/15 | - | 3/15 | 4/15 | - | 3/15 | 4/15 | 3/15 | 5/15 |
Score value | 0.07 ± 0.26 | 0.20 ± 0.41 | 0.07 ± 0.26 | 0.20 ± 0.41 | - | 0.20 ± 0.41 | 0.27 ± 0.46 | - | 0.20 ± 0.41 | 0.27 ± 0.46 | 0.27 ± 0.59 | 0.33 ± 0.49 | |
Vacuolation, glomerulus, focal, minimal | - | - | 1/15 | - | - | - | - | - | - | - | - | - | |
Score value | - | - | 0.07 ± 0.26 | - | - | - | - | - | - | - | - | - | |
Cyst, cortex/medulla, focal, minimal to moderate | 3/15 | - | - | - | 1/15 | - | - | - | - | - | - | - | |
Score value | 0.40 ± 0.91 | - | - | - | 0.07 ± 0.26 | - | - | - | - | - | - | - | |
Cast, renal tubule, cortex, focal, minimal | 2/15 | - | - | - | 1/15 | - | 1/15 | 1/15 | - | - | 1/15 | 2/15 | |
Score value | 0.13 ± 0.35 | - | - | - | 0.07 ± 0.26 | - | 0.07 ± 0.26 | 0.07 ± 0.26 | - | - | 0.07 ± 0.26 | 0.13 ± 0.35 | |
Basophilia, tubule, cortex, focal, minimal | - | - | - | - | 1/15 | - | - | - | - | - | - | - | |
Score value | - | - | - | - | 0.07 ± 0.26 | - | - | - | - | - | - | - | |
Dilation, tubule, cortex, focal, minimal | - | - | - | - | - | - | 1/15 | - | - | - | - | - | |
Score value | - | - | - | - | - | - | 0.07 ± 0.26 | - | - | - | - | - | |
Adrenals | Hyperplasia, subcapsular, cortex, focal, minimal | 2/15 | 1/15 | 1/15 | - | 1/15 | - | 1/15 | - | - | - | 2/15 | - |
Score value | 0.13 ± 0.35 | 0.07 ± 0.26 | 0.07 ± 0.26 | - | 0.07 ± 0.26 | - | 0.07 ± 0.26 | - | - | - | 0.13 ± 0.35 | - | |
Accessory adrenocortical nodule, focal, minimal to mild | - | 2/15 | - | - | - | - | - | - | - | 1/15 | 1/15 | - | |
Score value | - | 0.20 ± 0.56 | - | - | - | - | - | - | - | 0.07 ± 0.26 | 0.07 ± 0.26 | ||
Testis | Degeneration, germ cell, focal, moderate | - | - | - | - | - | - | 1/15 | 1/15 | - | - | - | - |
Score value | - | - | - | - | - | - | 0.20 ± 0.77 | 0.20 ± 0.77 | - | - | - | - |
Days | Day 2 | Day 15 | Day 29 | Day 91 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Dose (VG/kg) | - | 5 × 1011 | 5 × 1012 | - | 5 × 1011 | 5 × 1012 | - | 5 × 1011 | 5 × 1012 | - | 5 × 1011 | 5 × 1012 |
AAV8 antibody/total no. of animals | 0/15 | 0/15 | 0/15 | 0/15 | 15/15 | 15/15 | 0/15 | 15/15 | 15/15 | 0/15 | 15/15 | 15/15 |
Range (μg/mL) | - | - | - | - | 5.21 ± 2.38 | 10.99 ± 4.51 | - | 11.70 ± 5.51 | 32.79 ± 12.10 | - | 24.78 ± 25.15 | 42.50 ± 22.36 |
FIX antibody/total no. of animals | 0/15 | 0/15 | 0/15 | 0/15 | 1/15 | 0/15 | 0/15 | 0/15 | 4/15 | 0/15 | 0/15 | 4/15 |
Range (μg/mL) | - | - | - | - | 7.61 | - | - | - | 3.60 ± 5.48 | - | - | 0.57 ± 0.31 |
Bethesda inhibitor/total no. of animals | 0/15 | 0/15 | 0/15 | 0/15 | 0/15 | 0/15 | 0/15 | 0/15 | 0/15 | 0/15 | 0/15 | 0/15 |
Titer (BIU) | - | - | - | - | - | - | - | - | - | - | - | - |
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Chou, S.-C.; Huang, C.-P.; Su, Y.-H.; Yu, C.-H.; Yang, Y.-L.; Wang, S.-C.; Lin, Y.-H.; Chen, Y.-T.; Li, J.-Y.; Chang, Y.-T.; et al. 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. Int. J. Mol. Sci. 2025, 26, 6073. https://doi.org/10.3390/ijms26136073
Chou S-C, Huang C-P, Su Y-H, Yu C-H, Yang Y-L, Wang S-C, Lin Y-H, Chen Y-T, Li J-Y, Chang Y-T, et al. 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. International Journal of Molecular Sciences. 2025; 26(13):6073. https://doi.org/10.3390/ijms26136073
Chicago/Turabian StyleChou, Sheng-Chieh, Cheng-Po Huang, Ying-Hui Su, Chih-Hsiang Yu, Yung-Li Yang, Ssu-Chia Wang, Yi-Hsiu Lin, Yen-Ting Chen, Jia-Yi Li, Yen-Ting Chang, and et al. 2025. "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" International Journal of Molecular Sciences 26, no. 13: 6073. https://doi.org/10.3390/ijms26136073
APA StyleChou, S.-C., Huang, C.-P., Su, Y.-H., Yu, C.-H., Yang, Y.-L., Wang, S.-C., Lin, Y.-H., Chen, Y.-T., Li, J.-Y., Chang, Y.-T., Chen, S.-Y., & Lin, S.-W. (2025). 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. International Journal of Molecular Sciences, 26(13), 6073. https://doi.org/10.3390/ijms26136073