A single combination gene therapy treats multiple age-related diseases

Abstract

Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual's health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes (fibroblast growth factor 21 [FGF21], αKlotho, soluble form of mouse transforming growth factor-β receptor 2 [sTGFβR2]) delivered using adeno-associated viruses and explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure. Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.

Keywords: AAV; age-related diseases; combination therapy; gene therapy.

Fig. 1.

Systemic AAV delivery of combination gene therapy reverses symptoms of obesity for mice on an HFD. (A) Venn diagram of the combinations of gene therapies explored as well as the color coding for all subsequent graphs. FK, FGF21 + αKlotho. (B) Weight of mice over time injected with a control vector AAV:GFP on an ND or an HFD, and mice on an HFD injected with individual or all combinations of AAV:FGF21, AAV:sTGFbR2, or AAV:αKlotho. n = 10 for AAV:GFP ND mice, and n = 12 for AAV:GFP and all therapy-treated HFD mice. (C) Phenotype exhibited by mice on an HFD with control vector (Right) and AAV:FGF21 vector (Left). (D) Long-term effect on mice receiving an HFD of the AAV:FGF21 therapy up to 250 d postinjection (n = 4). (E) Percentage weight change of 18-mo-old mice on an ND with starting weight ∼40 g (n = 25 for each group). (F) CT scan of mice observing bone density after administration of AAV:FGF21 (n = 2 for each group). (G) Quantified muscle mass of MRI of whole mouse (n = 2 for each group). Statistical tests in B and E are 2-way ANOVA. Statistical tests in F and G are 2-sided t tests. Error bars represent SEM. C, control; F, FGF21; K, αKlotho; T, sTGFβR2; TF, sTGFβR2 + FGF21; TFK, sTGFβR2 + FGF21 + αKlotho; TK, sTGFβR2 + αKlotho. *P < 0.01 compared with AAV:GFP-HFD; **P < 0.0001 values compared with AAV:GFP-HFD.

Fig. 2.

Systemic AAV delivery of combination gene therapy reverses symptoms of diabetes for mice on an HFD. (A) GTT of mice fasted overnight for 8 h. Blood glucose measured at 0, 15, 30, 60, and 120 min after oral gavage of 50 mg of glucose. (B) Area under the curve (AUC) of the GTT. (C) ITT of mice fasted for 6 h. Blood glucose measured at 0, 15, 30, 60, and 120 min after subcutaneous injection of 0.5 IU/kg insulin. (D) AUC of the ITT. (E) HOMA-IR. (F) HOMA-β. (G) Fasting insulin measurements of mice fasted for 8 h overnight before GTT. n = 10 for AAV:GFP ND mice, and n = 12 for AAV:GFP and AAV:FGF21 HFD mice. Statistical tests in B and D–G are 1-way ANOVA. Error bars represent SEM. C, control; F, FGF21; K, αKlotho; T, sTGFβR2; TF, sTGFβR2 + FGF21; TFK, sTGFβR2 + FGF21 + αKlotho; TK, sTGFβR2 + αKlotho; FK, FGF21 + αKlotho. *P < 0.05 values compare HFD-fed control and therapy-treated mice; **P < 0.01 values compare HFD-fed control and therapy-treated mice; ^†P < 0.0001 values compare HFD-fed control and therapy-treated mice.

Fig. 3.

Systemic AAV delivery of combination gene therapy mitigates renal damage due to UUO. (A) Representative MTS kidneys for mice that underwent UUO surgery at day 7 for control, AAV:αKlotho, AAV:FGF21, and AAV:FGF21 + AAV:sTGFbR2 therapy groups. (B) Quantification of renal medullary atrophy of different therapy group kidneys. Photoshop was used to calculate the area of atrophy by tracing inner and outer edges and measuring pixel area. If there was a discontinuity in the shape edge, an ellipse was used for approximation. n values are as follows: control (C) = 7, sTGFβR2 (T) = 8, αKlotho (K) = 6, FGF21 (F) = 8, TK = 8, FK = 6, TF = 7, TFK = 6. (C) Representative images of SMA- and WGA-stained kidneys. (D) Quantification of the ratio of SMA- to WGA-stained kidney sections. n values: C = 5, T = 7, K = 7, F = 8, TK = 5, FK = 7, TF = 9, TFK = 7. All images were taken at 10×, stitched together using Zen Zeiss software, and analyzed using custom MATLAB software that used color thresholding to separate different color pixels. Statistical tests in B and D are 1-way ANOVA. P values compare each therapy group with AAV:GFP. Error bars represent SEM. *P < 0.05; **P < 0.01; ^†P < 0.001. FK, FGF21 + αKlotho.

Fig. 4.

Systemic AAV delivery of combination gene therapy stops progression of heart failure in an AAC mouse model. (A) FS quantification of ECHOs at baseline and 7, 30, and 90 d postsurgery. n = 10 for control (C), sTGFβR2 (T), sTGFβR2 + FGF21 (TF), and sTGFβR2 + FGF21 + αKlotho (TFK). n = 13 for sTGFβR2 + αKlotho (TK). (B) Representative ECHOs of mice at 3 mo post-AAC surgery. (C) Quantification of EF from ECHO. (D) Heart weight (HW) relative to body weight (BW) measured on day 90 post mortem. n for C = 6, n for T = 7, n for TF = 9, n for TK = 9, and n for TFK = 8. (E) Representative image of MTS hearts taken at 10× and stitched together. (F) Quantification of MTS-stained hearts. n for C = 10, n for T = 10, n for TF = 10, n for TK = 13, and n for TFK = 10. All images were taken at 10×, stitched together using Zen Zeiss software, and analyzed using custom MATLAB software that used color thresholding to separate different color pixels. Full ECHO data, including wall thickness, can be found in SI Appendix. Statistical tests in A and B are 2-way ANOVA, with P values representing comparison with AAV:GFP control mice over time. Statistical tests in D and F are 1-way ANOVA. Error bars represent SEM. F, FGF21. *P < 0.05; **P < 0.005; ^†P < 0.0002.