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A single combination gene therapy treats multiple age-related diseases

gene therapy aav combination therapy age-related diseases

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#1 Engadin

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Posted 05 November 2019 - 05:08 PM


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F U L L   T E X T   S O U R C E :    PNAS

 

 

 

 

Significance
 
Human and animal longevity is directly bound to their health span. While previous studies have provided evidence supporting this connection, therapeutic implementation of this knowledge has been limited. Traditionally, diseases are researched and treated individually, which ignores the interconnectedness of age-related conditions, necessitates multiple treatments with unrelated substances, and increases the accumulative risk of side effects. In this study, we address and overcome this deadlock by creating adeno-associated virus (AAV)-based antiaging gene therapies for simultaneous treatment of several age-related diseases. We demonstrate the modular and extensible nature of combination gene therapy by testing therapeutic AAV cocktails that confront multiple diseases in a single treatment. We observed that 1 treatment comprising 2 AAV gene therapies was efficacious against all 4 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.
 
Despite the interconnected nature of age-related diseases (1⇓–3), preventing or treating the sum of their diverse pathologies cannot be achieved by modulating a single genetic pathway. Also, while alteration of a single longevity-associated gene using transgenic mice has been shown to improve health span and extend life span by up to 30% (4⇓–6), acting on insight gained from such transgenic or loss-of-function models to generate practical therapies for adult nontransgenic animals has met with little success (7, 8). For instance, there are a number of traditional small molecule therapies that aim to influence longevity gene pathways, yet none are Food and Drug Administration (FDA) approved, and the possibility of related side effects is a concern (9⇓–11). Furthermore, traditional methods by their nature largely ignore the relation between age-related diseases, narrowly influencing a particular pathway involved in the pathogenesis of a single disease. An alternative approach that may relieve the bottleneck between antiaging transgenics and therapeutics is the delivery and direct modulation of longevity gene expression via adeno-associated virus (AAV)-mediated gene therapy (12). Even so, targeting gene therapy to a single pathology cannot correct or prevent the deterioration of health span that results from multiple age-related diseases and not just one.
 
In this work, we developed and tested 3 AAV-based gene therapies and administered them to adult nontransgenic mice for the treatment of 4 age-related diseases. The 3 genes involved in these therapies were fibroblast growth factor 21 (FGF21), αKlotho, and transforming growth factor-β1 (TGFβ1). These 3 genes were chosen due to their known beneficial role in aging and specific disease states (4⇓–6). FGF21 and αKlotho are circulating factors produced by the liver and kidney, respectively (5, 13⇓–15), and TGFβ1 is a secreted factor with expression that is not limited to a particular organ (16). FGF21 has established roles in metabolism and glucose handling (17), αKlotho is a known regulator of intracellular calcium and provides protection in heart and kidney pathologies (18, 19), and TGFβ1 signaling plays an important role in age-related hypertrophic cardiomyopathy, immune recruitment, and extracellular matrix formation (20). Although these 3 genes have known roles in various age-associated disease states, it remains unknown whether their simultaneous perturbation would provide an additive, synergistic, or deleterious phenotype in any given disease.
 
 
Results
 
We selected AAV as the gene therapy delivery method due to its safety, low immunogenicity, ease of manufacturing, ability to infect dividing and nondividing cells, and a growing number of successful human clinical trials (21⇓–23). We began by creating 3 separate AAV8 vectors to overexpress mouse FGF21, a soluble form of mouse transforming growth factor-β receptor 2 (sTGFβR2) that binds and represses TGFβ1 (24), and mouse αKlotho (Methods and SI Appendix, Fig. S1A). The AAV8 serotype was chosen as the delivery vector due to its high infection rate of the liver (25), an organ well known for its ability to produce high levels of secreted proteins (26) and the natural tissue for endogenous FGF21 expression (4). Following the generation and injection of each virus, we verified overexpression of the corresponding transgenes directly or from their downstream effect in mouse plasma using enzyme-linked immunosorbent assay (ELISA) and western blots (Methods and SI Appendix, Fig. S1 B–D) and found up to a 17-fold increase in FGF21, a 95% decrease in circulating TGFβ1, and an ∼10× increase in circulating αKlotho. We also performed full necropsies on mice injected with our therapies, and no remarkable pathological findings were noted, suggesting no harmful effects compared with control mice.
 
Obesity afflicts more than 1 in 3 adults of the US population and is responsible for an overall decrease in health and increased risks for cancer, heart disease, and neurological deterioration among many others (27). In light of FGF21’s reported role in metabolism and fat homeostasis, we hypothesized that sustained overexpression of FGF21 could counter metabolic dysregulation resulting from a high-fat diet (HFD), which is an established model for obesity and type II diabetes in mice (28). It has also been observed that αKlotho can help regulate high blood glucose in diabetes models (29) and that TGFβ1 signaling and other inflammatory pathways also impact obesity and disease (30⇓–32). Accordingly, we sought to investigate if a synergistic advantage could be achieved through the coexpression of αKlotho, FGF21, and sTGFβR2. Mice were fed an HFD for 3 mo, which yielded an average weight increase of 15 g (56%) per mouse compared with mice fed a normal diet (ND) (Methods). Of note, the mice were maintained on an HFD throughout the experiment (pre- and postinjection) to accurately reflect the reticent nature of human dietary habits. HFD mice were infected with AAV:FGF21 (F), AAV:sTGFβR2 (T), and AAV:αKlotho (K) individually or in combination (Fig. 1A). An AAV:GFP © vector was used in the control groups. Recipients of the AAV:FGF21 therapy, regardless of any other treatment, experienced a complete reversal of the obese phenotype within 40 d postinjection that was maintained throughout the study (3 mo), despite the continued HFD (Fig. 1 B and C). To further investigate how permanent this phenotype was, we also kept mice that received only the AAV:FGF21 therapy on an HFD for 8 mo and did not observe any weight reversal (Fig. 1D). It is unclear whether, at the applied AAV:FGF21 dose, any synergism could possibly be observed given the overwhelming effect of FGF21 alone. A slightly diminished effect from both AAV:sTGFβR2 and AAV:αKlotho in combination with AAV:FGF21 was observed, although not statistically significant.
 
 

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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. © 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.

 

 

To evaluate if our therapy could also mitigate age-related obesity, 18-mo-old aged mice on an ad libitum ND were used. These mice tend to naturally experience increased adiposity and weighed on average 40 g. We injected all 3 constructs individually or in combination into these mice, resulting in a return to a lean body weight of 30 g for mice that received AAV:FGF21 alone or in combination within 100 d postinjection, which was maintained until at least the 150-d mark (Fig. 1E). Interestingly, we witnessed a decrease in weight in all therapy groups that received AAV:αKlotho as well. AAV:αKlotho alone and in combination with AAV:sTGFβR2 was able to achieve up to 15% weight loss in naturally occurring age-related obesity but did not show any weight loss effects in middle-aged mice fed an HFD.

 
To further evaluate the effect of AAV:FGF21 on mice fed an HFD, the animals were placed in metabolic chambers, and their activity, food intake, O2 consumption, and CO2 production were measured. Significant increases in both O2 consumption and CO2 production were observed, indicating a higher metabolic rate compared with the HFD AAV:GFP control mice (SI Appendix, Fig. S2). The respiratory exchange ratio (RER) was also found to shift from the dysregulation caused by the HFD, where lipids are the predominant fuel source, toward normal levels, where carbohydrates are principally metabolized (33, 34) (SI Appendix, Fig. S2C). Notably, the AAV:FGF21 mice did not display an increase in activity or a decrease in food consumption, strongly suggesting that the observed weight reduction was due solely to metabolic changes (SI Appendix, Fig. S2C). While we did not investigate to what extent fat absorption contributed to the phenotype (due to a decrease in bile production) (35), the marked changes in CO2 and O2 produced and consumed, respectively, suggest that it is largely due to metabolic effects. Computer-aided tomography (CT) and MRI were used to confirm that the mice given AAV:FGF21 (individually) did not lose bone or muscle mass compared with HFD controls, further confirming that weight loss was due to fat loss (Fig. 1 F and G).
 
Mice fed a prolonged HFD are also known to acquire a type II diabetes phenotype with poor glucose handling (36). Type II diabetes affects 30.3 million people and is a leading risk factor for heart diseases, kidney disease, and stroke (37). Therefore, to investigate the effect of these therapies using a second disease model, a glucose tolerance test (GTT), an insulin tolerance test (ITT), a pyruvate tolerance test (PTT), and fasting blood glucose measurements were performed. GTT is used to assess how quickly an oral bolus of glucose can be cleared from the blood, ITT is used to evaluate the sensitivity of the animals to insulin, and PTT is used to ascertain the ability of the liver to produce glucose. Results of these assays showed that the AAV:FGF21 therapy alone completely mitigated the diabetic phenotype to varying degrees in combination with AAV:αKlotho and/or AAV:sTGFβR2, displaying an enhanced glucose response and recovered insulin sensitivity comparable with that of ND mice without affecting glucose production in the liver (Fig. 2 and SI Appendix, Fig. S3 A and B). While there are trending differences in the GTT curve for therapies AAV:αKlotho and AAV:sTGFβR2 individually or in combination, none are statistically significant without AAV:FGF21. The homeostatic model assessment for insulin resistance (HOMA-IR) and the homeostatic model assessment for β-cell function (HOMA-β) use combined fasting glucose and insulin levels to assess the overall function of this endocrine system. On testing, HOMA-IR showed improved function in HFD mice following treatment with all therapies and combinations compared with HFD AAV:GFP controls (Fig. 2 E and F). Interestingly, we saw a similar trend in the ability of the non-FGF21 therapies to improve insulin–glucose handling in the HOMA-IR and weight loss in old ND mice (Figs. 1E and 2E), suggesting that the HFD may “overpower” the weight loss effects of some of these therapies. However, the HFD was not able to completely abrogate the therapies’ effect on this endocrine system as seen in the HOMA-IR. All 3 therapies provided a substantial and lasting effect following a single administration as opposed to administering them as biologics, whereupon the observed effect is temporary due to its short half-life (i.e., FGF21) (38).
 
 
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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. © 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.
 
 
Kidney failure and renal fibrosis are a major concern regarding the aging population in the United States, with more than 661,000 people either on dialysis or recipients of a kidney transplant (39). Over 38% of patients who experience kidney failure, in fact, eventually die from a cardiac event (39). αKlotho and TGFβ1 have been shown to be key factors in the progression of kidney failure in mice, and FGF21 has been shown to protect against chemotherapeutic kidney damage (18, 40⇓⇓–43). The third disease model used to evaluate the single and combination therapies used unilateral ureteral obstruction (UUO), an established means of simulating progressive renal fibrosis, which is a feature of renal disease (44). We injected mice with single and combination gene therapies 1 wk prior to disease induction via UUO, and kidneys were harvested and analyzed for fibrosis and remodeling 1 wk after the UUO procedure. Whole-kidney images stained with Masson’s Trichrome stain (MTS) showed that overexpression of αKlotho was able to prevent deterioration of the renal medulla and thinning of the renal cortex compared with controls (Fig. 3 A and B). Surprisingly, the largest mitigation of medullary atrophy was due to the combination AAV:sTGFβR2 and AAV:FGF21, which performed significantly better than AAV:αKlotho at preventing renal medullary atrophy, with only 6.4% atrophy compared with 22.5%, respectively (P < 0.05) (Fig. 3 A and B). While kidney sections obtained from mice displayed only a slight increase in fibrosis at 7 d postsurgery, this is in line with earlier findings (18) that reported a trending difference between αKlotho transgenic and wild-type mice at day 7 that became significant at day 14 (Fig. 3A and SI Appendix, Fig. S4B). Myofibroblasts are key mediators of extracellular matrix formation and express α-smooth muscle actin (αSMA) (45). We stained kidney sections for this marker and observed lower αSMA expression compared to UUO controls. The AAV:FGF21 + AAV:sTGFbR2 therapy group had the largest effect with a 59% (P < 0.001) reduction in αSMA staining (Fig. 3 C and D). Surprisingly, we found that the AAV:FGF21 seemed to have a greater effect on medullary atrophy and αSMA than AAV:αKlotho or AAV:sTGFβR2. Also unpredictably, the 2 groups that contained both AAV:FGF21 + AAV:αKlotho were worse than combinations of AAV:sTGFβR2 + AAV: αKlotho or AAV:sTGFβR2 + AAV:FGF21.
 
 
 
 
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