New intranasal and injectable gene therapy for healthy life extension

Abstract

As the global elderly population grows, it is socioeconomically and medically critical to provide diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that the adeno-associated virus (AAV) vector induced overexpression of certain proteins, which can suppress or reverse the effects of aging in animal models. In our study, we sought to determine whether the high-capacity cytomegalovirus vector (CMV) can be an effective and safe gene delivery method for two such protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. We report CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Specifically, this treatment significantly improved glucose tolerance, physical performance, as well as preventing body mass loss and alopecia. Further, telomere shortening associated with aging was ameliorated by TERT and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with quality of life and an increased health span.

Keywords: TERT; aging; cytomegalovirus; follistatin; gene therapy.

Fig. 1.

Construction and verification of MCMV_TERT and MCMV_FST. (A) TERT-3′ and FST-3′ FLAG constructs. (B) Expression of TERT (∼131 kDa) or FST (∼41 kDa) proteins in MCMV_TERT- or MCMV_FST-treated NIH/3T3 cells. (C) PFU assay growth curve of MCMV_LUC (WT), MCMV_TERT, and MCMV_FST in NIH/3T3. n = 3 per group. Total photon counts are represented in log₁₀ scale. Data are presented as mean ± SEM. (D) Luciferase signal in vivo 3 d after IP inoculations with mock, WT, MCMV_TERT, and MCMV_FST. (E) Detection of TERT by ELISA in serum of treated 8-mo-old mice over 1 mo. Two-way ANOVA with Tukey’s posttests. P < 0.001 TERT-IN vs. WT-IN group at the same time point; P < 0.001 TERT-IP vs. WT-IP group at the same time point. n = 3 per group. Data are presented as mean ± SEM.

Fig. 2.

MCMV_TERT and MCMV_FST significantly extend lifespan. (A) Survivorship curve comparison, eight mice per group. The survival curve of mice in each group was determined by a Kaplan–Meier survival curve. χ² test, P < 0.001 TERT-IP vs. WT-IP and TERT-IN vs. WT-IN group at the 50% survival probability; P < 0.001 FST-IP vs. WT-IP and FST -IN vs. WT-IN group at the 50% survival probability. n = 8 per group. (B) C57BL/6J mice and human age equivalence at the start of experimental treatment. (C) TERT and (D) FST proteins by ELISA in blood serum from 24-mo-old mice. Two-way ANOVA with Tukey’s posttests. P < 0.001 TERT-IN vs. WT-IN group at the same time point; P < 0.001 TERT-IP vs. WT-IP group at the same time point. n = 3 per group. Data are presented as mean ± SEM. (E) The fold increase of TERT and FST mRNA levels in organs of MCMV_TERT- and MCMV_FST-treated mice by RT-qPCR in comparison to WT-treated mice. n = 3 per group. Data are presented as mean ± SEM.

Fig. 3.

MCMV_TERT-treated mice have longer telomeres. Telomere-FISH images of kidney (A) and muscle (B) tissue sections from 24-mo-old mice in indicated groups. Sections were stained with a CY3-labeled peptide nucleic acid probe complementary to telomeric repeats. The images show that TERT-treated mice have higher telomere fluorescence signal intensities compared to mice in the other groups. Telomeres, red; DAPI, blue. The mean fluorescence signal intensities in quantification of telomeres in the image A of kidney (C) and image B of muscle (D) tissue sections of treated mice in indicated groups. The error bars show SD. (E) Relative telomere lengths in organs from 24-mo-old mice vs. an 8-mo-old control was determined by RT-qPCR. The 36B4 gene was used for normalization (57). The relative telomere length was calculated by ΔCT value as described previously (58). Two-tailed unpaired t test. ***P < 0.001, TERT-IN vs. WT-IN group; P < 0.05, P < 0.01 FST-IN vs. WT-IN group. n = 3 per group. Data are presented as mean ± SE. NS, not significant.

Fig. 4.

MCMV_TERT and MCMV_FST dramatically improved physical and physiological conditions. (A) Hair and body appearance after 8 mo of treatment. (B) Biweekly body weight averages of surviving mice in each group. Treatment interruption (red arrow) and reinitiating (green arrow). n = 8 per group. Data are presented as mean ± SEM. (C) Average number of climbing attempts in 3 min. Two-tailed unpaired t test. P < 0.001 TERT-IP vs. WT-IP and TERT-IN vs. WT-IN group. n = 3 per group. Data are presented as mean ± SEM. (D) Beam crossing average execution time. Two-tailed unpaired t test. P < 0.001 TERT-IP vs. WT-IP and TERT-IN vs. WT-IN group; P < 0.001 FST-IP vs. WT-IP and FST -IN vs. WT-IN group. n = 3 per group. Data are presented as mean ± SEM. (E) Glucose tolerance test. Two-way ANOVA with Tukey’s posttests. P < 0.001 TERT-IP vs. WT-IP and TERT-IN vs. WT-IN group at the same time point; P < 0.001 FST-IP vs. WT-IP and FST -IN vs. WT-IN group at the same time point. n = 3 per group. Data are presented as mean ± SEM. (F) HbA1c levels in mock-, WT-, MCMV_TERT-, and MCMV_FST-treated mice. Two-tailed unpaired t test. P < 0.001 TERT-IP vs. WT-IP and TERT-IN vs. WT-IN group; P < 0.001 FST-IP vs. WT-IP and FST -IN vs. WT-IN group. n = 3 per group. Data are presented as mean ± SEM.

Fig. 5.

MCMV_TERT and MCMV_FST prevent mitochondrial deterioration in mice. (A and B) Representative EM images from the heart and skeletal muscle of untreated young mice and 24-mo-old mice treated with mock, WT, MCMV_TERT, and MCMV_FST (IN groups are shown). (Scale bar, 500 nm.) Quantitative analyses of the number of mitochondria with connected cristae and mitochondria area are on the Right of A and B. Two-tailed unpaired t test. ***P < 0.001 TERT vs. WT group; ^###P < 0.001 FST-IN vs. WT group. NS, not significant. n = 20 per group. Data are presented as mean ± SEM.