Gene engineering is the most powerful existing tool for life extension. Mutations in certain genes result in up to 10-fold increase in nematode lifespan and in up to 2-fold increase in a mouse life expectancy. Gene therapy represents a unique tool to transfer achievements of gene engineering into medicine. This approach has already been proven successful for treatment of numerous diseases, in particular those of genetic and multigenic nature. More than 2000 clinical trials have been launched to date.
We propose developing a gene therapy that will radically extend lifespan. Genes that promote longevity of model animals will be used as therapeutic agents. We will manipulate not a single gene, but several aging mechanisms simultaneously. A combination of different approaches may lead to an additive or even a synergistic effect, resulting in a very long life expectancy. For this purpose, an animal will be affected by a set of genes that contribute to longevity. In addition, a gene therapy of all major age-related pathologies will be developed to improve the functioning of individual organs and tissues in old age. As a result, we will develop a comprehensive treatment that will not only dramatically extend lifespan, but will also prevent the decrepitude of the body. Experiments will be conducted in old mice. Thus, in case of success, the developed method of aging treatment can be quickly moved to clinical trials.
The goal of the project is to develop a complex gene therapy that will drastically increase mouse lifespan and prevent tissue pathology in old age, coupled with the safety assessment of the treatment.
11 genes that are most promising in terms of life extension (table 1) will be used as targets for gene therapy. We will affect both the biological aging mechanisms, common to all the cells of the organism, as well as the primary neuroendocrine center, that regulates the whole organism’s longevity – the hypothalamus. The expression increase or decrease of these genes in animal models was shown to result in boosted longevity. If the increase in expression of a particular gene is necessary for longevity, we will deliver this gene into the body. If, on the other hand, longevity depends on the inhibition of a certain gene’s expression, we will introduce a genetic construct that encodes small RNAs that inhibit the expression of the target gene. Two out of 10 genes have previously been used for gene therapy of aging: the lifespan of mice was increased by 20% (Zhang et al., 2013, Bernardes de Jesus et al., 2012). In addition, we will deliver 8 genes that prevent the individual tissue function disruption in old age. Each of these genes separately has previously been successfully used for gene therapy of one of the age-related diseases in rodent models (table 2).
Therapeutic genes will be introduced into the body using viral vectors – the most powerful method of delivering genetic constructs. This novel therapy that utilizes all the genes simultaniously will be used for radical life extension and for fighting decrepitude. Furthermore, each of the therapeutic genes will be tested individually. All the experiments will be conducted in 2-year old mice.
The experiments will be conducted in the following groups of experimental animals:
- Simultaneous impact of 11 genes, known to extend life expectancy (table 1) and 8 genes that prevent the development of age-related diseases in various tissues (table 2)
- Simultaneous impact of 11 genes, known to boost longevity (table 1)
- Simultaneous impact of 8 genes that prevent the development of age-related pathologies in different tissues (table 2)
- The impact of each of the 10 genes that extend lifespan, individually (11 groups of animals) (table 1)
- Exposure to a combination of the 10 most effective geroprotectors
- Old animals without impact
- Young animals without impact
First of all, the efficiency of the delivery of therapeutic genes into the cells and duration of gene expression will be tested. If a tissue-specific therapy is needed, the specificity of the therapeutic construct delivery to the target tissue will be studied as well.
All groups of mice will be regularly tested for aging markers, and also the blood and adipose tissue transcriptome, proteome and metabolome will be analyzed. All age-related histological and physiological changes will be studied. Behavioral test will be performed to analyze cognitive ability and locomotor activity in mice. The average and maximum lifespan of mice will be determined. In addition, a detailed study of side effects will be performed. Mice will be compared with old mice of the control group as well as with young mice.
Table 1. Target genes for life extending gene therapy.
|Target gene||The impact on gene expression||Therapeutic effect|
|Effects on the hypothalamus|
|NF-кВ||Expression inhibition||The inhibition of NF-KB transcription factor causes an increase in hormone production by the hypothalamus with during aging and hypothalamus rejuvenation
|UCP2||Overexpression||Uncoupling protein 2 elevates the temperature of the hypothalamus, which is accompanied by a slight decrease in the overall body temperature and increased longevity|
|Systemic effect on most body cells|
|TERT||Overexpression||The catalytic subunit of the telomerase extends the end regions of chromosomes – the telomeres, which increase the replicative potential of cells and longevity of the body|
|Repetitive sequences of the genome, encoding retrotransposons||Expression inhibition||Inhibiting retrotransposon expression leads reduced genetic instability in old age|
|CRTC1||Expression inhibition||Inhibiting TOR-kinase, which promotes cell growth and proliferation, leads to increased life expectancy|
|FOXO3||Overexpression||A transcription factor that triggers stress response and promotes longevity|
|TFEB||Overexpression||A transcription factor that activates autophagy and leads to longevity|
|ELAVL1||Overexpression||RNA – binding protein HuR stabilizes mRNA of factors regulating the cell cycle. The overexpression of HuR leads to rejuvenation of senescent cells|
|SIRT6||Overexpression||The overexpression of sirtuin 6 – a NAD + -dependent deacetylase, leads to an increase in life expectancy|
|AMPK genes||Overexpression||AMPK overexpression triggers stress response and promotes longevity|
|Effect on senescent cells|
|HSV-TK||Overexpression||Herpes virus thymidine kinase promotes the transformation of a non-toxic prodrug into a toxic product. Thus, exposure to the prodrug induces death of senescent cells|
Table 2. Target genes for gene therapy of age-related pathologies. Overexpression of these genes is necessary for the treatment of senile tissue decrepitude.
|Target gene||Tissue and delivery method||Therapeutic effect||Genetherapyresearchlinks|
|VEGF||Systemic delivery into the blood||Vascular endothelial growth factor enhances angiogenesis (blood vessel formation)||Wang et al., 2004|
|BMP2, BMP7||Systemic delivery into the blood||Bone morphogenetic proteins enhance bone formation and the fracture healing process||Yue et al., 2005; Wang et al., 2008|
|IL-2 gene||Systemic delivery into the blood||A cytokine that stimulates an immune response||Fayad et al., 2004|
|CREB||Hippocampus||A transcription factor that in the hippocampus leads to the improvement of long-term memory formation||Mouravlev et al., 2006|
|IGF-1||Systemic delivery to the CNS||Insulin-like growth factor-1, whose delivery to the central nervous system (CNS) causes improvement of locomotor activity||Nishida et al., 2011|
|ecSOD||Penis||Extracellular superoxide dismutase improves erectile function by reducing oxidative stress||Bivalacqua et al., 2003|
|GDNF||Hypothalamus||Glial-derived neurotrophic factor that reduces obesity when delivered to the hypothalamus||Tumer et al., 2006|
|PVALB||Heart||A Ca2+- binding protein, that causes improvement of the hearts diastolic function||Schmidt et al., 2005|
Project authors: Anastasia Shubina, Mikhail Batin, Maria Konovalenko and Alexey Moskalev.
- Bernardes de Jesus B., Vera E., Schneeberger K., Tejera A.M., Ayuso E., Bosch F., Blasco M.A. Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer // EMBO Mol Med. – 2012. – .v.4(8). – P.691-704.
- Bivalacqua T.J., Armstrong J.S., Biggerstaff J., Abdel-Mageed A.B., Kadowitz P.J., Hellstrom W.J., Champion H.C. Gene transfer of extracellular SOD to the penis reduces O2-* and improves erectile function in aged rats // Am J Physiol Heart Circ Physiol. – 2003. – v.284(4). – H1408-21.
- Fayad R., Zhang H., Quinn D., Huang Y., Qiao L. Oral administration with papillomavirus pseudovirus encoding IL-2 fully restores mucosal and systemic immune responses to vaccinations in aged mice // J Immunol. – 2004. – v.173(4). –P.2692-8.
- Mouravlev A., Dunning J., Young D., During M.J. Somatic gene transfer of cAMP response element-binding protein attenuates memory impairment in aging rats // Proc Natl Acad Sci U S A. – 2006. – v.103(12). – P.4705-10.
- Nishida F., Morel G.R., Hereñú C.B., Schwerdt J.I., Goya R.G., Portiansky E.L. Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats // Neuroscience. – 2011. – v.177. – P.195-206.
- Schmidt U., Zhu X., Lebeche D., Huq F., Guerrero J.L., Hajjar R.J. In vivo gene transfer of parvalbumin improves diastolic function in aged rat hearts // Cardiovasc Res. – 2005. – v.66(2). – P.318-23.
- Tümer N., Scarpace P.J., Dogan M.D., Broxson C.S., Matheny M., Yurek D.M., Peden C.S., Burger C., Muzyczka N., Mandel R.J. Hypothalamic rAAV-mediated GDNF gene delivery ameliorates age-related obesity // Neurobiol Aging. – 2006. – v.27(3). – P.459-70.
- Wang H., Keiser J.A., Olszewski B., Rosebury W., Robertson A., Kovesdi I., Gordon D. Delayed angiogenesis in aging rats and therapeutic effect of adenoviral gene transfer of VEGF // Int J Mol Med. – 2004. – v.13(4). – P.581-7.
- Wang Q-L., Han Q-L., Kang J., Gou S.-H., Wang L.-Zh. Polyethylenimine-mediated BMP-7 gene transfection promotes fracture healing in elderly rats // Academic Journal of Second Military Medical University. – 2008. – v.28(5). – P.514-518.
- Yue B., Lu B., Dai K.R., Zhang X.L., Yu C.F., Lou J.R., Tang T.T. BMP2 gene therapy on the repair of bone defects of aged rats // Calcif Tissue Int. – 2005. – v.77(6). – P.395-403.
- Zhang G., Li J., Purkayastha S., Tang Y., Zhang H., Yin Y., Li B., Liu G., Cai D. Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH // Nature. – 2013. – v.497(7448). – P.211-6.