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The siRNA-mediated inhibition of the mTOR complex outperforms rapamycin in mounting antitumor immunity in a mouse model of melanoma

In the era of COVID-19 coronavirus, we've come to know of the pandemic's taking a heavy toll on the elderly.  The older individuals are paying a heavy price as those over 65 y account for the majority of those who perished.  For these individuals, much of the COVID-19 induced mortality was associated with the pre-exising 'underlying conditions'.  The underlying conditions referred to the diseases affecting blood circulation, cancer, diabetes, etc.  These disorders are commonly manifested illnesses that fall within the spectrum of aging-associated disorders.


The mechanism of aging has been studied from multiple angles using various animal models (albeit largely not understood).  The accumulation of genetic mutations has been implicated as a major cause of aging.  These spontaneously occurring mutations are thought to occur during the process of DNA replication or due to faulty DNA repair.  In the case of germ-line tissues (for reproduction), short-lived organisms such as flies (Drosophila) or mice exhibit higher mutation rates than long-lived mammals like the humans.  A similar phenomenon was observed in somatic tissues, which showed an increase in mutation frequency with age in mice, resulting in large genomic rearrangements (Vjig et al., 2002).  As such, molecules such as reactive oxygen species (ROS), which can crosslink DNA, or reducing sugars (ex. glucose, fructose), which form covalent links with structural proteins like collagen (stiffens blood vessels), are implicated in aging.

An alternate view portrays a more "programmed" mechanism of aging.   For instance, at the genetic level, specific methylation changes in DNA may occur with aging; alternatively, the length of telomeres may shorten as cells undergo senescence. 

Throughout the history, the ideal of reaching immortality has been a subject of intense fascination.  In the 1980s, researchers found that a single-gene mutation could increase the lifespan of nematode worm (C. elegans) significantly (Klass, 1983; Friedman, 1988).  This has led to the whole genome RNA interference screening (using siRNAs), which resulted in identifying >200 genes whose silencing (individually) increases lifespan (25-30% in flies, ~40% in mice) (Vjig et al., 2008).  Interestingly, many of the genes suppressed function in biochemical pathways that regulate cell growth (insulin or insulin-like growth factor-1 signaling), energy metabolism (ex. mitochondrial electron transport), etc., mimicking calorie-restriction.

                    

Over the years, pharmaceutical industries as well as the academic institutions have been actively engaged in finding novel therapeutics in natural product resources as diverse as the deep-sea environment or Amazon rainforest.  During 1960s, an expedition to Easter Island (i.e. Rapa Nui island located in South Pacific Ocean >2000 miles west of Chile) resulted in the discovery of an anti-fungal drug 'rapamycin' (named after the island) in 1972, which arrests cell cycle progression at G1 phase.  As it inhibits T cell proliferation, rapamycin has been approved by FDA (U. S. Food & Drug Administration) as an immunosuppressant (to suppress immune rejection of transplanted organs or coronary stents).

Subsequent research has uncovered that rapamycin forms a complex with FKBP12 protein, which in turn inhibits mTOR.   mTOR is a protein kinase that represents a core component of mTOR complexes 1 and 2.  It regulates various processes critical to cancer development such as cell proliferation, cell movement, etc.  The mTOR kinase integrates upstream signals (ex. nutrients, growth factors, energy level) with downstream events such as gene transcription or protein synthesis to impact autophagy (degradation of defective cell organelles), metabolism, growth, etc. that are critical for cell survival.  Subsequently, rapamycin (enterically delivered via feeding) was shown to extend the lifespan of worms, flies, and mice in the laboratory, generating an enormous amount of interest pharmaceutically (albeit FDA does not approve drugs that merely impede aging) (Robida‐Stubbs et al., 2012; Bjedov et al., 2010; Harrison et al., 2009; Livi et al., 2013). 

For vaccine induced immune response against pathogens like COVID-19 coronavirus or cancer, 'memory cells' are thought to play a critical role.  Immunologically, inhibition of mTOR by rapamycin led to converting activated CD8+ T cells into memory cells.  Nonetheless, as rapamycin is associated with immunosuppression, investigators at the University of Miami (USA) have developed an aptamer-guided siRNA targeting RAPTOR (a subunit of mTOR complex 1) and showed that it performs superior to rapamycin in mounting antitumor immunity against melanoma in a mouse model (Berezhnoy et al., 2014).

 

The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA).  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as to conjugate to peptides or antibodies.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

 

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References

Bjedov I, Toivonen JM, et al. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metabolism. 2010; 11:35‐46. PMID:  20074526

Berezhnoy A, Gilboa E, et al. Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity.  J Clin Invest. 124:188-97 (2014).  PMID: 24292708

Friedman DB, Johnson TE. A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility.  Genetics.  118:75-86 (1988). PMID: 8608934

Harrison DE, Strong R, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. 2009; 460:392‐395.  PMID:  19587680  

Hua H, Kong Q, et al.  Targeting mTOR for cancer therapy.  J Hematol Oncol.  12:71 (2019).  PMID: 31277692

Klass MR.  A method for the isolation of longevity mutants in the nematode Caenorhabditis elegans and initial results.   Mech Ageing Dev. 22:279-86 (1983).  PMID: 6632998

Livi CB, Sharp ZD, et al. Rapamycin extends life span of Rb1+/- mice by inhibiting neuroendocrine tumors.  Aging (Albany NY).  5:100-10 (2013).  PMID: 23454836

Robida‐Stubbs S, Glover‐Cutter K, , et al.  TOR signaling and rapamycin influence longevity by regulating SKN‐1/Nrf and DAF‐16/FoxO.  Cell Metabolism. 15:713‐724 (2002).  PMID: 22560223

Vijg J, Dollé ME. Large genome rearrangements as a primary cause of aging.   Mech Ageing Dev.  123:907-15 (2002). PMID: 12044939

Vijg J, Campisi J.  Puzzles, promises and a cure for ageing.   Nature.  454:1065-71 (2008).  PMID: 18756247