AGINGSCIENCES™ – Anti-Aging Firewalls™

The flood of telomere/telomerase research news has gotten to be so great that I have to be finicky in selecting items reported in this blog.  That having been said, I think the new finding reported here is an important one when viewed in context.   

The 2009 study Telomeres Acquire Embryonic Stem Cell Characteristics in Induced Pluripotent Stem Cells was important in that it showed that reversion of cells to iPSC status fully restores telomerase activity to iPSCs, equivalent to that in embryonic stem cells (ESCs). “We show here that telomeres are elongated in iPS cells compared to the parental differentiated cells both when using four (Oct3/4, Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming factors and both from young and aged individuals.  We demonstrate genetically that, during reprogramming, telomere elongation is usually mediated by telomerase and that iPS telomeres acquire the epigenetic marks of ES cells, including a low density of trimethylated histones H3K9 and H4K20 and increased abundance of telomere transcripts. Finally, reprogramming efficiency of cells derived from increasing generations of telomerase-deficient mice shows a dramatic decrease in iPS cell efficiency, a defect that is restored by telomerase reintroduction.”  Further, the 2009 publication Balancing Out the Ends during iPSC Nuclear Reprogramming discusses how “telomere length maintenance and long-term proliferative capacity of iPSCs is dependent on telomerase,” and concludes “Although a number of hurdles must still be cleared before iPS-based cell therapy becomes practical, the results (cited above) suggest that reprogramming of telomerase and telomeres may not be one them.”

The new this-week finding provides evidence that reverting cells to induced iPSC status fully restores their ability to express telomerase, even in a case when the original cells are seriously compromised in terms of telomere maintenance capability.  The 17 February 2010 online publication Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients states “Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues^{1, 2, 3}. Patient-specific induced pluripotent stem (iPS) cells⁴ represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT)^{5, 6, 7}. We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3′ deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.” 

Dyskeratosis congenita (DC) “is a rare progressive congenital disorder which results in what in some ways resembles premature aging (similar to progeria). — Specifically, the disease is related to one or more mutations which directly or indirectly affect the vertebrate telomerase RNA component (TERC).”  Apparently several different telomerase-related mutations can lead to DC  – see ref and the associated list of citations.  A 2008 publication Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita explains “Most of the mutations so far identified in patients with classical dyskeratosis congenita impact either directly or indirectly on the stability of RNAs. In keeping with this effect, patients with dyskerin, NOP10, and now NHP2 mutations have all been shown to have low levels of telomerase RNA in their peripheral blood, providing direct evidence of their role in telomere maintenance in humans.”   

The amazing thing about the latest study is that activities of two of the four new genes introduced during cell reprogramming appear to override the effect of the mutated gene or genes that defines the genetic defect that causes DC.  The reverted iPS cells appear to be capable of expressing telomerase and reproducing indefinitely, unlike the original DC cells which cannot express telomerase and die after a few generations.    

The iPSCs generated from DC patients continue to have the mutated genes that created the DC disease.  In fact, they should be like the patient’s original ESCs.  Therefore, there is no guarantee that cells those iPSCs differentiate into will have a capability to express telomerase; I suspect they won’t.  If the iPSCs are going to be used as a therapy for DC, they should be corrected first by splicing out the mutated genes and replacing them with good ones.  See the blog post Treating genetic diseases with corrected induced pluripotent stem cells. 

Besides possibly opening the way to a new therapy for DC, the finding provides further evidence that iPSCs could possibly close the loop in the stem cell supply chain enabling extraordinarily long lives.  See the blog post The stem cell supply chain – closing the loop for very long lives. The essence of the Stem Cell Supply Chain Breakdown theory of aging is that with aging the various pools of somatic (adult) stem cells in the body become depleted and those adult stem cells that are left are less prone to differentiate.  I am talking about mesenchymal and hematopoietic stem cells among others.  Adult stem cells like all other cells are differentiated from our original embryonic stem cells (ESCs).  Unlike ESCs or iPSCs, however, the adult stem cells express less telomerase and have limited replicative lifespans.  The result is that tissue renewal via replacement of normal tissue cells with differentiated somatic stem cells declines with age leading to the symptoms of aging.  So, I have speculated that if we can take a few normal body cells, revert them to iPSC status, multiply them in culture, correct them genetically if necessary,  and then re-introduce them into our bodies so they differentiate to replace the somatic stem cells in their niches, we could create cell renewal that is now a once-through (open loop) process that runs down with age into a continuously operating (closed loop) process that might go a long way towards eliminating aging.  The new finding confirms that reverting cells to iPSC status also gets them off to a roaring start generating telomerase just like ESCs do, and can do that even when the original cells have broken telomerase-generating genes in the case of DC. 

The idea of greatly enhancing longevity by closing the loop in the stem cell supply chain is of course a theory.  We will not know if it will work until it is tried.  The challenges that have to be overcome appear to be 1.  Reverting cells to iPSC status in substantial quantities and in ways that do not introduce foreign genes such as genes from a virus vector into the cell’s DNA, 2. Correcting the DNA in iPSCs for any mutational defects as pointed out above, and 3.  Introducing the iPSCs into the body in such a way that they differentiate in a controlled manner into somatic stem cells in the respective adult stem cell niches.  There has been significant progress on the first challenge as reported in the blog posts Footprint-free” iPSCs – and a crazy wager offer, Update on induced pluripotent stem cells, and Progress in closing the stem cell supply chain loop.  We appear to be moving along but much remains to be learned, particularly regarding the third challenge.

A number of important genome-wide association studies (GWASs) have come to my attention in the last few weeks.  And I anticipate that the current steady stream of them will very soon become a roaring river.  These are studies that sort through genomes of large numbers of people looking for systematic gene variation differences, say comparing genomes of people affected with a disease with genomes of people not so-affected.  “Association analysis:  A method of genetic analysis that compares the frequency of alleles between affected and unaffected individuals; a given allele is considered to be associated with the disease if that allele occurs at a significantly higher frequency among affected individuals(ref).”  Association studies may also compare the genomes of specific samples of people (such as aged Ashkenazi Jews living in Brooklyn or older women from Tanegashima island) or the genomes of disease tissues (such as from specific kinds of cancers) against the general human genome to determine possibly causal correlations between genomic variations and effects, such as extended longevity or the presence of a disease. 

I have already created a number of blog entries reporting on GWAS studies.  My focus here is on the general characteristics of GWAS studies, why they are important, why we will see more and more on them, and where they will lead us.

GWAS studies and why they are important

“If I saw a doctor, he would just find something wrong with me.”  Those are words my stepmother Ann told to me last Friday.  I was fortunate to be able to spend a good amount of time with her and Terry, Ann’s son and my half-brother, in the course of a visit to New York last week.   Ann lives independently in her own apartment in the upper West side of the City and, at age 92, it appears that there is nothing wrong with her.  She gets around easily walking in the city, does her own shopping, is mentally lucid and curious, loves to talk about things going on in the world, goes to movies, theatre and opera, and often sees Terry who lives only a few blocks away.  She had no trouble navigating the massive hallways and stairways of the Museum of Natural History for several hours with us and did not seem to get a bit tired. 

Except for a minor cold now or then, Ann never gets sick. She has a very positive attitude towards life and seems never to experience stress.  Ann was born in a small mining town in Iowa and spent her youth there.  She Moved to Des Moines at about 19, and then later to Detroit, to a 90-acre farm in Yale Michigan, and then to New York.   Ann tells me that when she was about 11 she came across copies of a now long-defunct Macfadden health magazine which strongly influenced her to have healthy eating habits.  She mostly avoids junk food.   Ann takes no medicines. She started taking a multivitamin pill and fish oil only this year.  Other than that she has taken no supplements.   

So, speaking as somebody who has seen many doctors and takes many supplements, I do infer a few things about longevity from the example of Ann. 

1.     Ann must be a winner in the longevity genetic luck-of-the-draw.  Her mother lived to 96 and her maternal grandmother to 92.  On the other hand all seven of her siblings have passed away, and all were younger than Ann.   Her genome must contain a good pro-longevity combination of genes.  I think I could convince Ann to let her genome be sequenced if I could find a reputable longevity-oriented genetics researcher interested in finding out what is keeping her going.

2.     Ann is aging significantly more slowly than many of us.  The rate of aging is not the same for everybody.  Many people are run-down, sick and old at 50 or 60.  Ann is in good physical and mental shape and at 92 probably has a number of years still to go in good health.  Of course, I personally want and would love that.

3.     Ann’s longevity and health has nothing to do with medical progress or getting good medical advice or taking the latest drugs.  She has steered clear of those things all her life. 

4.     Longevity implies not getting the diseases of old age, not managing them, not curing them once you get them.  The same genetic activation pathways that lead to long lives keep us healthy.  This appears to be a lesson learned by researchers at the Kenyon Lab at the University of California, the people who did some of the original research on extending the lifespan of nematodes. “Our work has now led to the discovery that mammalian aging is also regulated hormonally by insulin and IGF-1 endocrine system and has catalyzed a fundamental shift in the way scientists view the aging process, from one that is inevitable and intractable to one that is plastic and subject to regulation. Our findings have important disease implications, since these long-lived mutants have been found to be resistant to many age-related diseases. This raises the possibility of a new therapeutic strategy based on the ability to postpone the onset of age-related disease by slowing the aging process itself(ref).”

5.     Successful aging might mean a lot fewer encounters with the medical establishment because a lot fewer sickness will come up.  Successful anti-aging strategies might make us like Ann.  Instead of senior citizens requiring 3-5 medical appointments a week, a yearly checkup might do.  Most medical practice is repair-shop in nature, dealing with managing or curing sicknesses that have emerged.  If sicknesses emerge a lot less, the need for doctors or hospitals recedes in importance. So, some people on a successful anti-aging track may develop the same attitude as Ann.  “Who needs a Doctor?”

6.     The annual health-care cost for Ann is zero.  Her medicare cost is zero.  If we could all extend our healthy lifespans by ten years it would be worth about ten trillion dollars in decreased health care costs and perhaps twice that much more in productivity gains. (Current US health care costs are something like 3 trillion dollars representing over 17% of gross domestic product(ref), and a disproportionally large slice of the cost is for people in the last 10 years of their lives.)

Longevity is by far the best area of investment for economic development.  With an increase of 10 years in our average healthy lifespan, we could quickly wipe out both the national budget deficit and the national debt.

MicroRNAs (miRNAs) are generating increased excitement among cancer, neurobiology and longevity researchers.  I wrote an introduction to MicroRNAs is in my earlier blog post MicroRNAs, diseases and yet-another view of aging, and readers might want to review that information before proceeding further here.  The purpose of this post is to zero in on some recent research findings in the areas of cancer and aging. 

Again, miRNAs are short strands of RNA encoded by genes which do not themselves encode proteins, proteins being the expression products of “regular” genes.  Instead, MiRNAs can profoundly affect the expression of genes that do encode proteins by post-translational operation on the messenger RNA produced by such genes.  “MicroRNAs downregulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation(ref).”  That is, miRNAs can halt or slow the creation of proteins of “regular”genes by damaging or interfering with their messenger RNA (mRNA) before it is converted into proteins or by interfering with the final protein-creation step.  See the earlier post for more detailed discussion and links on what miRNAs do, how many of them there are, their roles in stem cells, and how they enter into several disease processes.  I also mentioned the possible roles of miRNAs in aging in that post and promised I would elaborate on that topic in a subsequent post, which is this one.

One aspect of miRNAs emphasized in this post is that many of them are ancient and evolutionary conserved in species as different as nematode worms (Caenorhabditis elegans), flies, fish, and humans. This means that aging research done on species that only live a few days might well be applicable to humans. Nematodes are “–  simple enough to be studied in great detail. Strains are cheap to breed and can be frozen. When subsequently thawed they remain viable, allowing long-term storage. — In addition, C. eleagans is transparent, facilitating the study of cellular differentiation and other developmental processes in the intact organism. The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped out(ref).^[8][9]“ 

MiRNAs and cancers

Quoting from a January 2010 review study The role of let-7 in cell differentiation and cancer: “MicroRNAs (miRNAs or miRs) are small noncoding RNAs capable of regulating gene expression at the translational level. Current evidence suggests that a significant portion of the human genome is regulated by microRNAs, and many reports have demonstrated that microRNA expression is deregulated in human cancer. The let-7 family of microRNAs, first discovered in Caenorhabditis elegans, is functionally conserved from worms to humans. The human let-7 family contains 13 members located on nine different chromosomes, and many human cancers have deregulated let-7 expression. A growing body of evidence suggests that restoration of let-7 expression may be a useful therapeutic option in cancers, where its expression has been lost. In this review, we discuss the role of let-7 in normal development and differentiation, and provide an overview of the relationship between deregulated let-7 expression and tumorigenesis. The regulation of let-7 expression, cancer-relevant let-7 targets, and the relationship between let-7 and drug sensitivity are highlighted.” A 2008 publication indicates that the let-7 miRNA works by targeting the miRNA processing enzyme Dicer within its coding sequence.

It appears that miRNAs could play important roles in prognostication of cancer outcomes.  The February 2010 research publication A MicroRNA Expression Signature for Cervical Cancer Prognosis states “Invasive cervical cancer is a leading cause of cancer death in women worldwide, resulting in about 300,000 deaths each year. The clinical outcomes of cervical cancer vary significantly and are difficult to predict. Thus, a method to reliably predict disease outcome would be important for individualized therapy by identifying patients with high risk of treatment failures before therapy. In this study, we have identified a microRNA (miRNA)-based signature for the prediction of cervical cancer survival. miRNAs are a newly identified family of small noncoding RNAs that are extensively involved in human cancers. Using an established PCR-based miRNA assay to analyze 102 cervical cancer samples, we identified miR-200a and miR-9 as two miRNAs that could predict patient survival. A logistic regression model was developed based on these two miRNAs and the prognostic value of the model was subsequently validated with independent cervical cancers. — Our results suggest that both miR-200a and miR-9 could play important regulatory roles in cervical cancer control. In particular, miR-200a is likely to affect the metastatic potential of cervical cancer cells by coordinate suppression of multiple genes controlling cell motility.”

A stream of publications explores the role of the miRMA miR-34a in neuroblastoma. “miR-34a acts as a suppressor of neuroblastoma tumorigenesis by targeting the mRNA encoding E2F3 and reducing E2F3 protein levels(ref).” “A functional screen identifies miR-34a as a candidate neuroblastoma tumor suppressor gene(ref).”  Also see MicroRNA involvement in the pathogenesis of neuroblastoma: potential for microRNA mediated therapeutics, MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells, and The MYCN oncogene is a direct target of miR-34a.The 2008 publication Diagnostic and prognostic microRNAs in stage II colon cancer states “Functional studies showed that miR-145 potently suppressed growth of three different colon carcinoma cell lines. In conclusion, our results suggest that perturbed expression of numerous miRNAs in colon cancer may have a functional effect on tumor cell behavior, and, furthermore, that some miRNAs with prognostic potential could be of clinical importance.”  And the report of a November 2009 study indicates “Human colon cancer profiles show differential microRNA expression depending on mismatch repair status and are characteristic of undifferentiated proliferative states.”A December 2009 study Gene networks and microRNAs implicated in aggressive prostate cancer reports “Prostate cancer, a complex disease, can be relatively harmless or extremely aggressive. To identify candidate genes involved in causal pathways of aggressive prostate cancer, we implemented a systems biology approach by combining differential expression analysis and coexpression network analysis to evaluate transcriptional profiles using lymphoblastoid cell lines from 62 prostate cancer patients with aggressive phenotype (Gleason grade 8) and 63 prostate cancer patients with nonaggressive phenotype (Gleason grade 5). From 13,935 mRNA genes and 273 microRNAs (miRNA) tested, we identified significant differences in 1,100 mRNAs and 7 miRNAs with a false discovery rate (FDR) of <0.01. We also identified a coexpression module demonstrating significant association with the aggressive phenotype of prostate cancer (P = 3.67 x 10–11). The module of interest was characterized by overrepresentation of cell cycle–related genes (FDR = 3.50 x 10–50). From this module, we further defined 20 hub genes that were highly connected to other genes. Interestingly, 5 of the 7 differentially expressed miRNAs have been implicated in cell cycle regulation and 2 (miR-145 and miR-331-3p) are predicted to target 3 of the 20 hub genes. Ectopic expression of these two miRNAs reduced expression of target hub genes and subsequently resulted in cell growth inhibition and apoptosis. These results suggest that cell cycle is likely to be a molecular pathway causing aggressive phenotype of prostate cancer.”The concept of sets of hub genes that are highly interconnected with other genes is an important one characterized in this article. Hub genes were discovered in the nematode and are evolutionary conserved across many species including humans.  “–their normal function is to act as genetic buffers, minimizing the effects of mutations in other genes. — “RNAi (RNA interference) in the nematode is a particularly simple and elegant method to study the effects of genetic variation,” explained Dr Andy Fraser, Principal Investigator at the Wellcome Trust Sanger Institute, “and the worm is ideally suited to high-throughput analysis. If we want to understand how genes work in concert, we have to look at the activities of combinations of genes in living organisms.” — “We found specific roles for many new genes in individual known pathways that are key in human disease but, more important, identified a class of hub genes, which affect many different pathways. We predict that other classes of hub genes will be identified using this method.”

The 2009 study Profiling of 95 microRNAs in pancreatic cancer cell lines and surgical specimens by real-time PCR analysis reports “The expression of miRNAs for pancreatic cancer tissue samples or cancer cell lines was normalized to U6 RNA and compared with those in relatively normal pancreatic tissues or normal human pancreatic ductal epithelial (HPDE) cells. Human pancreatic tissue with chronic pancreatitis also was included for analysis. RESULTS: In the initial analysis, the expression of most 95 miRNAs was substantially changed in pancreatic cancer tissues (n=5) and cell lines (n=3) compared with relatively normal pancreatic tissues and HPDE cells. However, each pancreatic cancer tissue or cell type had a substantially different profiling pattern with other cases or cell types as well as chronic pancreatitis tissue, indicating the individual diversity of pancreatic cancer. Further analysis was performed on 10 pancreatic cancer cell lines and 17 pairs of pancreatic cancer/normal tissues. Eight miRNAs were significantly upregulated in most pancreatic cancer tissues and cell lines, including miR-196a, miR-190, miR-186, miR-221, miR-222, miR-200b, miR-15b, and miR-95. The incidence of upregulation of these eight genes between normal control subjects and tumor cells or tissues ranged from 70-100%. The magnitude of increase of these miRNAs in pancreatic cancer samples ranged from 3- to 2018-fold of normal control subjects.”

The above-quoted studies are just a sampling of the extensive work going on investigating the roles of miRNAs in various types of cancers.  The outcomes of this work are likely to be 1.  a lot of new fundamental insights into cancer processes, 2.  a new generation of miRNA-based diagnostic and prognostic tests related to specific cancers and 3. At some point, new miRNA-based cancer therapies.  I point out that this discovery work is greatly empowered by the availability of new-generation highly efficient analysis technology like that embodied in the Quantimir Small RNA Quantitation System.

MiRNAs and aging

It is fascinating that miRNAs known as lin-4 and lin-14 appear to govern how long tiny Caenorhabditis elegans worms (nematodes) live.  The 2007 publication A developmental timing microRNA and its target regulate lifespan in Caenorhabditis elegans relates “The microRNA lin-4 and its target, the putative transcription factor lin-14, control the timing of larval development in Caenorhabditis elegans. Here, we report that lin-4 and lin-14 also regulate life span in the adult.  Reducing the activity of lin-4 shortened life span and accelerated tissue aging, whereas overexpressing lin-4 or reducing the activity of lin-14 extended life span. Lifespan extension conferred by a reduction in lin-14 was dependent on the DAF-16 and HSF-1 transcription factors, suggesting that the lin-4-lin-14 pair affects life span through the insulin/insulin-like growth factor-1 pathway. This work reveals a role for microRNAs and developmental timing genes in life-span regulation.”  Actually, lin-4 and lin-14 were the first two miRNAs to be intensely studied(ref).  A great history on the discovery of miRNAs going back to 1979 can be found here.

The question of course, is whether there are evolutionary-conserved homologs of lin-4 and lin-14 that control human longevity, a bit of a push since these two genes are involved in larval development of nematodes.  But it is not necessarily an impossible push.   The first item under cancers above points out how “The let-7 family of microRNAs, first discovered in Caenorhabditis elegans, is functionally conserved from worms to humans.”  A great many other miRNAs are similarly evolutionary conserved  and are found in diverse species including worms, flies, zebrafish, mice and humans(ref).  It appears that when evolution finds a biological pathway that works for certain essential life functions in primitive species, when possible it carries this pathways forward with minimum required tinkering as it evolves higher species.  This seems to be a general principle in genomics where many genes are the same in us and in yeast cells. 

An interesting 2007 blog comment quotes research indicating that there seems to be no aging-related effect on gene expression for 256 miRNAs in lung tissues.  “ — we have used a highly-sensitive, semi-quantitative RT-PCR based approach to measure the expression profile of 256 of the 493 currently identified miRNAs in the lungs from 6 month (adult) and 18 month (aged) old female BALB/c mice. We show that, despite the characteristic changes in anatomy and gene expression associated with lung aging, there were no significant changes in the expression of 256 miRNAs. Conclusion: Overall, these results show that miRNA transcription is unchanged during lung aging and suggests that stable expression of miRNAs might instead buffer age related changes in the expression of protein-encoding genes.” 

Some researchers think we can take a lesson from the situation for Caenorhabditis elegans and look for miRNAs that are directly related to aging pathways.  We know that the insulin/insulin-like growth factor pathway, the one affected in the nematode by the lin-4-lin-14 pair, is also very important for longevity in humans.  It is the pathway affected by calorie restriction and by taking resveratrol. 

The 2009 publication Epigenetic Control of MicroRNA Expression and Aging suggests that with aging miRNA expression tends to increase creating increased quieting of target genes, slowly shutting down our protein-making and gene expression.  “Our recent results with several model systems show that upon aging, there is a trend of up-regulation of microRNA expression, with concomitant inverse down-regulation of target genes. This review addresses molecular mechanisms that may provide the underlying control for this up-regulating trend, focusing on activation by various microRNAs’ own promoters, through binding with pivotal transcription factors, stress response, methylation of clustered DNA domains, etc.  Thus, epigenomic control of aging may be due in part to heightened promoter activation of unwanted microRNA expressions, which in turn down-regulate their target gene products. Overriding and dampening the activation of these noncoding RNAs may prove to be a new frontier for future research, to delay aging and extend healthy life-span.”  This suggests another mechanism behind the 13^th theory of aging covered in my treatise, Programmed Epigenomic Changes.  Why, exactly, there is increasing expression of miRNA genes with age is not clear to me, however. 

The 2007 publication MicroRNA, the putative molecular control for mid-life decline  suggests “– possible derailment of these negative regulators for gene expression (miRNAs)in mid-life may be the putative force inducing molecular frailty in individual cell signaling, and in time leading to tissue-wide dysfunction.”  

In my earler post on miRNAs I mentioned a somewhat different take on miRNAs and aging.  “Quoting again from the Gen article, “Eugenia Wang, Ph.D., professor at the University of Louisville, has proposed that miRNAs have a critical role in “a universal or system-specific programmatic shift of signaling control” that occurs at mid-life and brings about a decline in cellular health status associated with aging, which may precipitate increased risk of late-life diseases. In her presentation, she will review the hypothesis that the changes in expression of most if not all aging-related genes are controlled by underlying hubs and the belief that miRNAs, acting as molecular master switches, are candidate hubs.”   This seems more or less consistent with the ideas in the previous paragraph.   

Another line of research applicable to both cancers and longevity has looked at at our old friend, the P53 tumor-suppressor gene and its relationship to miRNAs.  The 2009 publication The p53 Pathway Encounters the MicroRNA World has this to say: “Recently, microRNAs (miRNAs) have been reported to be directly transactivated by p53. Equally, p53 and components of its pathway have been shown to be targeted by miRNA thereby affecting p53 activities. In this review, we focus our discussion on the biological and pathological roles of miRNAs in the p53 pathway.”  P53 is of course a key apoptosis gene and when it is incorrectly turned off cancers can result.  When it comes to molecular biology of cells, it seems to me that often everything has to do with everything else. 

In summary, while many researchers think miRNAs are implicated in aging, there are several quite different hypotheses about what they might be doing.  As far as I can tell we don’t yet have a “smoking gun” for the  aging control in humans like lin-14-Lin4 miRNAs are for nematodes. 

The 2009 publication MicroRNAs in C. elegans Aging: Molecular Insurance for Robustness? suggests that there may be a lot more for us to learn about aging by studying the impacts of miRNAs on lifespans in those little critters. “It has been proposed that miRNAs ensure biological robustness, and aging has been described as a progressive loss of system and cellular robustness, but relatively little work to date has addressed roles of miRNAs in longevity and healthspan (the period of youthful vigor and disease resistance that precedes debilitating decline in basic functions). The C. elegans model is highly suitable for testing hypotheses regarding miRNA impact on aging biology: the lifespan of the animal is approximately three weeks, there exist a wealth of genetic mutations that alter lifespan through characterized pathways, biomarkers that report strong healthspan have been defined, and many miRNA genes have been identified, expression-profiled, and knocked out. 50/114 C. elegans miRNAs change in abundance during adult life, suggesting significant potential to modulate healthspan and lifespan. Indeed, miRNA lin-4 has been elegantly shown to influence lifespan and healthspan via its lin-14 mRNA target and the insulin signaling pathway. 27 of the C. elegans age-regulated miRNAs have sequence similarity with both fly and human miRNAs. We review current understanding of a field poised to reveal major insights into potentially conserved miRNA-regulated networks that modulate aging.”  

Our understanding of how miRNAs work in humans is just getting off the ground.  As the last publication suggests back to the nematode may be a good strategy for now.  Let us fully exploit the fact that evolution carries biological machinery forward from species to species, including possibly machinery that controls aging.  If we can thoroughly investigate various combinations of miRNAs already suspected or known to keep C. elegans young,  and find homologs in humans of the same miRNAs, they may well keep us young too. We already know that the normal lifespans of nematodes of 20 days can be extended to 125 days by genetic manipulation(ref).  If that life extension factor of more than six could be translated to humans, it would mean normal lifespans of around 480 years.

The idea of people living hundreds of years has about as much credibility today as the idea of the world not being at the center of the universe had in 1540.  Intellectually and in terms of our laws, institutions and actions, we are just not ready for radical life extension.  I will illustrate this point with a short piece of fiction.

The story of the X pill

A team of distinguished university researchers uses molecular engineering to create a substance X that appears to activate an evolutionary genetic pathway affecting the expression of hundreds of aging-related genes.  When tested, the substance doubles the life of laboratory mice and rats.  Based on solid theoretical considerations, it appears that X could be the basis for making an anti-aging pill for humans.  Extrapolating up from the mice and rats, it appears in theory that people who start taking the X pill on a daily basis in their 40s will double their average life spans.   They will, the reasoning goes, continue to age but at a much slower rate.  They will still get the diseases and problems of old age but, on the average, much later.  At age 110 they should be about as healthy as people are today at 55.

So, the researchers raised venture capital to start up a biotech company to manufacture X pills and sell them to the public.  What happened?

1.     Everybody including the researchers recognized that the anti-aging effectiveness of the X pill can’t be established for a long-long time.  There is no guarantee that X will work in humans as it does in mice and rats.  Taking the X pills seems to result in healthier biomarkers and possibly longer telomeres, but this can’t be confirmed until there are 10 years or more of history.   And, those factors by themselves do not guarantee longer lives.  The new X biotech company is trying the pills on large monkeys, but it will also take 30-40 years to establish that the pills double the average monkey life spans.  In 15-20 years it might be noticed that people taking the X pills continue to look younger or have less deadly diseases.  But it will take 150 – 200 years to unambiguously establish that most people taking the X pills live twice as long.  And this can only happen if data is gathered on the pill-taking cohort for all that time.

2.     For the same reason, for a long time it will be impossible to know for sure that taking the pills is safe.  How could anybody be sure that some deadly side effect won’t kick in after 5-10 years?

3.     The realization soon dawned that the X pills cannot be developed, tested, approved, and sold as a drug.  Drugs are approved by the FDA for disease indications and aging is not a disease.  The X substance will not cure diseases.  Instead, its action is to prevent many diseases from occurring in the first place, and there is no drug category for that.  Once this was realized, it became very difficult for the X biotech company to raise additional development capital.

4.     The managers of the X biotech company came to realize a horrible truth: because X is a new and strange synthetic chemical, it is possible that the X pills cannot be legally sold for human consumption in the US and most other advanced countries.  Fortunately, a graduate school assistant working for X discovered in the literature that members of a rare species of deep-sea urchin have tiny trace amounts of X in them.  This fact allows selling X pills as a “natural substance” dietary supplement.  Whew!  That was a close one!

5.     A clinical trial of the X pill is out of the question.  If there were such a trial, it would have to last 30 years just to get early indications.  Nobody is prepared to pay the hundreds of millions of dollars required for such a trial, particularly the new X biotech company which is running on a shoestring.

6.     For the first two reasons listed above, the majority of doctors recommend their patients not to take X pills.  They rightfully say that substance X is unproven, unapproved by the FDA and potentially dangerous.  They think they are following the basic medical precept “First, do no harm.”  For patients who have had an episode of cancer or heart disease, the warnings from the doctors are particularly stern and ominous.

7.     Some prominent religious figures start a crusade against taking the X pills.  Their point is that “extending human life would go against God’s natural order.”  This is a mixed blessing for the X biotech company.  On the one hand it gives them press exposure.  On the other hand it leads many pious followers to eschew the X pill

8.     Big pharmaceutical companies mount a well-funded PR campaign warning people against taking the X pill.  They do not want to see large numbers of people taking x pills for good business reasons.  They are not afraid that it won’t work; they are afraid that it will work.  The ideal pharmaceutical drug from a commercial viewpoint is not a cure for a serious disease, but is a drug that must be taken continuously to prevent or blunt the symptoms or ravages of such a disease, year after year.  That is a prerequisite for a blockbuster drug that rakes in $10 billion or more a year.  An X pill that results in many fewer cases of disease happening could be commercially disastrous to many big pharma companies.  So, they subsidize doctors to write articles and go on TV opposing the X pills.

9.     The big hospital centers that mass-process patients with life-threatening cardiovascular and cancer problems also see their businesses threatened, including cardio surgeons, chemotherapists, radiation therapists, hospital administrators and insurance company executives, people who depend on a steady stream of diseased patients in order to earn from a half-million to more than a million dollars a year. They join the big pharma companies in the anti X pill chorus.

10.                        The big research-sponsoring establishments like the National Cancer Institute and those who feed in their troughs also have a vested interest in fighting the availability of such a pill.  The War on Cancer going back to 1971 has produced marginal results for the hundreds of billions of dollars spent on research.  In 2008 the NCI spent $4.83 billion on 5,380 research grants.  Until genomics came along, the required basic science platform for discovering what really goes on in cancers was simply not there.  Although cancer deaths are down, much of the progress has been due to public health measures like decline in cigarette smoking.  Yet, the cancer research establishment is not only going on but is growing.  If an X anti-aging pill came along that postpones when most cancers happen by 70 years, imagine what an embarrassment that would be for everyone in the cancer research establishment.  Thousands of researchers with their own pet theories about cancer and more thousands working in the cardiovascular, diabetes and neurological disease areas start throwing Olympic-sized swimming pools full of ice water on the X pill idea.

11.                        As sales of the X-pill increase, the anti X-pill factions decide they have to do something to stop it.  They join together in a successful lobbying effort to “regulate” the X pill which means halting its sale and handing rights to future anti-aging drugs to big pharma.  People who want to continue taking X pills will have to smuggle them in from Bulgaria.  And then they will for-sure not know what they are getting.

This little story brings out several important points about the current reality.  Several dietary supplements in the anti-aging firewalls Supplement Regimen are similar to the X-pill in that they theoretically produce healthful and life-extending results, they can demonstrably extend the lives of small animals by 20% to 40%, but what they will do for humans is not yet very clear.  Telomerase-activators like cycloastragenol or TA-65 have a significant life-extending potential but what they actually do for humans remains under a cloud of uncertainty.  Companies like TA Sciences and Sirtris Pharmaceuticals are facing many of the same problems faced by the fictional X pill company.

There is a major need to prepare the society for the possibility of radical life extension:

1.     There is a clear need for educating medical professionals as to progress in developing anti-aging strategies and therapies, and for getting them used to the possibility of radical life extension.  The same is true for all concerned parties: lawmakers, institutional administrators, and the public at large.

2.     There is a need for a massive shift in government health research funding, away from simply finding chemical and surgical cures for diseases, towards promotion of longevity and disease prevention via public health measures and epigenomic and genomic enhancements of individuals.  Public money spent over the years on longevity research is a tiny drop in the bucket compared to money spent of cancer and other disease research, despite the incredible leverage on incidences of the same diseases increased longevity would provide. And significant sums of money need to be set aside for long clinical trials and population studies to help evaluate the effectiveness of emerging anti-aging therapies. 

3.     Above all, there is a need for a major shift in general perspective regarding life extension FROM more and more doddering, sick, non-functional, non-contributing individuals drawing social security, filling nursing homes, having automobile accidents and driving health care costs ever-higher, TO more and more healthy, creative, fully-functional working individuals in their 70s, 80s, 90s and beyond who are not getting the diseases of old age, and who are more than doing their part to contributing to our society in every way.

The press has been making a big deal of research made public a few days ago that correlates a genetic defect in one of the key telomerase-producing genes TERC with shorter telomeres later in life.  This link leads to 23 news stories on the research.  The abstract of the study itself Common variants near TERC are associated with mean telomere length reads succinctly:  “We conducted genome-wide association analyses of mean leukocyte telomere length in 2,917 individuals, with follow-up replication in 9,492 individuals. We identified an association with telomere length on 3q26 (rs12696304, combined P = 3.72 x 10(-14)) at a locus that includes TERC, which encodes the telomerase RNA component. Each copy of the minor allele of rs12696304 was associated with an approximately 75-base-pair reduction in mean telomere length, equivalent to approximately 3.6 years of age-related telomere-length attrition.“  This site graphically shows key data produced by the study. 

 The study says that people who possessed the gene variation (minor allele of rs12696304) had shorter telomere lengths, equivalent to 3.6 years of aging.  People who had two copies of the variation had telomere lengths expected for people 7.2 years older.  The implication is that people with the gene defect age faster.  I think the study is a good piece of research to add to the aging puzzle but that it is not the big-deal breakthrough suggested in the news headlines because:

–         The result is what would be expected all along; a defect in a telomerase-making gene results in the production of less telomerase resulting in telomere lengths being less than expected.  Some of the headlines make declarations like Scientists find genetic link to ageing  which are rather misleading.  The link of telomerase genes to aging has been known for decades now. “The existence of a compensatory shortening of telomere (telomerase) mechanism was first predicted by Soviet biologist Alexey Olovnikov in 1973^[1], who also suggested the Telomere hypothesis of ageing and the Telomere relations to cancer. Telomerase was discovered by Carol W. Greider and Elizabeth Blackburn in 1985 –(ref).”  We knew back in 1998 that there was Severe growth defect in mouse cells lacking the telomerase RNA component.

–         Several other gene variations also lead to telomere dysfunction or shortening and/or accelerated aging.  See, for example, the blog posts Werner Syndrome – another model for aging, Progerin, HGPS and a possible new theory of aging and Hoyeraal-Hreidarsson Syndrome and telomere dysfunction.Gene variations that lengthen telomeres or extend lifespans tend to grab me more.  For example, a report on how adding extra copies of a telomerase gene and a P53 gene can extend the life of mice by 26% to 40%(ref).

The idea raised by this study of people aging at variable rates depending on their genes is an interesting and probably valid one though definitely not new. Children born with a mutation in the LMNA gene look old and wizened and are experiencing the ravaging diseases of old age when they are only 14 or 15.  See the blog entry Progerin, HGPS and a possible new theory of aging.  Personally, I believe lifestyle and behavioral factors affect the rate of aging along with the genetic ones.  That is the point of the lifestyle and dietary supplements regimens suggested in my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY.

When my guest-bathroom toilet flap valve recently gave out due to old age, I purchased and tried out four different “one size fits all” replacement flap valves, shopping at Home Depot and different hardware stores.  They are very simple devices but none quite fit and all left the toilet leaking.  The process required four different shopping trips and left me with a non-functioning toilet for 10 days.  Then I did the smart thing and found out the brand of my toilet (an Elger) and went to a plumbing supply house and purchased an Elger toilet retrofit kit.  It worked perfectly.  The solution was personalized to the particular design and dimensional specifications of my toilet.

Physicians are trained to diagnose what is going on in an individual and to prescribe a treatment tailored to that individual and his disease condition.  Modern diagnostic tests and instruments such as MRIs have been making this process ever-more precise.  However, until now it has not been possible to make treatment choices based on the molecular, genetic and epigenetic makeup of a given patient.  We are all genomically and epigenomically vastly more different from one another than an Elger toilet is from other toilets.  So, as far as therapies go, medicine has too-often proceeded on the basis of “one size fits all” when the facts have often shown “one size misfits most.”  Finding the right prescription medicine can be like finding the right toilet flap valve, a matter of trial and error and high cost and inconvenience.

According to an article Personalized Medicine Realizing Its Promise by Edward Abrahams, Ph.D. “On average, a drug on the market works for only 50% of the people who take it. The consequences in terms of quality and cost of care are significant, leaving patients to contend with their disease and their medical bills as they switch from one drug to another until they find an effective therapy.”  Trial-and-error of drugs is particularly prevalent in some fields like psychiatry.  A psychiatrist is often unable to make a definitive diagnosis at first between the various shades of depression, bipolar and related mental disorders.  Instead the psychiatrist will keep prescribing different drugs until one or more work to control the symptoms.  Then, based on the drug that works, a diagnosis can be pronounced. 

Now, in the emerging context of personalized medicine, customization of treatment can depend not only on observable diseases conditions but also on the molecular, genomic and epigenomic makeup of the particular patient.  There is increasing evidence, for example, that combinations of genetic and epigenetic markers can be useful in diagnosing mental disease conditions. Genomic, epigenomic and other diagnostic tests are coming on the market which will tell whether a drug treatment or medical procedure is likely to work or not.   For people on an expensive year-long drug therapy, such a test could save their life by indicating that a blockbuster drug is probably not going to work and by preventing exposure to its side effects.  For the government and society, such tests could in aggregate mean hundreds of billions of dollars saved in irrelevant treatments and unnecessary hospital stays.

A special report in Gen provides some examples:

“For example, Genentech/Roche’s Avastin costs $50–$100,000 per year of treatment but works in fewer than 50% of patients. Given that Avastin may generate $12 billion in peak sales, this low rate of efficacy translates into billions of dollars in misdirected healthcare spending. A test for Avastin response, such as that in discovery by BG Medicine, could save the system as much as $6 billion per year if all nonresponders could be removed from the treatment pool. Assuming that a test of this sort is introduced at the beginning of 2013 and is 100% adopted, cumulative savings of $40 billion could be realized by 2019.”  That is not small change. 

“Genomic Health’s Oncotype Dx is a test with compelling cost-saving potential. It is used to predict chemotherapy benefit for patients who have node-negative, estrogen receptor positive (node-, ER+) breast cancer. By averting unnecessary chemotherapy, the test has been shown to save about $2,000 per patient. Extending this cost savings to the roughly 100,000 new cases of node-, ER+ breast cancer in the U.S. each year, this test could save the U.S. healthcare system up to approximately $200 million a year or about $2 billion over the 10-year time horizon under legislative consideration.” Adding up the savings for drug after drug after drug could chop an enormous slice over our annual health care costs.

With the development and adoption of appropriate diagnostic tests, the aggregate effect on health care costs and patient wellbeing could be enormous.  One approach is the development and use of specific tests associated with specific drugs or treatment procedures.  For example.  before prescribing Avastin, the oncologist could order the test being developed by BG Medicine. A better approach that will no doubt be implemented in the longer run involves building databases of patient-specific data, such as their entire genome.  In a recent blog entry, I pointed out that the cost of sequencing the entire genome of a patient is heading down to the $1,000 level probably this year and will be probably at the $100 level within four years or less. If a patient has his or her genome already laid out in such a database, many bad-choice drug treatments could be avoided by a simple computer check against the database, just like transfusions of the wrong types of blood are often avoided now.

Also, the existence of such a database could signal disease susceptibilities and the advisability of preventative actions.  “Over 1,300 genetic tests exist that signal inherited susceptibility to conditions as wide-ranging as hearing loss and sudden cardiac arrest. While not every test has a therapeutic option, a genetic diagnosis often permits targeted prevention or mitigation strategies(ref).”  One example is looking for BRCA1 and BRCA2 genetic mutations indicating a hereditary propensity for breast and ovarian cancer. “Women with BRCA1 or BRCA2 genetic risk factors have a 36% to 85% lifetime chance of developing breast cancer, compared with a 13% chance among the general female population.  For ovarian cancer, women with certain BRCA1 or BRCA2 gene mutations have a 16% to 60% chance of disease, compared with a 1.7% chance among the general population. The BRCA1 and BRCA2 genetic test can guide preventive measures such as increased frequency of mammography, prophylactic surgery, and chemoprevention(ref).

The bottom line for the government is to effectively control health care costs, do everything you can to further implementation of personalized medicine and the building of patient-specific genomic and epigenomic databases..

When I wrote my first blog entries on epigenomics and epigenetics eleven months ago(ref)(ref) , it was clear that these were active areas of extremely interesting academic research.  However, my impression was that it would be years before the knowledge being accumulated could be put to practical work in medicine.  I was wrong.  Not only has the depth and amount of research in these areas increased significantly but epigenetics has entered the commercial medical marketplace in the forms of new therapeutic drugs and diagnostic tools.  This blog entry provides an update.

Background

An introduction to epigenetics and epigenomics and their relevance to aging is in the Feb 2009 blog entry Epigenetics, Epigenomics and Aging.  And, of course, the 13th theory of aging in my treatise is Programmed Epigenomic Changes.  “Epigenetics is the study of changes in phenotype and gene expression arising from mechanisms other than changes in a gene’s DNA sequence.– Over 25 years ago, Surani et al. showed that certain regions of a cell’s genome carry markers over and above the actual gene sequence. This imprint conveys information on differential gene expression, and therefore, shapes the fate of the cell.  Epigenetic information is passed from one cell to another, but the epigenetic code can change through life by interacting with environmental factors. Moreover, unlike gene-sequence mutations, epigenetic changes may be reversible(ref).”

Since cells of all types in an individual have the same DNA, epigenetic information is what gives a cell memory as to the type of cell it is, and many events in the lifetime of a cell are also encoded epigenetically.  And some epigenetic information can be inherited.  The main mechanisms of encoding epigenetic information are DNA methylation and chromatin modifications, such as histone acetylation.  See the blog posts DNA methylation, personalized medicine and longevity and Histone acetylase and deacetylase inhibitors. 

“DNA methylation involves the addition of a methyl group to the 5 position of cytosine (one of the four bases of DNA), which occurs in the context of CpG (cytosine followed by guanine) dinucleotides. This modification can be inherited through cell division. DNA methylation is typically removed during zygote formation and reestablished through successive cell divisions during development. DNA methylation is a crucial part of normal organismal development and cellular differentiation in higher organisms. DNA methylation stably alters the gene expression pattern in cells such that cells can “remember where they have been”; in other words, cells programmed to be pancreatic islets during embryonic development remain pancreatic islets throughout the life of the organism without continuing signals telling them that they need to remain islets. In addition, DNA methylation suppresses the expression of viral genes and other deleterious elements which have been incorporated into the genome of the host over time. DNA methylation also forms the basis of chromatin structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA. DNA methylation also plays a crucial role in the development of nearly all types of cancer(ref).^[1]”

Of particular interest from the viewpoint of DNA methylation in mammals are the so-called CpG islands, gene promoter sites that are not methylated when a gene is being expressed.  “CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates.^[2]  — “Unlike CpG sites in the coding region of a gene, in most instances, the CpG sites in the CpG islands of promoters are unmethylated if genes are expressed(ref).”

Testing for DNA methylation

A standard approach has been developed for checking out the DNA methylation patterns of humans known as bisulfite sequencing.  “Bisulfite sequencing is the use of bisulfite treatment of DNA to determine its pattern of methylation. — Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single- nucleotide resolution information about the methylation status of a segment of DNA.”  Once a DNA sample has been pre-treated with bisulfite, it can be sequenced just like any other DNA.  Software comparison(ref) of a sequenced output against that for a normal human genome will reveal the GpC promoter sites that are methylated.  Thus, orders-of-magnitude increases in power of gene sequencing such as discussed in the blog post My personal longevity – the race between death-stalker and life-prolonger equally benefit testing for DNA methylation. 

Some sequencing platforms have been used extensively for bisulfite sequencing for methylation patterns, for example the Illumina GoldenGate bead array platform. “Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases(ref).”

The June 2009 report CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics provides a snapshot of the technology at that time, a picture that is rapidly changing with the arrival of new ever-more powerful sequencing platforms.

Several methylation studies have been concerned with basic science.  An example is described in a report dated February 3, 2010 International Team Maps Methylation Changes During Cellular Differentiation.  “Using bisulfite sequencing with the Illumina Genome Analyzer, researchers from Singapore and the US mapped and compared DNA methylation patterns in human cells during three progressive stages of differentiation: embryonic stem cells, skin-like cells derived from embryonic stem cells, and primary neonatal skin cells. In the process, the team identified shared and cell type-specific methylation patterns, providing insights into how gene regulation shifts during development.  — With these comprehensive DNA methylome maps, scientists now have a blueprint of key epigenetic signatures associated with differentiation,” co-corresponding author Chia-Lin Wei, a researcher affiliated with the Genome Institute of Singapore and the National University of Singapore, said in a statement.”

DNA methylation and diseases

“DNA methylation is widespread and plays a critical role in the regulation of gene expression in development, differentiation, and diseases such as multiple sclerosis, diabetes, schizophrenia, aging, and cancers (Li et al. 1993; Laird and Jaenisch 1996; Egger et al. 2004). Methylation in particular gene regions, for example in promoters, can inhibit gene expression (Jones and Laird 1999; Baylin and Herman 2000; Jones and Baylin 2002). Recent work has shown that the gene silencing effect of methylated regions is accomplished through the interaction of methylcytosine binding proteins with other structural components of chromatin (Razin 1998), which, in turn, makes the DNA inaccessible to transcription factors through histone deacetylation and chromatin structure changes (Bestor 1998).  Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as the most frequent mechanism for gene inactivation in cancers (Esteller 2002; Herman and Baylin 2003 Changes in DNA methylation are recognized as one of the most common forms of molecular alteration in human neoplasia (Baylin and Herman 2000; Balmain et al. 2003; Feinberg and Tycko 2004))(ref).”
 

Many studies have been performed using high-throughput bisulfate sequencing to identify methylation patterns of disease conditions, so many that I can cite only a few examples here.

A December 22 2009 report Twin Epigenetics Study IDs Methylation Differences in Lupus.  “– Epigenetic changes, specifically differences in DNA methylation, may contribute to environmental factors involved in systemic lupus erythematosus risk, according to an online study in Genome Research today. — A Spanish, German, and American research team used bead arrays and targeted bisulfite sequencing to compare DNA methylation patterns in the genomes of more than a dozen sets of identical twins who were discordant for SLE or two other autoimmune diseases. While they did not detect methylation differences for two of the conditions, the team did detect intriguing epigenetic changes when one twin had SLE and the other did not. — “Our study suggests that the effect of the environment or differences in lifestyle may leave a molecular mark in key genes for immune function that contributes to the differential onset of the disease in twins,” Esteban Ballestar, a researcher at the Bellvitge Biomedical Research Institute in Barcelona, said in a statement.”

The September 2009 study Epigenetic silencing of death receptor 4 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in gliomas had the purpose “To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy.”  The conclusion is “DR4 promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies.”

Other recent research reports on the epigenetics of disease processes include The ADAMTS12 metalloprotease gene is epigenetically silenced in tumor cells and transcriptionally activated in the stroma during progression of colon cancer, New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling, Epigenetic profiling reveals etiologically distinct patterns of DNA methylation in head and neck squamous cell carcinoma, Aberrant DNA methylation is a dominant mechanism in MDS progression to AML, Large-Scale Profiling of Archival Lymph Nodes Reveals Pervasive Remodeling of the Follicular Lymphoma Methylome, Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation, and Epigenetic Profiles Distinguish Pleural Mesothelioma from Normal Pleura and Predict Lung Asbestos Burden and Clinical Outcome. And this is just a starter list.

Epigenetic drugs

A Feb 1 2010 report in Gen Epigenetics Offers Strategies for New Drugs states “According to a recent report from Business Insights, “Innovations in Epigenetics: Advances in Technologies, Diagnostics & Therapeutics,” epigenetic medicine is already here. — The company puts the epigentic market at over $560 million, derived from the sale of three anticancer products (Dacogen™ from Eisai, Vidaza® from Celgene, and Zolinza® from Merck), which target two epigenetic pathways—DNA methyltransferase (DNMT) and histone deacteylase (HDAC). — Approximately 30 epigenetic drugs are under development by more than a dozen biotechnology companies, according to the Business Insights study. These drugs focus mainly on the treatment of cancer, and neurodegenerative and infectious.”  The $560 million market mentioned above is only for drug sales.  It does not include epigenetics R&D expenditures and the market for epigenetic disease tests.  Whatever the market size is now, I estimate it will double in size every 12 to 18 months being driven by the factors that drive Giuliano’s Law until it gets up into the hundreds of billions of dollars in less than 15years.  Epigenetics and epigenomics are destined to be very big biz.

Epigenetic diagnostic tools

Finally, as DNA methylation patterns are discovered and confirmed for more and more cancers and other diseases, and as the costs of bisulfite epigenomic sequencing continue to drop, it is likely that we will see many methylation diagnostic and predictive tests being integrated into mainline medical practice.  There is evidence, for example, that cancers develop in stages and that methylation tests can provide predictors of premalignant conditions long before the cancers themselves emerge. “One of the advantages of using epigenomic biomarkers is that, in most cases, DNA methylation changes precede clinical symptoms. “If there is a small abnormality that is not yet an invasive cancer, but a precancerous lesion, or a small tumor, many genes will have abnormal methylation, and that is probably true in many tissues,” says Dr. Baylin(ref) .

A number of small biotech companies are moving into this diagnostic area.  As reported in Gen the first of this month “Epigenomics specializes in DNA-methylation technologies and biomarkers and is developing a number of cancer diagnostics based on differences in DNA methylation between healthy and diseased tissue. Constellation Pharmaceuticals is focused on developing therapeutics based on epigenetics. It is currently establishing a preclinical pipeline and developing a technology platform for histone modification. Initial applications will be in oncology. Finally, Epizyme is looking at histone methyltransferases and is developing a pipeline of inhibitors for cancer.”

Like the computer industry was in 1959, the genetic, genomic, epigenetic and epigenomic areas are multifaceted and messy.  They involve far-out ideas, scientific research, hype, university gurus, technology industry developments, entrepreneurial ventures, commercial activities in big companies and sizeable investments. Some approaches and companies will succeed, others will fail.  Some will succeed for a while and then fail.  That is what progress looks like.  Epigenetics, genomics and sequencing are built on the back of the computer and communications revolution.  Without extremely powerful and cheap computers, genetic or epigenetic sequencing would be impossible.  This time around, one net result will be that people will live longer.

Can big multinational corporations buy and own rights to what I do with my nose, my liver, my heart, my little right toe? Possibly, because they can buy or license patent rights for many of my genes.  That is the way it has been for a number of years now.   I have always thought that having someone else hold rights to my natural body parts is completely absurd.   I should be able to do anything with my genes that I want.  But what do I know about such matters?  I am not a lawyer.

Finally, patentability of genes is being challenged in the courts.  According to a Feb 2 news highlight in Gen “Today, the American Civil Liberties Union (ACLU) and the Public Patent Foundation (PUBPAT) will deliver oral arguments asking the court to rule that patents on two human genes associated with breast and ovarian cancer are unconstitutional and invalid. The groups charge that the patents stifle diagnostic testing and research that could lead to cures and that they limit women’s options regarding their medical care. — The lawsuit Association for Molecular Pathology et al. v. U.S. Patent and Trademark Office et al. was originally filed on May 12, 2009, in the U.S. District Court for the Southern District of New York on behalf of breast cancer and women’s health groups, individual women, and scientific associations representing approximately 150,000 researchers, pathologists, and laboratory professionals. — The lawsuit was filed against the PTO as well as Myriad Genetics and the University of Utah Research Foundation, which hold the patents on the genes BRCA1 and BRCA2. The lawsuit charges that patents on human genes violate the First Amendment and patent law because genes are “products of nature” and therefore can’t be patented.”

“The plaintiffs include Breast Cancer Action, The American College of Medical Genetics, the Association for Molecular Pathology, the College of American Pathologists, the American Society for Clinical Pathology, individual researchers, patient advocacy groups, genetic counselors, and individual women(ref).”

So, how did genes get to be patentable in the first case, and what are the main issues involved?  For genes to be patentable, they have to be viewed as inventions.  And they have been so-viewed because attorneys have successfully argued that it has taken inventions to identify them.  Basically, “the US Patent and Trademark Office (USPTO) and the European Patent Office (EPO) have treated isolated and purified nucleotide sequences as if they were the same as man-made chemicals(ref).”  The decision to allow genes to be patented has engendered much controversy and endless opinion papers.  See this list for some of them. 

The economic argument for gene patentability is of course that it provides economic incentive for discovery and invention.  The biotech and pharma companies and universities that hold the gene patents will want to hold onto them.  Increasingly, however, voices are questioning the wisdom of gene patentability and whether it gets in the way of scientific progress, public health and patient care. 

A recent position paper starts out: “Concerns about human gene patents go beyond moral disquiet about creating a commodity from a part of the human body and also beyond legal questions about whether genes are unpatentable products of nature. New concerns are being raised about harm to public health and to research. In response to these concerns, various policy options, such as litigation, legislation, patent pools and compulsory licensing, are being explored to ensure that gene patents do not impede the practice of medicine and scientific progress.  Although gene patents have been granted worldwide for several years, the wisdom of this action is now being questioned.  Lawsuits, proposed legislation, international protests and even patent-office proposals have recently been initiated to eliminate, undermine or otherwise challenge the scope of patents on human genes. The challenges come from various interested parties — people from whom patented genes have been isolated, researchers who wish to undertake genetic epidemiological studies or to develop gene therapies, clinicians and health-care providers who cannot afford expensive licensing fees for genetic tests and policymakers who want to ensure that the patent system actually meets its goal by encouraging invention. Evidence is mounting that gene patents are inhibiting important biomedical research, interfering with patient care and provoking criticisms from international trading partners.” 

My guess is that the case may take 5-10 years to work its way through the courts and may well end up in front of the Supreme Court.  And who knows how they will choose to look at the situation.  In terms of property rights or interstate commerce?  Meanwhile we will have to live with gene patentability. I realize that in working out on the treadmill or taking supplements that either activate or inhibit certain of my genes, I am probably violating somebody or the other’s patent.  So are you.  Bah!

The track record of clinical trials involving human embryonic stem cells (hESCs) is worse than miserable.  A dozen years ago, it was thought that for sure by now in 2010 hESCs would be used in all kinds of regenerative medicinal applications.  Instead, they remain stalled at the gate while other types of stem cells are being used to produce all kinds of intereting results.  In the blog post It’s a long way to stem cell treatment I discussed how the clinical trial of Geron’s proprietary hESC-based product GRNOPC1 was being delayed for the second time in August 2009 because of FDA caution(ref).  That trial, still being delayed, involves the use of hESCs for treating severe spinal cord injuries.

It is very tempting to blame lack of progress in development of hESC therapies on government banning of funding of most embryonic stem cell research during the Bush era.  I personally think the ban was a terrible idea, anti-science and anti-humanistic.  While the ban has been a factor, however, I do not think it had a major impact on worldwide progress in hESC research.  And, simply put, other kinds of stem cells have turned out to be more exciting and more easy to work with.  The stubborn challenges associated with hESC therapies appear to be 1.  possible immune system rejection or reactions because the cells are not derived from the patient (the cells are not autologous), and 2.  assuring that the stem cells differentiate into the desired target cell types and only those types.  More-specialized patient-derived stem cells like haemopoietic and mesenchymal stem cells do not run the risk of immune system rejection and their differentiation can be directed more easily.  And, autologous induced pluripotent stem cells (iPSCs) seem to be able to do everything that hESCs can do without immune system rejection.

There was news last week that GRNOPC1 is emerging again in a new clinical context, this time for treating Alzheimer’s disease.  The report Geron to Study Its hESC Product in Alzheimer Disease with University of California in Gen states “Geron and researchers from the University of California have decided to work together to assess the company’s human embryonic stem cell (hESC) product, GRNOPC1, for Alzheimer disease. The work will be jointly funded by the firm and a university discovery research and training grant. — Geron and the University of California team will now evaluate GRNOPC1 in models of Alzheimer disease. The study is designed to assess whether memory shows recovery after transplantation of GRNOPC1.  — The research will be led by Frank M. LaFerla, Ph.D., director of the Institute for Brain Aging and Dementia at the University of California, Irvine.”

The interest follows in large part from promising mouse studies.  “There are striking parallels between recent data on mouse stem cells in Alzheimer’s disease models and what we know about GRNOPC1(ref).” Dr. LaFerla and his colleagues published a research report in August 2009 entitled Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease.  That report cited data demonstrating “that defects in memory were improved by glial cells derived from mouse neural stem cells transplanted into the hippocampus of rodent models of Alzheimer disease. — GRNOPC1 contains the precursors to human glial cells, which matured and repaired the lesion site in rodent models of spinal cord injury. Additionally, the improvement in memory and the increase in synaptic density observed after injection of neural stem cells were found to be mediated, at least in part, by the neurotrophic factor BDNF, which is secreted from the transplanted cells. GRNOPC1 has been found to secrete BDNF as well as other neurotrophic factors(ref).”  “Taken together, our findings demonstrate that neural stem cells can ameliorate complex behavioral deficits associated with widespread Alzheimer disease pathology via BDNF(ref).”

If and when GRNOPC1 is to be used in a clinical trial for Alzheimer’s disease is yet to be determined as, apparently, whether and when the clinical trial of GRNOPC1 for severe spinal cord injury will be resumed.  The great original glow of hESC-based therapies is now dim and continues to fade while the prospects for other stem cell therapies seem to grow brighter and brighter.