Aptamers and Clinical Applications

  • Gov Lists 28 Clinical Studies, Mostly Ocular, for Aptamers
  • Only 3 On This List Are Currently Active Studies
  • NOXXON Pharma’s Mirror-Image L-RNA Aptamer is in the Clinic for Cancers

Devoted readers of Zone In With Zon who have photographic memories—or anyone who simply uses this blog’s search engine—will know that my first blog on aptamers touted these nucleic acids as being better than antibodies in many applications. That deliberately provocative proclamation was followed by a second blog on aptamers featuring top-cited publications, and also included aptamer studies which cited TriLink products in the methods section. The present, third blog on aptamers focuses entirely on clinical applications.

In the interest of full disclosure, picking this blog’s titled topic was catalyzed, so to speak, by my being invited to contribute a chapter devoted to clinical aspects of aptamers for a book on nucleic acids therapeutics to be published in 2019. After this book becomes available, I’ll be able to comment on its well-known co-editors, many contributors, and comprehensive contents. However, for now, here are “sneak previews” of some items that will be covered in my chapter.

Overview of Aptamer Functional Versatility

Taken from genelink.com

Aptamers are highly structured nucleic acids that bind to a specific target molecule. RNA or DNA aptamers are usually selected from a very large pool (aka library) of random sequences, and can be comprised of either natural and/or chemically modified nucleotides. Clinical applications of aptamers, with or without chemical modifications, are all predicated on target-specific binding.

However, as depicted below for diverse examples, aptamers can function as therapeutic agents per se, or as targeting agents. Direct agency of an aptamer drug can include binding to either extracellular protein, cell-surface protein, or viral surface protein, wherein each target is associated with a specific disease or clinical indication of interest for achieving therapeutic intervention. These three targets are also addressable by conventional small molecules or antibodies; however, it was recognized early on that aptamers might provide advantages in specificity or cost, respectively.

Cartoon depicting various modes for RNA aptamers (shown as stem-loop structures) functioning as therapeutic agents (left panel) or as cell-targeting agents (right panel). Figure from Poolsup & Kim with permission.

Use of aptamers as targeting agents is depicted as conjugates for delivery of either a nanoparticle loaded with drug, therapeutic oligonucleotide, such as short-interfering RNA (siRNA), or small molecule drug, such as a cytotoxic agent. Although the present blog is devoted to the first modality, namely, aptamers as therapeutic (i.e. clinical) agents, a review by Poolsup & Kim can be consulted for information on the second mode, namely, aptamers as targeting agents.

Aptamer Drugs Listed in ClinicalTrials.Gov

ClinicalTrials.gov is my “go to” website for obtaining reliable information on clinical investigations in general. This freely accessible website is a comprehensive registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. This trove of information, which is provided as a service of the U.S. National Institutes of Health, is currently comprised of 261,814 clinical research studies in all 50 states and in 201 countries.

Basic information for each study listed in ClinicalTrials.gov includes the sponsor, participating investigators and hospitals, aptamer drug, targeted disease(s), and type of study (aka Phase), i.e. safety (Phase 1), efficacy (Phase 2), and better than standard of care (Phase 3). Geographical mapping on global, country, or state/regional bases for participating hospitals is also provided.

Taken from aidsinfo.nih.gov

The ClinicalTrials.gov website is relatively easy to navigate, and has built-in help “buttons” as well as dropdown menus as aids to find and filter the extensive amount of available information. My search results from ClinicalTrials.gov several months ago using the term “aptamer” provided information for 32 different clinical studies, which when sorted by medical conditions gave 87 items covering a wide range of diseases, with ocular being the most prevalent general category. However, my reading of procedural details for all 32 studies revealed that, in 4 studies, samples from subjects treated with non-aptamer drugs were to be used for in vitro investigations such as screening for aptamer biomarkers. The remaining 28 clinical studies involve aptamer drugs per se, about which I’ll comment below.

Status of Aptamer Studies in ClinicalTrials.Gov

Among the various ways the search results for 28 aptamer drug studies in ClinicalTrials.gov can be sorted for perusal and commentary, I chose to use study “status,” which ClinicalTrials.gov defines as follows:

  • Completed—clinical study has ended normally, and participants are no longer being examined or treated (that is, the “last subject, last visit” has occurred).
  • Terminated—clinical study has stopped recruiting or enrolling participants early and will not start again and participants are no longer being examined or treated.
  • Withdrawn—clinical study stopped before enrolling its first participant.
  • Active—clinical study is ongoing (that is, participants are receiving an intervention or being examined), but potential participants may not be being currently recruited.

Taken from doctorabidi.com

These categories can be used as filters to easily group studies for perusal, which when I did so, led to several general observations. The overwhelming majority of the 17 Completed clinical studies involve ocular diseases, of which age-related macular degeneration (ARMD) was a frequently studied indication. ARMD is the leading cause of blindness for people over the age of 55 years, in the U.S. and Europe, and occurs as either “dry” or “wet” ARMD, as described elsewhere.

While ARMD is a significant medical problem, my guess as to why it was chosen for initial clinical studies with aptamers is that clinical development had fewer practical challenges. Ocular administration required much smaller amounts of drug, which made manufacturing scale-up less problematic, and there were likely less toxicity issues to worry about. In any case, the sponsors included Eyetech Pharma, Ophthotech Corp, Achemix and NOXXON.

Terminated or Withdrawn studies, which numbered 8, were also mostly ARMD-related but also included aptamer for hemophilia and acute myeloid leukemia sponsored by Baxalta and Antisoma Research. Perhaps most important are the Active studies, which were only 3 in number, and I’ve put into tabular form here with some basic information including the National Clinical Trials (NCT) identification number that is searchable in any search engine. You can click on each NCT number to access detailed information provided in ClinTrials.gov; however, following are some snippets for each of these Active Studies.

Table of Active Studies Listed in ClinicalTrials.Gov in 2017

Sponsor (Study ID) Aptamer Drug Disease(s) Type of Study
Eyetech Pharma (NCT01487044) pegaptanib sodium (Macugen) Diabetic Macular Edema (DME) Phase 1
Ophthotech Corp (NCT02686658) ARC1905 (Anti-C5 Aptamer), Zimura® Dry ARMD Phase 2

 

Ophthotech Corp (NCT02591914) E10030 (Anti-PDGF Pegylated Aptamer, Fovista®) ARMD Phase 1

Pegaptanib structure taken from DrugBank. Light blue nucleotides are 2’-hydroxyl, dark blue nucleotides are 2’-fluoro, and red nucleotides are 2′-O-methyl.

Pegaptanib sodium (aka Macugen), which originated at NeXstar Pharma and was later taken on by Ophthotech via Eyetech, is a 5’-PEG (40kDa)/3’-inverted dT blocked 2′-fluoro/2′-O-methyl modified 27-mer RNA aptamer targeted against vascular endothelial growth factor (VEGF), and is the only FDA-approved aptamer drug. This Active Study is a single-center trial at the Retina Institute of Hawaii, and involves loading of intravitreal pegaptanib bi-weekly during the initial treatment period for diabetic macular edema (DME) when levels of VEGF, its protein target, are the greatest. This is followed by gradually extending the administration frequency to monthly as homeostasis ensues for the treatment of DME. This study was registered in 2011 and is currently recruiting patients.

ARC1905 (aka Anti-C5 Aptamer and Zimura) is reported to be a 38-mer aptamer sequence that was originally identified from a 2′-fluoro-pyrimidine-modified library, and all but three purines could be replaced by 2′-O-methyl purine nucleotides. For in vivo applications, an inverted dT and a 40 kDa PEG moiety were added to the 3′ and 5′ end, respectively, according to a review. Opthotech’s study of ARC1905 assesses the safety and efficacy of intravitreous administration in subjects with geographic atrophy secondary to dry ARMD. This study was registered in 2016 and is currently recruiting participants in numerous locations in the U. S. as well as some sites in Hungary.

Ophthotech’s E10030 (aka ARC127, Fovista), which is an anti-PDGF-BB aptamer, is reported to be a 36-mer published in 1996 that was derived from a DNA library, and was only modified with a 3′-inverted dT moiety to inhibit exonuclease degradation. Further shortening and post-SELEX modifications (2′fluoro pyrimidines, 2′-O-methyl purines, internal hexyl-linkers, all wherever possible) is reported to improve nuclease resistance and resulted in a 32-mer that was later named ARC126, and then ARC127 when pegylated at the 5′ terminus. Ophthotech’s Active Study of E10030 is an open-label trial located at Retinal Consultants of Arizona for safety, tolerability and development of subfoveal fibrosis by intravitreal administration of altering regimens of this aptamer drug and anti-VEGF therapy in subjects with neovascular ARMD. This study is ongoing but not recruiting patients.

Conclusions and Prospects

Oligonucleotides aptamers as possible therapeutics were scientifically “birthed,” so to speak, nearly 30 years ago, shortly after the “birth” of antisense oligonucleotides for therapeutics. Consequently, on a relative basis, I think it’s telling that my search of ClinicalTrials.gov lists only 28 studies of aptamer drugs compared to 152 studies found for “antisense.” In addition to this difference in total number of studies for these two classes of oligonucleotide drugs, filtering the data by Study Phase gave 7 aptamer studies as Phase 3 (all ocular diseases) compared to 14 antisense studies as Phase 3 (various diseases). The number of FDA-approved or soon-to-be-approved drugs mirrors the same inequality: only one (Macugen) for aptamers and a handful for antisense (see Ionis Pharma, Alnylam Pharma, and Sarepta Therapeutics).

Having said this, I hasten to add that I’m not “anti-aptamers” and instead “pro-antisense.” In fact, I’m “bullish” on both strategies for drug development, and expect more clinical progress with aptamers as new chemistries are pursued. Past focus on 2′-fluoro/2′-O-methyl modified oligonucleotide as aptamers represents, in effect, exploration of very limited structural diversity, which can now be expanded to hydrophobic SOMAmers and thioaptamers, or C5-modified dU X-Aptamers.

Finally, let’s consider the graph shown here, which I constructed using PubMed search results for the number of annual publications indexed to “clinical development of aptamers” as a search phrase. To me, there is clearly a trend toward increased numbers of such publications with time. Factors could arise which negatively impact this trend going forward, but absent that eventuality the future looks promising.

As usual, your comments are welcomed.

Footnote

There are additional Active Studies not registered in ClinicalTrials.gov, such as those with NOXXON’s “Spiegelmers,” which are mirror-image L-nucleic acids stable toward nuclease degradation in vivo. NOXXON states that its lead candidate, NOX-A12, which is an L-RNA aptamer, “is under development as a combination therapy for multiple cancer indications

Taken from iomers.net

where its impact on the tumor microenvironment is intended to significantly enhance the effectiveness of anti-cancer treatments without adding significant side effects for patients. NOX-A12 is currently in a Phase 1/2 trial in metastatic pancreatic and colorectal cancer patients who are not expected to respond to checkpoint inhibition alone. NOX-A12 has also completed two Phase 2a trials, one in chronic lymphocytic leukemia and the other in in multiple myeloma.”

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Transfer RNA (tRNA) Fragments Are Connected to Diseases

  • Specifically Formed tRNA Fragments (tRFs) can Repress Expression by RNAi 
  • Specific tRFs are Associated with Cancer and Other Diseases
  • Chemical Modifications in tRFs Pose a Challenge for Sequencing 

Researching new, trending topics for Zone in with Zon rewards me in several ways, including learning about important subject matter that I only vaguely knew about, or had been completely unaware of. The present blog is about tRNA fragments (tRFs), which was totally new subject matter for me that I found to be very interesting and worth sharing here.

But before getting to biological formation and functions of tRFs, I want to mention what led me to this intriguing class of RNA molecules. In a nutshell, TriLink’s R&D team decided to “brainstorm” on how its expertise in chemically modified RNA might be leveraged into new product offerings beyond its current lines of modified oligo RNA and modified messenger RNA (mRNA). Since tRNAs were long known to have numerous types of chemical modifications, as detailed elsewhere, TriLink’s R&D started to think about tRFs for reasons outlined below.

Biogenesis of tRFs

Formation of tRNA is a complex process. Initially, tRNA is transcribed in the form of a precursor (pre-tRNA) containing 50-nt leader and 30-nt trailer sequences, and in some cases introns in the anticodon loop. Pre-tRNAs then undergo various types of RNA processing steps to ultimately form mature tRNAs. During tRNA maturation, the 50-nt trailer is processed by RNase P, the 30-nt trailer is removed by RNase Z, and following 30-trailer removal, the 30-nt end of all human tRNAs is modified by enzymatic addition of the universal CCA triplet, as depicted here.

Pre-tRNA (left) and mature tRNA (right); adapted from Anderson & Ivanov FEBS Lett (2014)

Also depicted here are specific types of enzymatic cleavage reactions of mature tRNA by ribonucleases Dicer and angiogenin (ANG) that lead to formation of 5’-tRFs and 3’-CCA tRFs, as well as 5’-halves and 3’-halves. These tRFs derived from mature tRNAs, as well as tRFs from pre-tRNAs that will not be discussed here, have now been extensively characterized by high-throughput short RNA sequencing methods. Among new advances in this sequencing methodology, TriLink’s recent PLOS One publication of its innovative CleanTag™ sample prep procedure has already been viewed an impressive ~6,000 times since appearing online only ~14 months ago as of this writing.

Mature tRNA (adapted from Anderson & Ivanov)

It should be noted that tRFs are not restricted to humans but have been shown to exist in multiple organisms. Two online tools are available for those wishing to learn more about tRFs: the framework for the interactive exploration of mitochondrial and nuclear tRNA fragments (MINTbase) and the relational database of Transfer RNA related Fragments(tRFdb). MINTbase also provides a scheme for the naming of tRFs called tRF-license plates that is genome independent. A recent publication by Kim et al. is a good lead reference for various functions of tRFs, some of which include the following.

Possible Roles of tRFs in Human Diseases

In a review of this subject, Anderson & Ivanov emphasize that, while production of tRFs have been observed in several types of human diseases, it remains to be determined whether these tRFs contribute to disease pathogenesis. Landmark findings regarding functions of tRFs were published by a team, including Andrew Fire—2006 Nobel Laureate for  RNA interference (RNAi)—titled Human tRNA-derived small RNAs in the global regulation of RNA silencing that provided compelling evidence demonstrating that human tRFs can enter RNAi pathways. These findings by Fire & coworkers are now recognized as a previously unknown nexus of RNAi translational repression pathways involving tRFs and microRNAs (miRNAs) depicted here.

Schematic representation of the biogenesis of miRNAs and tRFs associated with Argonaute (AGO) proteins. Taken from Shigematsu & Kirino Gene Regul Syst Bio (2015)

tRFs and Cancer: In 2009, Lee et al. reported that a specific tRF, designated as tRF-1001, is highly expressed in a wide range of cancer cell lines but much less in tissues, and its expression in cell lines was tightly correlated with cell proliferation. Furthermore, siRNA-mediated knockdown of tRF-1001 impaired cell proliferation. Since that discovery, various research groups have similarly found specific tRFs associated with different types of cancer, as recently detailed by Croce & coworkers, who concluded the following:

“We found that tRNA-derived small RNAs (tsRNAs) [i.e. tRFs in this blog] are dysregulated in many cancers and that their expression is modulated during cancer development and staging. Indeed, activation of oncogenes and inactivation of tumor suppressors lead to a dysregulation of specific tRFs, and tRFs-knock out cells display a specific change in gene-expression profile. Thus, tRFs could be key effectors in cancer-related pathways. These results indicate active crosstalk between tRFs and oncogenes and suggest that tRFs could be useful [bio]markers for diagnosis or targets for therapy. Additionally, [overexpression of two specific tRFs] affect cell growth in lung cancer cell lines, further confirming the involvement of tRFs in cancer pathogenesis.”

Biomarkers in blood, which I’ve blogged about previously, are a “hot topic” in disease diagnostics because they offer a more general, less invasive and safer means of patient sample access compared to traditional tumor biopsies.

tRFs and Pathological Stress Injuries: Stress-related cellular damage is central to disease pathogenesis that can be induced by hypoxia, nutrient deprivation, oxidative conditions and metabolic imbalance. Dhahbi et al. sequenced short RNAs from mouse serum and identified abundant 5′-halves derived from a small subset of tRNAs, implying that these tRFs are produced by tRNA type-specific biogenesis. A survey of somatic tissues revealed that these tRFs are concentrated within blood cells and hematopoietic tissues, with very little in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of these 5′ tRNA halves change markedly with age, either up or down, and these changes were prevented by calorie restriction.

Taken from Mishima et al. J Am Soc Nephrol (2014)

In a study by Mishima et al., it was shown in vivo that oxidative stress leads to conformational changes in tRNA that thus allows ANG-mediated productin of tRFs. This stress-induced conformational change allows 1-methyladenosine nucleoside (m1A), a modification important for stabilizing the L-shaped structure of tRNA, to be recognized by an m1A-specific antibody, as depicted here. This antibody was used to show that renal injury and cisplatin-mediated nephrotoxicity (which both induce tissue damage via oxidative stress) generate tRFs. Similar results were obtained using m1A-based immunohistochemistry to directly visualize damaged areas of kidneys, brain and liver. Mishima et al. further demonstrated that these tRFS avoid degradation in the blood because they are associated with circulating exosomes, which are extracellular vesicles packed with proteins and nucleic acids.

tRFS and Neurodegenerative Diseases: As detailed in the above mentioned review by Anderson & Ivanov, ANG mutants possessing reduced ribonuclease activity were reported in 2006 to be implicated in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS; aka Lou Gehrig disease), which is a fatal neurodegenerative disease that I have blogged about. In 2012, a subset of ALS-associated ANG mutants was also found in Parkinson’s Disease (PD) patients. Recombinant ANG is neuroprotective for cultured motor neurons, and administration of ANG to a standard mouse model for ALS significantly promotes both life-span and motor function.

Concluding Comments on Analysis of tRFs

Although I started this blog by refering to the fact that mature tRNAs are extensively modified by a wide variety of nucleobase and ribose chemical modifications, these modifcations were not further mentioned. That is because sample prep for short RNA sequencing uses reverse transcription to form cDNA that is then PCR amplified before sequencing, and it is widely acknowledged (e.g. Cozen et al.) that certain chemical modifications in RNA can interfere with reverse transcription. Thus, aside from reported use of demethylases to first remove interfering methyl groups from m1A, N1-methylguanosine, N3-methylcytosine, and N2,N2-dimethylguanosine, sequenced tRFs exclude many tRFs having chemical modifications that prevent reverse transcription.

Recognizing the need for alternative methods of determining structures of chemically modified tRFS, Limbach & Paulines have recently proposed the possibility of developing mass spectrometric (aka mass spec) approaches in a publication provocatively titled Going global: the new era of mapping modifications in RNA. I think this is a great idea, and hope that the mass spec community will soon address this challenge.

As usual, your comments are welcomed.

ADDENDUM

After writing this blog, Eng et al., who investigated the mosquito Aedes aegypti—the primary vector of human arboviral diseases caused by dengue, chikungunya and Zika viruses—reported the following:

Aedes aegypti mosquito. Taken from wcvb.com

“[A]single tRF derived from the precursor sequences of a tRNA-Gly was differentially expressed between males and females, developmental transitions and also upon blood feeding by females of two laboratory strains that vary in midgut susceptibility to dengue virus infection. The multifaceted functional implications of this specific tRF suggest that biogenesis of small regulatory molecules from a tRNA can have wide ranging effects on key aspects of Ae. aegypti vector biology.”

Click here to read my past blogs about Zika virus.

Long Noncoding RNA (lncRNA) Revisited

  • Publications Dealing with lncRNAs Show Exponential Growth
  • Evidence for Involvement of lncRNAs in Cancer is Increasing
  • Value of lncRNAs as Biomarkers Has Been Validated

Several years ago, I posted a blog about long noncoding RNAs (lncRNAs), which are defined as non-protein coding transcripts in the range of ~200 nt to ~100 kb long. Interest in lncRNA—and other types of noncoding RNA such as microRNA (miRNA) and short interfering RNA (siRNA)—is fueled in large part by a collective scientific desire to uncover and understand the existence and function of all forms of RNA dark matter, so named by analogy to dark energy in cosmology. The lncRNA component of RNA dark matter is certainly generated from transcription of noncoding (formerly “junk”) DNA, but much has yet to be elucidated about function.

As depicted below, lncRNA (red) may act as (a) decoys to release proteins from chromatin, (b) scaffolds for grouping protein complexes, (c) guides to recruit proteins or (d) transcriptional enhancers by bending chromatin. Not shown is lncRNA acting as an antagonist for other regulatory noncoding RNAs, namely miRNA, which can be studied by next-generation sequencing methods such as TriLink’s CleanTag approach.

Taken from Bohla et al. Dis Markers (2017)

Numerical support for upward trending interest in lncRNA is provided by the chart shown here for the number of annual publications on lncRNA. This chart was produced by using data I found in PubMed for 2000-2016, which clearly show a relatively flat rate of ~150 papers per year from 2000-2007, and then an exponential increase to ~2,220 papers in 2016.

Although it’s not possible to say for sure what catalyzed this marked upturn in lncRNA publications, searching the 2005 literature in Google Scholar led to finding the following top-5 cited publications, which from titles alone could be likely scientific co-catalysts:

In any case, when I did keyword searches of the ~13,000 publications on lncRNA, roughly one-third (~4,700) were related to cancer, and many (~1,800) dealt with biomarkers primarily for (~1,400) cancer, but also including cardiovascular diseases, diabetes, epilepsy, general anxiety disorder, inflammatory bowel diseases, etc. Given that cancer is a major medical problem for all countries to deal with, and knowing that early detection of cancer by finding better biomarkers is critically important, this blog revisits lncRNA in the context of cancer and biomarkers.

State-of-the-Art Technologies to Explore lncRNA

At the risk of over simplification, advances in RNA sequencing (RNA-Seq)—enabled largely by high-throughput instrumentation from Illumina, PacBio, and Thermo Scientific—has revolutionized the field of molecular biology by revealing that up to ~75% of the human genome is actively transcribed, and that most of this transcriptome consists of lncRNA. Bioinformatic analyses, which are way beyond my expertise, have played a key role in sorting out lncRNA from mRNA and other RNA species. Interested readers can consult Cobos et al. as a recent lead reference to learn more about these bioinformatic methods.

Following are links to a constellation of additional experimental techniques that are available for exploring lncRN, and can be perused in detail later:

Although these methods are employed to shed light on lncRNA cellular localization, structure, interaction networks and functions, interested readers should consult a review by Salehi et al. and research paper by Goyal et al. for discussions of the advantages and disadvantages of these techniques. For example, Goyal et al. note that many lncRNA are derived from bidirectional promoters, or overlap with promoters, or bodies of sense or antisense genes. In a genome-wide analysis, they found only 38% of 15,929 lncRNA loci are safely amenable to CRISPR applications, while almost two-thirds of lncRNA loci are at risk to inadvertently deregulate neighboring genes. For several representative lncRNAs, it was found that CRISPR—but not RNAi by siRNA or antisense oligos—also affects their respective neighboring genes.

In closing this section on methods, readers who follow my blogs know that I’m a big fan of nanopore sequencing, about which I’ve commented in several previous posts. Oxford Nanopore Technologies has recently announced advances in its nanopore technologies that now allow sequencing of an RNA strand directly, rather than analyzing the products of reverse transcription and PCR reactions. My scientific “crystal ball” sees use of nanopore sequencing of lncRNAs in the not too distant future.

Long Noncoding RNA in Cancer and as Biomarkers

According to Bohla et al., lncRNAs are now known to function as regulatory factors for numerous, important cellular processes, such as growth, differentiation, and cell death. In addition, lncRNAs are involved in controlling alternative splicing, regulation of gene expression at the posttranscriptional level, chromatin modification, inflammatory pathologies, and—when deregulated—various types of cancer. Lnc2Cancer is a manually curated, interactive database of cancer-associated lncRNAs with experimental support that provides a high-quality and integrated resource for exploring lncRNA deregulation in various human cancers. In my opinion, Lnc2Cancer is definitely worth perusing.

The figure shown here indicates various types of cancers (black) for which lncRNAs (names of which are in green, blue or red) have been implicated. Searching PubMed or Google Scholar using any of the lncRNA names will provide a host of publications to peruse.

Taken from Vitiello et al. Cellular Oncology (2015)

For example, searching PubMed for the term “HOTAIR” gives ~475 publications, in chronological order starting with the most recent. By contrast, searching Google Scholar for the terms “HOTAIR lncRNA” gives ~6,100 articles ranked by “relevance,” which is explained elsewhere as being heavily influenced by citation frequency. Readers interested in more details can consult Lin & Yang, who review the mechanisms by which lncRNAs regulate cellular responses to extracellular signals, and discuss the clinical potential of lncRNAs as diagnostic indicators, stratification markers and therapeutic targets of combinatorial treatments.

As was mentioned in the introduction, there are numerous publications aimed at using these and other cancer-associated lncRNAs as biomarkers. Among the main advantages of lncRNAs that make them suitable as cancer diagnostic and prognostic biomarkers, high stability while circulating in body fluids, especially when included in exosomes or apoptotic bodies, is noted by Bohla et al. Despite abundant quantities of ribonucleases in different body fluids, lncRNAs protected in exosomes or apoptotic bodies can be detected in whole blood, plasma, urine, saliva and gastric juice. These lncRNAs as biomarkers are obtainable by non- or minimally invasive methods, which are well tolerated by patients compared to conventional biopsies.

Taken from liquid-biopsy.gene-quantification.info

Challenges for use of lncRNAs as biomarkers include development of convenient, low cost yet robust isolation methods, and accurate quantitation of relatively low copy numbers, which heretofore has relied on an amplification step, such as enzymatic conversion into cDNA followed by PCR. However, single-molecule detection approaches have evolved to obviate the need for amplification.

For example, NanoString Technologies now offers the nCounter® lncRNA Assay for validation of lncRNA discoveries, which can then be followed by use for biomarker quantification. As depicted below in the left panel, single molecules of lncRNAs (red) can be detected at the same time as mRNAs green and blue), if so desired, using sequence-specific probes each having a fluorescence-based “barcode” identifier. The right panel depicts extension of this approach to identify lncRNA-protein interactions by inclusion of antibody precipitation. This digital-counting assay allows researchers to select up to 800 lncRNAs for analysis in a single multiplexed reaction, which is quite impressive, in my opinion.

Taken from nanostringxt.com

Taken from Cesano J Immunother Cancer (2015)

Readers interested in more details for application of nCounter® for analysis of biomarkers are referred to a recent publication by Permuth et al. dealing with pancreatic ductal adenocarcinoma (PDAC), which is an aggressive disease that lacks effective biomarkers for early detection. Briefly, these researchers hypothesized that circulating lncRNAs may act as diagnostic markers, and used nCounter® technology to measure the abundance of 28 candidate lncRNAs in pre-operative plasma from a cohort of pathologically-confirmed PDAC cases of various grades of severity and non-diseased controls. Results showed that two lncRNAs aided in differentiating PDAC from controls, and an 8-lncRNA signature had greater accuracy than standard clinical and radiologic features in distinguishing ‘aggressive/malignant’ PDAC that warrant surgical removal from ‘indolent/benign’ PDAC. In my opinion, these findings seem very promising for use with PDAC and, by conceptual extension, to other cancers.

Temozolomide (TMZ). Taken from sigmaaldrich.com

As a final example of lncRNA biomarkers for cancer, MALAT1 (pictured above) has been recently reported by Chen et al. as a prognostic factor in glioblastoma multiforme (GMF), and induces chemoresistance to temozolomide (TMZ). The significance of these findings is that GBM is the most malignant brain tumor with limited therapeutic options, and that TMZ is first-line chemotherapy for GBM. These researchers first used deep-sequencing and bioinformatic methods to identify lncRNAs showing different expression levels in TMZ-resistant and non-resistant patients. RT-qPCR was then performed in tissues and serum samples, and lncRNA MALAT1 was shown to discriminate between responding patients from non-responding patients.

Closing Comments

If you’re are interested knowing much more about lncRNAs, then lncRNABlog.com is a great website for you to visit and subscribe to, if you want to keep current on all manner of lncRNA research and industry news. This interactive blog, which posts abstracts and images from the latest lncRNA publications, allows readers to post comments that allow you to join in the “conversation” or simply follow what others are thinking about these articles.

Also provided are links to a host of different online tools for lncRNA research and development, as well as “what’s happening” in terms of upcoming lncRNA events or conferences. Those of you currently seeking a new position may find the jobs postings to be helpful.

Finally, there are there plenty of pop-up advertisements and commercial banners, but these are also informative about lncRNA products and services that are available.

As usual, your comments are welcomed.

The Scientist’s Top 10 Innovations in 2017—and Jerry’s Top Pick

  • Multiplexed Assays Are Trending
  • Miniaturization is Big—Pun Intended
  • Jerry’s Top Pick is an Amazing 4 Inch Cube

Taken from the-scientist.com

Welcome to my first blog of the New Year, 2018! My New Year’s resolution is to “double down” by giving my best effort to provide interesting and informative content about what’s trending in nucleic acid research. Having said that, and in keeping with tradition, this first blog of the year pairs my comments on the Top 10 Innovations in 2017, as reported by the The Scientist, with my personal “fav” for the best new product launched last year.

So, with an imaginary loud flourish of trumpets, read on to learn about The Scientist’s 10 winners that I’ve listed from 10th to 1st place—to build your interest—after which I comment on my personal fav.

Top 10 Innovations in 2017 Reported by The Scientist

10. TrueCut Cas9 Protein v2 from Thermo Fisher Scientific is a next-generation CRISPR-Cas9 protein engineered to deliver maximum editing efficiency across a range of genes and standard cell lines, as well as stem cells, T cells, and primary cells.

With regard to this product, I think it’s worth mentioning that TriLink offers mRNA-encoded Cas9, as an alternative to delivering Cas9 protein per se. Also, my several past blogs on CRISPR can be accessed here.

9. TSQ Altis Triple Stage Mass Spectrometer from Thermo Fisher Scientific robustly and reliably quantitates most analyte types, even in complex samples such as plasma and tissue, thus enabling wide applicability, including forensic toxicology and clinical research.

8. Chromium from 10x Genomics for profiling single-cell gene expression, enables deep profiling of complex cell populations, is provided as a complete droplet-based system of reagents, barcodes, hardware and software for sample prep prior to high-throughput sequencing.

7. Edit-R crRNA Library—Human Genome from Dharmacon provides users with an arrayed library of synthetic crRNA guides in a “one-well-per-gene” format, with four distinct guides per gene for redundancy to improve statistical power.

6. HiBiT Protein Tagging System from Promega is a new detection system for quantifying proteins in or on a cell of interest, using a small and easily integrated 11-amino-acid tag (HiBiT) that interacts with a complementary large 156-amino-acid component leading to bioluminescence.

5. SR-X Ultra-Sensitive Biomarker Detection System from Quanterix offers more than 80 different assays to test samples (e.g. blood, serum, cerebral spinal fluid, single-cell lysates) for cytokines or other markers of neurodegeneration or neuroinflammation, and more.

4. Blaze from Intabio is a system for detecting and identifying protein isoforms that aims to save pharma companies time in lab prep work for QC of biologics manufacturing. Launch will be “within the next few months” and “pricing is still yet to be set,” according to The Scientist.

3. QGel Assay Kit for Organoids from QGel provides fully synthetic extracellular matrix to reproducibly grow research organoids, which are miniaturized and simplified version of an organ produced in vitro.

2. i-STAT Alinity from Abbott is a handheld, cartridge-based blood-testing device for user-friendly point-of-care assays on a blood sample of just several drops—including glucose levels and hematocrit—with results directly delivered to a patient’s medical record for clinicians within 2-10minutes. Alinity is available in about 50 countries, but Abbott is waiting for a few more assays to be cleared by the US Food and Drug Administration before selling it stateside.

Regarding this product, I should mention that my several past blogs on point-of-care can be accessed here.

1. IsoCode Chip from IsoCode is new single-cell technology allowing researchers to characterize cells based on the proteins they secrete—as many as 42 different cytokines, chemokines, and other types of molecules. IsoCode chips contain thousands of long microchambers that house only single cells. Within each microchamber, 15 spatially separated slots contain up to three different antibodies targeting specific secreted proteins; upon binding, each antibody fluoresces in a different color to distinguish the proteins. This provides the ability to simultaneously profile thousands of individual T cells or immune cells at one time.

Taken from The Scientist Dec 2017

IsoCode chips come in 10 different panels, ranging from 24 to 42 antibodies per panel, at a cost of $500–$600. The automated IsoLight imaging and workflow platform can be purchased starting at $200,000. But the IsoCode chips can also be paired with other fluorescence microscopy systems.

Jerry’s Fav for the Best Innovation in 2017

My personal pick for this honor goes to the world’s first on-site Legionella DNA test to prevent Legionnaires’ disease, which was released this past November by the Canadian company Spartan Bioscience. According to a press release, it is the first on-site DNA test for Legionella bacteria and it can detect and quantify Legionella in only 45 minutes, compared to 10-14 days for off-site sample analysis using traditional culturing methodology. The system pictured here consists of a coffee-cup-sized portable DNA analyzer called the Spartan Cube, which employs a single-use disposable test cartridge.

Taken from ctvnews.ca

This innovative product stood out for me because it brings together the following topics that I have separately blogged about:

  • Reoccurring outbreaks of potentially fatal Legionnaire’s disease, such as that which recently hit New York City, and also shut down Disneyland.
  • Decentralized analytical testing for more rapid “sample-to-answer” applications on-site, i.e. in the field—wherever that might be—or at point-of-care in hospitals, clinics, and elsewhere.
  • Miniaturization and simplification of qPCR using cleverly engineered devices, such as the Spartan Cube, in conjunction with single-use disposable test cartridges.

Legionella is a common environmental bacterium that can infect the cooling towers of Heating, Ventilation, and Air Conditioning (HVAC) systems of large buildings. Infected cooling towers release aerosolized water droplets contaminated with Legionella into the surrounding air. Globally, there are hundreds of thousands of office-building towers, hospitals, hotels, shopping malls, and other large buildings at risk for infection by Legionella. Weekly testing with the Spartan system can rapidly detect Legionella bacterial growth early, and thus allow cleaning and decontamination of the cooling tower before Legionella reaches dangerous levels to human health.

In addition, and also importantly, traditional culture test methods can underestimate the Legionella concentration on site. The Centers for Disease Control and Prevention (CDC) found that Legionella culture can underestimate actual Legionella levels by a factor of 10 or more. Culture incorrectly reported that water samples were negative for Legionella an average of 11.5 percent of the time when in fact they were positive.

Paul Lem. Provided by Paul Lem

According to Paul Lem, M.D., CEO of Spartan Bioscience, who I contacted about cost to customers, “the price for the Cube and Legionella tests is $5-10K/building/year, depending on the building. It’s a subscription model.”

Regarding my further inquiry about testimonials to date, Dr. Lem provided a copy of a November 26, 2017 article reported in The Globe and Mail which quotes him as saying that “several property managers are testing the device at close to 100 properties, including BGIS and Ottawa’s KRP Properties, owned by tech entrepreneur [Sir] Terry Matthews.”

The article also states that “the market could be worth billions of dollars globally, encompassing office buildings, malls, hospitals, schools, theme parks, spas and so on. ‘Nobody really knows because the market doesn’t really exist yet,’” said Lem.

For the sake of increased public safety toward exposure to Legionella, let’s all hope that this application of the amazing Spartan Cube is indeed very successful. And, moreover, that 2018 is a great year for other nucleic acids-based innovations, many of which I look forward to blogging about here.

As usual, your comments are welcomed.

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RNA Epigenetics – Part 3

  • 2′-O-Methylation Sites in Human mRNA Sequenced with Base Precision
  • N3-Methylcytidine Discovered in Human mRNA
  • “The More You Know, the More You Know You Don’t Know”—Aristotle

A few years ago, I posted a blog titled In Search of RNA Epigenetics: A Grand Challenge, which commented on new discoveries of how chemically modified bases in mRNA were enzymatically added and removed, as mechanisms of cellular regulation. Since these processes occur post-transcriptionally, i.e. after mRNA is polymerized from DNA template, they are by definition epigenetic. The Greek prefix epi- (“over, outside of, around”) in epigenetics implies features that are “on top of” or “in addition to” the traditional genetic basis for inheritance.

That earlier blog (Part 1), featured N6-methyladenosine (m6A) and 5-methylcytidine (m5C), and quoted an expert’s comment that biological functions of pseudouridine (Ψ), 2’-O-methyl (2′-OMe), and potentially other modifications in mRNA and various non-coding RNAs need to be elucidated. I subsequently blogged about profiling Ψ in mRNA in Part 2. In this post, Part 3, I will share recent developments on 2’-OMe and introduce N3-methylcytidine (m3C) as the most recently discovered modification in mammalian mRNA.

Taken from Liu & He J Biol Chem (2017). See comment section below for correction of pictured stereochemistry kindly provided by Prof. Fritz Eckstein on Dec 14, 2017.

Mapping 2′-O-Methylation Sites in Human mRNA with Base Precision

This section heading is taken from the title of a July 2017 publication in Nature Methods by an international team that included Chuan He, who I featured in Part 1 of this series. These investigators noted that existing methods for locating 2’-OMe sites are underpowered for detection in relatively rare RNA molecules, such as mRNA, or at low abundance within a given RNA molecule.

To address these challenges, they developed a conceptually distinct approach based on the different chemical properties of nucleosides with 2′-OH and 2′-OMe, as well as combining enrichment with detection of a positive signal (rather than the lack of signal) to produce a suitably sensitive sequencing method. This method, named Nm-seq, leverages oxidative cleavage of ribose 2′,3′-vicinal diols by periodate to expose, enrich and map 2’-OMe sites in the transcriptome without bias and with single-nucleotide precision, as depicted below. Stepwise details for this biochemical methodology can be found below in the Addendum, so let’s skip to what was found using Nm-seq.

Taken from tud.ttu.ee

Stepwise details for this biochemical methodology can be found below in the Addendum, so let’s skip to what was found using Nm-seq:

  • Total levels of 2′-OMe in HeLa mRNA by LC-MS/MS and obtained 2′-OMe /2’-OH molar ratios ranged from 0.012% for A to 0.15% for U, with G and C between these values, and covered 7,412 sites, typically in less structured regions.
  • The majority of 2′-OMe sites (95.7%) occurred in 2,398 RefSeq annotated genes, 95.9% of which were protein coding, with 77% of these 2′-OMe-modified gene harboring only one 2′-OMe site.
  • Most of the sites (70.3%) occurred in coding sequences, and the rest occurred in 5′ and 3′ untranslated regions (3% and 10.6%, respectively) as well as in introns (16.2%), suggesting that 2’-OMe is installed co-transcriptionally in the nucleus.
  • While 2’-OMe was found in all three codon positions, the 1st position had more methylation than expected by chance, and the 3rd had less, which is consistent with a recent study that showed a codon-position-dependent effect of 2’-OMe on translation.
  • Nm-seq applied to mRNA purified from HEK293 cells recapitulated the features observed in HeLa mRNA.

In my opinion, the biggest mystery in these findings is why nature evolved to include only one 2’-OMe modification in most of the mRNAs. It’s also worth pondering whether synthetic modified mRNAs, about which I’ve blogged, would function better when having 2’-OMe in such sites.

Discovery of N3-methylcytidine (m3C) in Mammalian mRNA

In September 2017, an international team reported compelling evidence for the discovery of m3C in mRNA from mice and humans, which previously had only been found in tRNAs. This discovery by Xu et al. is well worth reading for full appreciation as a tour de force of experimental methodology; however, a brief synopsis of key steps is as follows.

The team investigated three mammalian methyltransferases—METTL2, METTL6 and METTL8—the genes for which were each knocked out in mice and in human cell lines using CRISPR/Cas9 (about which I’ve previously blogged multiple times). Using liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS), they quantified m3C in tRNA fractions in brain and liver tissues from wild-type mice and knockout mutants. The results showed that METTL2 and METTL6 deficiencies led to a 35 and 12% reduction in m3C levels in tRNA, respectively. In contrast to these results, METTL8 deficiency did not produce a significant change in tRNA m3C levels. Instead, Xu et al. provide definitive proof through stringent purification of mRNA and quantification by LC-MS/MS that METTL8 acts on mRNA.

Specifically, tRNA was first removed from the total RNA sample by size-exclusion chromatography. The rest of the RNA fraction was subsequently subjected to poly(A) enrichment and rRNA depletion. mRNA extracted from mutant mice with a deficiency of METTL8 showed lower levels of m3C, with no noticeable changes observed for the m3C levels in tRNA.

Commenting on this work by Xu et al., experts Liu & He pointed out that there are “several immediate angles to explore.” Specifically, the locations of the METTL8-installed m3C modifications remain unknown. Furthermore, the functional roles of METTL8 and the m3C modifications, and whether these vary under different stress conditions or during cellular signaling remain to be elucidated. They add that “[i]dentifying and deciphering the roles of RNA modifications is more than just a biochemical treasure hunt: Defects of certain RNA-modifying enzymes are known to be associated with human diseases. Moving beyond the abundant RNAs to…mRNA and long noncoding RNA, coupled with the discoveries of chemical modifications such as…m3C methylations, is opening new directions in understanding RNA modification-mediated RNA processing and gene expression regulation.”

Taken from brittanica.com

In my humble opinion, the number of chemical modifications in mRNA has indeed increased quickly during the past decade, which adds to our factual data for RNA epigenetics while continuing to challenge our understanding of this exciting field through further experimentation. In this regard, I’m reminded of the following quote attributed to the ancient Greek philosopher and scientist Aristotle:

“The more you know, the more you know you don’t know.” 

Addendum:  Stepwise details for Nm-seq methodology

Nm-seq first exposes internal 2’-OMe (“Nm”) sites in RNA fragments (Step I below) by iterative oxidation–elimination–dephosphorylation (OED) cycles that remove unmodified 2′-OH nucleotides (one per cycle) in the 3′-to-5′ direction. Vicinal diols in these nucleotides are readily oxidized by sodium periodate to yield a dialdehyde intermediate that undergoes spontaneous β-elimination under mildly basic conditions. With the removal of a nucleoside, the resulting 3′-monophosphate is enzymatically dephosphorylated to allow another cycle to take place. Once a 2’-OMe is encountered, the progressive shortening process comes to a halt, as lack of vicinal diols prevents oxidation (Step II below).

The net result of the iterative exposure process is an enrichment of fragments ending with 2’-OMe, although those ending with 2’-OH still constitute the majority of 3′ termini. A final round of oxidation–elimination (OE) reaction, excluding dephosphorylation, is then performed to generate two types of 3′ ends that differ in their ligation compatibility (Step III below). While unmodified 2′-OH ends produce an unligatable 3′-monophosphate, 2’-OMe ends are resistant to oxidation and retain their ligatable 3′-OH group. In this way, fragments ending with 2’-OMe are preferentially ligated to the 3′ adaptor (note shown below) and further enriched by PCR amplification.

Interested readers should consult the Nm-seq publication by He & coworkers for further details on how this cyclic biochemistry, ligation, and amplification lead to libraries for sequencing and decipherable patterns indicating 2’-OMe positions.

Taken from He & coworkers Nature Methods (2017)

Advances in Nucleic Acid-Based Therapeutics Against Alzheimer’s

  • Dementia Develops in Someone in the World Every 3 Seconds
  • Nucleic Acid-Based Approaches for an Alzheimer’s Drug Are Advancing
  • Ionis Pharmaceuticals is First to Begin Clinical Trials with Antisense Oligonucleotide Targeting Tau

The concept of nucleic acid-based therapeutics, as originally conceived by Paul Zamecnik, goes back to his seminal publication in 1978 that I’ve blogged about previously. However, it wasn’t until the advent of automated synthesis of various types of modified oligonucleotide analogs that this “antisense” approach to drug development achieved critical mass of sustained attention. Since then, synthetic oligonucleotides and mechanisms of interference with mRNA expression have greatly expanded to now include a diverse “armory” of alternatives to combat diseases, including:

  • Antisense oligonucleotides (ASOs) that induce cleavage of mRNA by RNase H or other mechanisms
  • Short-interfering RNA (siRNA) that are incorporated into RISC that cleaves mRNA
  • Antagomirs that block microRNA (miR) binding to mRNA
  • Aptamers that bind to target proteins as drugs, or for targeted delivery of other types of drugs
  • Splice-switching oligonucleotides (SSOs) that hybridize with a pre-mRNA and disrupt normal splicing
  • Recently reported RNA-guided RNA-targeted CRISPR-Cas variants that can knockdown RNA systems much more specifically than siRNA

Investigating possible NA-based approaches to currently refractory, or other so-called ‘undruggable’ diseases, has attracted much needed interest from academics and pharma researchers, as well as implementation of GMP procedures. Recognizing this need, and the absence of a conventional drug to treat Alzheimer’s disease (AD)—the leading cause of dementia in adults—kindled my efforts to research the literature and write this blog.

What is AD?

Auguste Deter, who was a patient of German psychiatrist Alois Alzheimer, was the first described case of what became known as Alzheimer’s disease. Taken from Wikipedia.org

According to a U.S. National Institutes of Aging fact sheet, AD is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, and eventually the ability to carry out the simplest tasks. In most people with AD, symptoms first appear in their mid-60s. Statistics vary, but AD accounts for as much as ~80% of all dementia—an umbrella term for loss of cognitive functioning—which will develop in someone in the world every 3 seconds, and afflict close to 50 million people in 2017. This is based on estimates by Alzheimer’s Disease International.

Brains from bodies of deceased persons who had advanced AD exhibit overall “shrinkage,” macroscopically, compared to persons who did not show indications

Taken from fellowshipoftheminds.com

of AD. Microscopically, AD is characterized by accumulation of toxic “amyloid plaques,” which are sticky buildup that accumulate outside nerve cells, or neurons. Amyloid is a protein that is normally found throughout the body, but in AD the protein is cleaved by beta secretase (BACE1) and gamma secretase to yield amyloid beta (Aβ) peptides of 36–43 amino acids. Aβ molecules can aggregate to form flexible soluble oligomers that may exist in several forms. It is now believed that certain misfolded oligomers can induce other Aβ molecules to also take the misfolded oligomeric form, leading to a chain reaction akin to a prion infection.

Amyloid plaques. Taken from slideshare.net

Since 1993, when a variant of the apolipoprotein E (APOE) gene was found to be strongly associated with increased vascular and plaque Aβ deposits in late-stage AD patients, many researchers have probed APOE connections to Aβ. However, in September 2017 this investigative field was reportedly “stunned” by results published by Shi et al. showing that neurotoxic effects associated with APOE may result from a damaging immune response to a different protein named tau, encoded by the MAPT gene. This late-breaking scientific news is part of an ongoing story of AD molecular pathology that interested readers will want to follow.

“Tangles” of tau protein (green) are visible in a brain cell from someone who had Alzheimer’s disease. Taken from Underwood, Science (2017)

Recent Advances in Nucleic Acid-Based Drugs for AD

My strategy for researching this topic began with querying Google Scholar for “Alzheimer’s” articles since 2013 that are coupled with key words for each of the above nucleic acid-based drug modalities, namely, “antisense,” “siRNA,” etc. This led to literature ranked by Google Scholar according to citation counts. I then perused this information to select items for each of these modalities. I linked to the search results for readers interested in further digging into these topics.

Antisense Oligonucleotides

A report that caught my attention was by Lane et al. from Ionis (formerly Isis) Pharmaceuticals. This company (founded and still led by Stanley Crooke, about whom I’ve blogged) is widely acknowledged to be the leader in clinical development of antisense therapeutics. In a nutshell, Lane et al. hypothesized that, given the critical role for tau (see above) in transducing Aβ-linked neurotoxicity, reducing the synthesis of tau could have a therapeutic effect.

Ionis-MAPTRx, a 2′-O-methoxyethyl chimeric ASO, was found to reduce tau expression in transgenic mice and was tested in FDA/IND-enabling toxicology studies in rodents and non-human primates (NHPs). Intrathecal administration of the highest dose in NHPs resulted in a mean MAPT mRNA reduction of 77% in frontal cortex and 74% in hippocampus without dose-limiting side effects. Following up on this, I found that on October 13, 2017, Ionis announced initiation of a Clinical Study of Ionis-MAPTRx in patients with AD, thus earning a $10 million milestone payment from Biogen.

In another ASO approach, Farr et al. have further investigated their previously reported 20-mer phosphonothioate oligonucleotide (GAO) that had been shown to knockdown levels of glycogen synthase kinase (GSK)-3β, which is a multifunctional protein implicated in the pathological characteristics of AD, including neurofibrillary tangles, Aβ, and neurodegeneration. In the present study, they assessed the impact of peripherally administered GAO on learning and memory—measured by a T-maze (see picture)—in two different mouse models of AD, as well as knockdown of protein expression. GAO-treated mice showed improved acquisition and retention, along with decreased protein levels. They concluded that this study “supports peripherally administered GAO as a viable means to mediate GSK-3β activity within the brain and a possible treatment for AD.”

Taken from ratbehaviour.org

To learn about a T-maze, click here.

siRNA

SLN loaded with siRNA (green). Taken from precisionnanosystems.com

While there are several intriguing studies of ASOs associated with AD therapy, siRNA is much more common in the research and treatment of AD. The report that I found most interesting was published by Rassu et al. in 2017, and is titled Nose-to-brain delivery of BACE1 siRNA loaded in solid lipid nanoparticles for Alzheimer’s therapy. My interest stemmed from the focus on developing delivery technology to achieve “nose-to-brain” as a very convenient, non-invasive route for delivery using solid lipid nanoparticles (SLNs), which are viewed as promising technologies for drug-delivery.

In this report, the siRNA targeted the secretase BACE1, which has been widely investigated because of its involvement in forming neurotoxic Aβ peptides, as mentioned above. To increase the transcellular pathway in neuronal cells, a short cell-penetrating peptide derived from rabies virus glycoprotein known as RVG-9R was used, based on previous studies demonstrating that intravenous treatment with an RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Building on this prior knowledge, Rassu et al. optimized the molar ratio of RVG-9R and BACE1 siRNA, and investigated chitosan-coated and uncoated SLNs as a nasal delivery system capable of exploiting both olfactory and trigeminal nerve pathways.

Taken from researchgate.net

The positive charges from protonated amino (NH2) groups of the coating formulation ensured muco-adhesiveness to the particles, and prolonged residence-time in the nasal cavity. They studied cellular transport of siRNA released from the SLNs using the Caco-2 cells, which is a human epithelial colorectal adenocarcinoma cell line, as a model for epithelial-like phenotypes. It was found that siRNA better permeates the monolayer when released from chitosan-coated SLNs vs. uncoated SLNs or “naked” siRNA.

Chitosan. Taken from Wikipedia.org

Antagomir

MicroRNA-146a (miR-146a) is upregulated in the brains of patients with AD and induces activation of tau (see above). To determine whether reducing miR-146a could ameliorate tau-related AD pathologies, Wang et al. assessed its levels and the use of a miR-146a inhibitor (antagomir) in a validated mouse model of AD. The antagomir and solvent control were delivered into the hippocampus of these mice at three months of age and memory was tested in all mice using several types of mazes (see above) before extracting brain samples for quantitative RT-PCR using measurements of miR-146a and protein targets. In a nutshell, the overall results demonstrated that improvement of memory by intrahippocampal miR-146a antagomir was associated with the predicted alterations in the tau-related neural pathway, confirming that inhibition of miR-146a expression has a therapeutic effect in this mouse model of AD. It was concluded that “this data support (sic) the concept that miR-146a antagomir is a potential efficacious therapeutic target for the tau pathology of AD.”

Aptamers

Aptamer A1 structure reported by Liang et al.

In a study published by Liang et al. in 2015, systematic evolution of ligands by exponential enrichment (SELEX) with random-sequence libraries was used to obtain a DNA aptamer (A1). That aptamer is pictured below, and has been shown to have high-affinity binding to purified human BACE1 extracellular domain. They subsequently confirmed that A1 exhibited a marked inhibitory effect on BACE1 activity in an AD cell model, based on decreased concentrations of Aβ fragments and BASE1 protein. These investigators concluded that “these findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.”

Splice-Switching Oligonucleotides

According to Hinrich et al., apolipoprotein E receptor 2 (ApoER2), which is involved in long‐term potentiation, learning, and memory, has been proposed to be involved in AD, though a role for the receptor in the disease is not clear. ApoER2 signaling requires amino acids encoded by alternatively spliced exon 19. To test the role of deregulated ApoER2 splicing in AD, they designed a splice-switching oligonucleotide (SSO) that increases exon 19 splicing. Treatment of AD mice with a single dose of SSO corrected ApoER2 splicing for up to 6 months and improved synaptic function and learning and memory. They concluded that “these results reveal an association between ApoER2 isoform expression and AD, and provide preclinical evidence for the utility of SSOs as a therapeutic approach to mitigate AD symptoms by improving ApoER2 exon 19 splicing.”

CRISPR

Individuals heterozygous for the Swedish mutation of the amyloid precursor protein (APPswe) display an increased β-secretase cleavage leading to higher Aβ levels—both in brain and peripheral tissues, according to Gyorgy et al. They added that the mutation is a double base change adjacent to each other and has a dominant effect, which led them to hypothesize that the CRISPR system would selectively disrupt the mutated allele without affecting the wild-type (wt) allele.

In a nutshell, human APPswe fibroblasts and non-mutated control fibroblasts from subjects of the same family were grown in vitro, and then transfected with a Cas9 plasmid together with different guide RNAs (gRNAs) designed to bind either the mutated or non-mutated site with the mutation in the gRNA recognition sequence. Sanger sequencing was performed on cells that had been successfully transfected with CRISPR plasmids, and on such cells, both the APPswe mutant and wt alleles could be disrupted with gRNAs designed against the mutated and non-mutated sites, respectively. Moreover, these effects appeared to be highly specific as assayed by deep sequencing as they did not find any random mutations on the wt allele with the gRNA targeting the mutated site or vice versa.

This study reported in 2016 was said to “[provide] the first experimental evidence that the CRISPR/Cas9 method could be used to develop a novel treatment strategy against familial forms of Alzheimer’s disease caused by dominant mutations.”

Closing Comments

From the above sampling of publications reporting promising results for nucleic-acid-based therapeutic approaches to AD, I hope you will agree with me that it seems likely a clinically successful drug will prevail. What and when are uncertain, but I’m betting that something will be Ionis-MAPTRx , which is the most advanced clinical candidate to date.

Addendum

Taken from October 31, 2017 GEN

Alzheimer’s disease may move, cancer-like, from place to place in the body, lodging in the brain after originating in peripheral tissues, according to October 31, 2017 news in GEN. This cancer-like mobility was demonstrated through a technique called parabiosis—the surgical union of two specimens to allow them to share a blood supply, as shown here. This technique was used to keep pairs of mice together for several months, wherein normal mice, which don’t naturally develop AD, were joined to transgenic AD mice, modified to carry a mutant human gene that produces high levels of plaque-forming Aβ. It was reported that human Aβ originating from transgenic AD mice entered the circulation and accumulated in the brains of normal mice, forming cerebral amyloid angiopathy and Aβ plaques after a 12-month period of parabiosis.

Legionnaires’ Disease Outbreak in New York

  • First Identified as a New Pathogen 40 Years Ago, Legionella Persists
  • Legionella’s Life Cycle Involves “Biological Sanctuaries”
  • qPCR Proven to Outperform Antibody-Based Detection of Legionella

When I read about an outbreak of Legionnaires’ disease (LD) in New York City, baseball legend Yogi Berra’s famous quote, “It’s déjà vu all over again” immediately came to mind, along with the irony of Berra playing for the New York Yankees. So, if you’re much younger than me, you’ll likely not know why “It’s déjà vu all over again” and you may wonder who Berra was. You can read about him later elsewhere, but for now you should read on to learn about Legionnaires disease and why déjà vu is apropos.

History of LD

Notable positive events during 1976 in the United States included our Bicentennial Celebration, unveiling by NASA of the first space shuttle (the Enterprise), establishment of Apple Computer Company by Steve Jobs and Steve Wozniak, and Silly Love Songs by Paul McCartney and Wings ascending to #1 on the charts. While many of these events were the beginning of fabulous things to come, one proved to be the beginning of something catastrophic. American Legionnaires who gathered in Philadelphia, Pennsylvania for the Bicentennial were struck with a mysterious epidemic of fatal respiratory disease.

Taken from networks.org

Sadly, 182 members of the Pennsylvania American Legion were affected, and 29 individuals died after they returned from the convention in Philadelphia. The epidemiological and microbiological studies continued for months before scientists began to understand what had happened. Much of the basic framework of our knowledge of Legionnaires disease, as the epidemic came to be known, was developed by a team from the CDC and the Pennsylvania Department of Health, as detailed elsewhere.

Taken from case1study.wikispaces.com

The cause of the disease remained a mystery until 1977 when an investigative team led by J. E. McDade and C. C. Shepard (of the Leprosy and Rickettsia Branch, Virology Division, Bureau of Laboratories, CDC) reported on the isolation of a Gram-negative bacillus found in patient samples. As often done for naming pathogens after sources, the genus of this rod-shaped bacterium was aptly named Legionella. Legionella includes the species L. pneumophila, which caused the pneumonia-like illness medically named legionellosis, but commonly referred to as LD.

2017 LD Outbreak Hits New York City—Again

In June of this year, forty years after the first characterization of Legionella, it’s lethal infectivity reoccurred in an outbreak in the Upper East Side of the Manhattan borough of New York City, leaving one person dead and six other people sickened. According to a newspaper account, this outbreak occurred within 11 days, and may have been triggered by contacting contaminated water as has happened in other cases.

While this incident affected relatively few people compared to other previous outbreaks, including one in the Bronx borough of New York City in 2015 that killed 15 people and sickened more than 70, it’s a scary reminder of the persistence of Legionella in the environment. In this regard, it has been reported that 200 to 400 cases of the illness are recorded each year in New York, despite the monitoring of 6,000 water systems wherein Legionella can flourish in warm conditions. This environmental factor provides a segue into what genomic sequencing has revealed about Legionella.

Genomics-Based Insights on Legionella

The bacterial pathogen L. pneumophila is found ubiquitously in fresh water environments where it replicates within protozoan hosts. When inhaled by humans it can replicate within alveolar macrophages and cause severe pneumonia associated with Legionnaires disease. As detailed elsewhere, recent advances in genome sequencing has had a major impact on understanding of the pathogenesis, evolution and genomic diversity of Legionella.

A lipopolysaccharide cell wall and several outer membrane proteins are essential virulence factors. Central to the pathogenesis of L. pneumophila is its Type IV secretion system, which translocates over 270 effector proteins into the host cell, thus allowing this bacterium to manipulate host cell functions to its advantage and assures intracellular survival and replication.

Within aquatic media, as depicted below, Legionella exist as part of biofilms, which provide a protective environment—or biological sanctuary, if you will—wherein the bacteria exhibit marked increase in resistance to biocidal compounds and chlorination. Aside from the resultant difficulty of purging water systems to be free of Legionella, these bacteria can invade and multiply within protozoa (which are ubiquitous and include amoeba), thus providing yet another biological sanctuary. Protozoa are present in all aquatic or moist environments, and can be found in even the most inhospitable parts of the biosphere, thus providing further protection to Legionella.

Taken from Comas Nature Genetics (2016)

The actual infectious particle is not known but may include excreted legionellae-filled vesicles, intact legionellae-filled amoebae or free legionellae that have lysed their host cell. Transmission to humans occurs via mechanical means, such as air-conditioning units, taps and showerheads, as well as others listed by the World Health Organization (WHO).

Infection in humans occurs by inhalation of the infectious particle and establishment of infection in the lungs. After ingestion by macrophages, L. pneumophila have been found to inhibit acidification and maturation of its phagosome. Following a 6–10 hour lag period, the bacteria replicate for 10–14 hours until macrophage lysis releases dozens of L. pneumophila progeny.

It’s worth noting that, according to WHO, there is no direct human-to-human transmission of Legionella, which in my opinion is why incidence of LD remains relatively low.

Gardening Can Be Bad for Your Health—No Joke.

Unfortunately, there are other ways of contacting LD besides ingestion of tainted water. At the risk of sounding flippant, gardening can be seriously bad for your health because of contracting LD by breathing in aerosolized Legionella from contaminated soil. This is especially true in New Zealand, which has the highest incidence of LD in the world, according to a recent publication, with L. longbeachae being the most clinically relevant species. This infectious agent is predominantly found in soil and composted plant material. Most cases occur over spring and summer, and the people at greatest risk are those involved in gardening activities.

Taken from lawrieco.com.au

Some agricultural experts advocate smelling soil to assess its quality, stating that “[t]he smell of a soil can often reveal its state of health, sweet or offensive or plain bland,” and adding “the smell does not actually come from the dirt itself, but from soil microbes that inhabit a healthy soil environment. Sweet smelling soil has good levels of organic carbon which is vital to supporting the world of billions of beneficial bacteria and fungi in every cup of healthy soil.”

These soil sniffing experts, however, fail to consider the presence of pathogenic organisms including L. longbeachae. I, for one, will carefully avoid purposefully smelling any soil when gardening, and will instead be sure to wear a good mask capable of filtering out aerosolized Legionella, as you should too!

Nucleic Acid-Based Detection of Legionella

Rapid and effective diagnosis of LD is extremely important so that timely and appropriate therapy can be provided, thereby lowering the morbidity and mortality rates and reducing the health and economic costs associated with this disease. Surprisingly, diagnosis is reportedly established solely by time-consuming microbiological tests. Luckily, it looks like testing procedures could soon change for the better, thanks to PCR and NGS.

Taken from corisbio .com

Earlier this year, Christovam et al. assessed the accuracy of various detection tests in patients suspected of being infected with Legionella and in patients with laboratory-confirmed LD. Investigators analyzed urinary Legionella antigen detection, direct fluorescent antibody (DFA) staining, serological testing and PCR vs. culture analysis (the reference standard). The sensitivity and specificity for PCR were 83 % and 90 %, respectively, whereas DFA sensitivity and specificity were 67 % and 100 %, respectively. Moreover, PCR had high sensitivity and specificity for early diagnosis of LD.

Taken from letsfixit.co.uk

While the study results reported by Christovan seem promising, less definitive results have been reported. Krøjgaard et al., who compared culture and qPCR assays for the detection of Legionella in 84 samples from shower hoses and taps in a residential area before and after two decontaminations. Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc. was said to be “dubious.” However, the rapid detection of high concentrations of Legionella by qPCR was said to be valuable as an indicator of risk, although it may be false positive compared to culture results. Detection of a low number of bacteria by qPCR was said to be a strong indication for the absence of risk.

Not surprisingly, the advent of powerful next-generation sequencing (NGS) is emerging as a better method for genus-specific, sensitive and quantitative determination of Legionella. In 2017, Pereira et al. reported findings from a study using NGS to differentiate 20 pathogenic strains of Legionella in fresh water systems. A genome standard and a mock community consisting of six different Legionella species demonstrated that the reported NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. Comparison of quantification by real-time PCR showed consistency with the NGS data, thus indicating that NGS “provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method.”

Concluding Comments

Aside from providing a brief introduction and update on LD, my additional intent was to alert readers—without undue alarm—to the myriad circumstances in which Legionella can infect humans. According to the aforementioned list provided by WHO, the most common form of transmission of Legionella is inhalation of contaminated aerosols produced in conjunction with water sprays, jets or mists. Infection can also occur by aspiration of contaminated water or ice, particularly in susceptible hospital patients.

Researching transmission of Legionella in Google Scholar led me to find additional information (see links below) that you may find useful or interesting.

Thankfully, as I’ve said before, Legionella is not transmitted human-to-human. The scary aspect of Legionella, however, is that it’s continually mutating, which raises the specter of emergence of a strain that can spread within a human population. Let’s hope that this doesn’t happen and/or that modified mRNA vaccines can be quickly produced to combat that possibility.

As usual, your comments are welcomed.

Addendum

After finishing this blog, there was a Reuters news report on October 9, 2017 that Michigan’s top medical official, Dr. Eden Wells, will be charged with involuntary manslaughter for her role in the city of Flint’s water crisis, which was linked to an outbreak of LD that caused at least 12 deaths. Dr. Eden Wells would become the sixth current or former official to face involuntary manslaughter charges related to this crisis, which principally involved lead contamination in the city’s water supply.

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Your Meals, Wines and Much More are Personalized to Your DNA

  • Personalized DNA Sequencing for Lifestyle Guidance is all the Buzz
  • Vita Mojo Provides Meals to Match Your DNA Codes
  • Vinome Promises to Find Wines for Your Unique Palette
  • SlumberType Claims to Analyse Your DNA and Help You Sleep Better

Among the most significant trends in nucleic acids-based R&D these days is personalized medicine, which uses a person’s DNA sequence (or RNA expression profile) to guide the selection of the best available therapy for that person. This approach is opposite to the traditional strategy for drug development leading to “one treatment regimen for all patients.” Thus, as depicted below, major medical facilities now offer patients personalized cancer therapy based on molecular profiling that features analysis of each patient’s DNA and/or RNA markers.

Taken from pct.mdanderson.org

Scientific studies supporting advantages of nucleic acids-based personalized therapies tailored or customized for each individual are quite compelling, and definitely on the rise, based on my PubMed search results. Given this situation for medical therapies, you might wonder whether personalized nucleic acids-based strategies can be extended to other aspects of human biology, perhaps even to what each of us should eat or drink. Well, such wondering has already been done by others, and has recently led to genetic analyses moving from what I describe as “medicine to mainstream,” as you’ll now read.

Your Meals Personalised to Your DNA

Taken from foodsyoucan.co.uk

A chain of cafes in London is now catering to your body’s every whim—right down to its genetic makeup. Vita Mojo is the first in the world to create meals based on a customer’s DNA profile. It’s an avant-garde part of a huge trend for wellness and healthy eating, an industry worth $3.72 trillion—yes, trillion—across the world, according to a report by the Global Wellness Institute.

Vita Mojo customers first arrange to have their DNA analyzed, and then receive a profile of DNA markers which indicate food groups they should avoid and food groups to eat more of. The gene testing service provided by DNAFit, which was founded in 2013, costs $199 and works like this:

  • You receive a DNA collection kit from DNAFit to provide a saliva sample that is mailed to a company called Helix, which uses Illumina’s Exome+ assay to sequence all 22,000 protein-coding genes, as well as generating additional relevant genomic information.
  • DNAFit interprets your genetic data in terms of fitness or nutrition insights, to help you discover more about yourself and make better informed decisions about your wellness.
  • DNAFit provides you with actionable information about your genetics related to:
    • carbohydrate and fat response
    • lactose tolerance
    • genetic detoxification
    • anti-oxidant and omega-3 needs
    • vitamin B and vitamin D needs
    • alcohol and caffeine response

Most importantly, you also receive a 12-week personalised meal plan, recipes and shopping list. I assume this is the information used by Vita Mojo to provide you with your personalized meals. Vita Mojo, which is backed by the $7 billion French catering company Elior, is reportedly in the midst of raising more capital by crowd-funding, about which I have previously blogged. Helix located in San Carlos, California, lists Illumina among its investors in a deal reported elsewhere. Helix also lists numerous corporate partners, which segues into the next section featuring one of these partners with the clever name Vinome derived from vino (wine) and genome.

Your Wine Personalized to Your DNA

Taken from trendwatching.com

Truth be told, I first came across Vinome not via its partnership with Helix but in a Tweet that caught my attention because I enjoy wine, and follow new applications of sequencing. I was most curious about how drinking wine and sequencing were now being linked. In any case, the Tweet about Vinome led me to do some research about this new startup company that offers to personalize your wine drinking experience through sequencing your DNA, and is appropriately located in Healdsburg, which is in the heart of California’s premier wine country.

Here’s how it works:

  • Vinome cost $109.99, which includes $80 for the Helix test (sequencing of a saliva sample), and $29.99 for your Vinome Profile
  • Vinome analyzes 10 genetic markers related to smell and taste
  • The company then combines these DNA markers with your stated taste preferences to reveal your “vinome,” which is defined by the company as “your unique wine preference profile”
  • You can then join the Vinome wine club or shop its wine store to receive “boutique bottles specifically catered to your vinome results”

Vinome states that over 500 volunteers had their DNA sequenced, and then participated in blinded tasting of a spectrum of wines, answering questions about how much they liked the wines and what flavors they could taste. This data was then compared to DNA genetic markers reported in the scientific literature as important for taste and smell. The participants also answered a detailed questionnaire about their taste preferences for various foods and beverages. Vinome then developed an algorithm that combines this genetic data with taste preference information to deliver its personalized wine recommendations. Vinome works with about 50 boutique wineries and offers bottles generally priced from $18 to $50.

Not everyone, however, has hopped on the consumer sequencing band wagon. One media piece quotes a university professor and researcher as saying that “[i]t’s just completely silly. Their motto of ‘A little science and a lot of fun’ would be more accurately put as ‘No science and a lot of fun.’” Personally, I wouldn’t go so far as to say no science, but I do agree that much more correlative genetic and tasting data needs to be obtained to substantiate Vinome’s claims. Interested readers can later consult an entertaining—to me—account in Business Insider written Lydia Ramsey and titled I took a DNA test that claims to reveal the best wine for you — here’s the verdict.

Your Lifestyle Personalized to Your DNA

While researching the above stories, I happened to receive an email advertisement about Exploragen, which was described as “a new DNA lifestyle company to deliver useful DNA-based apps directly to consumers.” It went on to say that the apps from this California startup, which also utilizes the Helix platform, represent “the first online marketplace of DNA-powered consumer products [to] monitor sleep patterns and caffeine metabolism, optimize fitness, personalize cosmetics and much more.”

Taken from livescience.com

If you check out Exploragen’s website, as I did, you’ll find that the latter statements are somewhat misleading because the only app currently available is called SlumberType. This app is stated to “improve your nights and change how you feel during the day” by discovering how your DNA influences your sleep habits such as the quality of sleep and how long it takes you to fall asleep and stay asleep. SlumberType also promises to help you ‘find out how your sleep DNA relates to your diet, productivity, exercise, and caffeine consumption’.

I found some details about how SlumberType works buried in the FAQs on the website. SlumberType checks genetic variants that have been shown to be associated with sleep traits, including how long it takes you to fall asleep, how long you stay asleep, the quality of your sleep, and your genetic similarity to self-reported “morning” or “evening” persons. The SlumberType app is said to make it “simple for you to record your sleep/wake times, your morning and evening mood—plus other factors you choose—with just a few taps.” One important stated caveat is that genetic associations used by this product were originally discovered in European populations and may or may not be applicable to people from a different background.

Visitors to the website are encouraged to stay in touch as more apps are supposedly coming soon. While all of this sounds interesting, and potentially useful for some persons, I’m not convinced there will be enough early adopters to sustain Exploragen’s business model, but that’s just my humble opinion.

Closing Comments

Vinome’s use of genetic markers related to smell and taste led me to research this topic, and in so doing I found a lengthy scientific review article by Reed & Knaapila. This article is well worth a quick read to get a sense—pun intended—of what’s known about genetic markers and our senses.

In a nutshell, it’s a very complicated story because of the complexities and differences in sensory perceptions among individuals. To this point, I found the following hypothetical analysis by Reed & Knaapila to be a good example of how taste and smell genotypes may contribute to different perceptions of the same food (in this case a ham and cheese sandwich containing bread, onion, tomato, watercress, cheese, and ham).

Taken from Reed & Knaapila Prog Mol Biol Transl Sci (2012)

In this hypothetical case, sucrose in the onion will be detected by sweet receptors on the tongue, TAS1R3; glutamate in the tomato (perceived as a savory or umami taste) is sensed by the umami receptor, TAS1R3; bitterness of watercress is due to isothiocyantes detected by bitter receptors, TAS2R38; isovaleric acid in cheese has a “sweaty” odor detected by an olfactory receptor, OR11H7; ham contains androstenone having an odor called boar taint detected by OR7D4.

People with two positive alleles (+/+) perceive the compounds better than people with two negative alleles (−/−). Person 1 can taste the pleasant sweetness of the onion and the umami of the tomato but does not perceive the bitterness of the watercress or the unpleasant odors of the cheese or ham. Thus, Person 1 likes the ham sandwich more than Person 2.

Importantly, in my opinion, Reed & Knaapila note that “[p]eople eat what they like, but they also eat for many other reasons. Simple explanations of the links between sensory perception and food intake are misguided: Just as people do not choose art or music based solely on how well they can hear or see, we do not choose food based solely on the reactions of the tongue or nose. Although genetic differences determine what we can taste and smell (and at what concentration), our taste is ultimately determined by our experiences, learning, and culture, in an artistic sense, as well as in our likes and dislikes of food and drink.”

Bon appétit and à votre santé!

As usual, your comments are welcomed.

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Jerry’s Favs from the Recent 7th Cambridge Symposium

  • DNA Can Function as an Enzyme
  • RNA Polymerase Activity Without Proteins
  • Systemic Brain Delivery of Therapeutic Oligos

The 7th Cambridge Symposium on Nucleic Acids Chemistry and Biology took place September 3-6, 2017 at historic Queens’ College, Cambridge, which was founded in 1448 by Margaret of Anjou (who was then Queen of England by marriage to King Henry VI). Yours truly had the honor of participating in this event and presenting one of TriLink’s posters on the company’s new types of chemically modified mRNA for mRNA therapeutics. As done for other conferences I’ve attended on behalf of TriLink, I wish to share here my personal favorites among the many lectures, which are still fresh in my mind. However, I hasten to emphasize that, while choosing these “favs” is biased a bit by my scientific interests, all the lectures topics are worth looking at later by perusal of the symposium program of nearly forty presentations.

Taken from na-cb.co.uk

By the way, the symposium’s logo artistically depicts a DNA helix under a wooden bridge, better seen in the accompanying picture of the actual bridge over the River Cam at Queens’ College. Built in 1749, it has become known as the Mathematical Bridge for reasons you can read later, and appears to be an arch but is composed entirely of straight timbers. The historical connection between the DNA double helix and Cambridge is that in 1953 Watson & Crick proposed this now famous deoxynucleic acid structure as the molecular basis for genetics, which I’ll comment on again at the end of this post.

Taken from worldachitecture.org

Overview of the Symposium

Mike Gait. Taken from histmodbiomed.org

This symposium is the 7th in a popular conference series going way back to 1981 that brings together nucleic acids scientists across a broad area but with emphasis on chemistry, biochemistry and structure. Michael (Mike) Gait, who is at the Medical Research Council Laboratory of Molecular Biology in Cambridge, originated this series and has been a key organizer for all seven conferences. Participants come from all over the world and include professors, students, and companies—as well as Nobel Laureates (this year Jack Szostak of telomeres fame).

In addition to Mike, the organizing committee included Sir Shankar Balasubramanian, who was recently knighted for his contributions to next-generation sequencing and research on G-quadruplexes, the latter of which I featured here a few years ago. Other committee members were Rick Cosstick, Phil Holliger, and Chris Lowe.

Subject areas this year included:

  • Nucleic acids as therapeutics (including antisense, RNAi, aptamers, immune recognition, cell delivery)
  • RNA and DNA structures and their protein complexes (duplexes, quadruplexes, RNA and DNA enzymes, riboswitches, protein complexes + assemblies)
  • Nucleic acids chemistry applied to cells and cell mechanisms (genomes, evolution, repair, cell manipulation)
  • Nucleic acids as tools, structural assemblies and devices (nanostructures, cages, arrays, supra-molecular chemistry)

Marv Caruthers. Taken from colorado.edu

The showcased and highly prestigious Nucleic Acids Award was presented to Marvin (“Marv”) Caruthers in recognition of his seminal contributions to the synthesis of oligodeoxynucleotides (aka “oligos”) based on the use of phosphoramidite chemistry. This mechanistically elegant chemistry enabled much faster and more efficient coupling for automated synthesis of oligos, which fundamentally transformed all manner of basic and applied research with DNA. His award lecture was titled Synthesis, Biochemistry, and Biology of New DNA Analogues, some of which has been recently published. My previous several posts commenting on Marv, who has been a professor at the University of Colorado in Boulder since 1973, can be read later here.

Jerry’s Favs from the Symposium

To encourage inclusion of unpublished results and other types of “late breaking news” from the lab, the organizers forbade use of Twitter or other real-time social media, blogging, or taking pictures of slides being shown. Consequently, what I can say here is restricted to published papers related to my favs. Keeping this limitation in mind, here are my personal top-three talks that I consider to be tied (i.e., have equivalent scientific importance).

DNA Can Function as an Enzyme!

This lecture by Scott Silverman at the University of Illinois, Urbana-Champaign, dealt with DNA enzymes (aka deoxyribozymes), which were first reported in 1996 by Carmi et al., and are of interest because they expand enzymatic functionality from naturally occurring proteins to synthetic nucleic acids. DNA enzymes can be evolved in vitro starting with random sequences of DNA and applying suitable selection.

In a published account titled Pursuing DNA Catalysts for Protein Modification, Silverman has provided a lengthy and chemically detailed description of his use of in vitro selection to develop DNA catalysts for many different covalent modification reactions of peptide and protein substrates. While interested readers can consult Silverman’s account for various examples, it’s illustrative to consider the molecular design strategy depicted below that was used to evolve a synthetic DNA functional-equivalent of naturally occurring protein kinases that, by definition, carry out protein phosphorylation.

Taken from Silverman Acc Chem Res (2015)

In this case, modular deoxyribozyme design involved a stretch of 40 randomized bases (N = A/G/C/T) having a hairpin loop conjugated to a tyrosine (Tyr)-containing peptide on one end, and an ATP-binding aptamer on the other end. This was intended to experimentally assess whether it would be helpful to provide a predetermined small-molecule binding site in the form of an aptamer, which would cooperate functionally with an initially random catalytic region (N40) from the onset of selection. The selection outcome established that while modular deoxyribozymes that utilize a distinct predefined aptamer domain can indeed be identified, such DNA catalysts do not have any functional advantage relative to nonmodular analogues selected simultaneously for binding and catalysis, at least for this test case of tyrosine kinase activity using an ATP phosphoryl donor.

RNA Polymerase Activity Without Proteins!

By analogy to use of in vitro selection to evolve DNA enzymes from complex pools of random sequences of DNA, complex mixtures of unrelated RNA sequences can also be subjected to in vitro selection to evolve RNA enzymes (aka ribozymes). Indeed, as noted and cited in a talk by co-organizer Philipp Holliger, the emergence of an RNA catalyst capable of self-replication is considered a key transition in the origin of life in the prebiotic “RNA World” first hypothesized by Walter Gilbert in the 1980s. How such self-replicating (replicase) ribozymes emerged from the pools of short RNA oligomers arising from prebiotic chemistry and non-enzymatic replication, however, is unclear.

In a published version of Holliger’s talk addressing this important open question, his laboratory carried out an elegant series of experiments demonstrating that RNA polymerase ribozymes can assemble from catalytic networks of RNA oligomers that are each no longer than 30 nucleotides. Additionally, they found that entropically disfavored assembly reactions are driven by iterative freeze-thaw cycles. Such cooling (to freeze)-warming (to melt) cycles for aqueous solutions of RNA oligo reactants are notionally opposite to heating (to dissociate)-cooling (to hybridize) cycles used for amplification by PCR.

Interested readers can peruse Holliger’s publication for details about these novel findings, but for the purposes of this blog the schematic shown below depicts and describes the mechanism for assembly wherein relatively short RNA oligomers undergo serial ligations and “grow” into a self-replicating RNA polymerase. To me, these results provide an amazing glimpse backward in time to how the RNA World may have evolved!

Assembly of a RNA polymerase ribozyme (RPR 1234) from oligonucleotides devoid of pre-activation. (a) Schematic representation of the assembly trajectory involving (anti-clockwise from top left), ribozyme (blue) cleavage of a short 3′ tail (red) generating a 2′, 3′ cyclic phosphate (>p) (red dot), dissociation of the cleaved tail and strand exchange to cognate substrate (orange) followed by ligation of substrate 5′ OH with >p. (b) Network diagram of RPR 1234 assembly from 4 tailed fragments 1, 2, 3 and 4. Tailed input fragments can ligate to their cognate 5′fragments but must be cleaved (red lines) before ligation to 3′fragments. Taken from Holliger Nat Chem (2015).

Systemic Brain Delivery of Therapeutic Oligos!

Taken from igtrcn.org

A talk by Fazel Shabanpoor titled Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy described work that he had just published with a group of collaborators that included symposium co-organizer Mike Gait, whose lab interests have recently focused on cell-penetrating peptides. This was a fav for me because it had multiple interesting elements: (1.) systemic brain delivery, which is a widely recognized challenge; (2.) “weirdly” structured morpholino oligos, which have backbone structures quite unlike DNA that I’ve commented on here previously; and (3.) splice-switching antisense oligos (SSOs). The latter class of molecules (SSOs) base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Readers interested in SSOs—currently a “hot topic”—can consult a recent comprehensive review, while SSOs for spinal muscular atrophy (SMA) has been featured in previous blog here.

Shabanpoor’s lecture highlighted the fact that development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, Shabanpoor et al. investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching 20-mer phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. They identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript.

Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. It was concluded that this work provides proof of principle for the ability to select new “peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform” for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.

Parting Thoughts and The Eagle

I hope that you can now appreciate why these three lectures were my favs from the 7th Cambridge Symposium. Silverman’s conversion of DNA into an enzyme that can phosphorylate a protein is an exciting demonstration of the power of bio-organic chemistry to manipulate DNA to do things it can’t do naturally. Holliger’s demonstration of how RNA polymerase ribozymes may have evolved gives credence to the RNA World hypothesis, and indicates that this supposed prebiotic environment may have provided critical freezing and thawing cycles over many millennia of molecular evolution. In the present biotic world, humans afflicted with neuromuscular and neurodegenerative diseases may benefit from Shabanpoor & Gaits’ new peptide paradigms to enhance CNS delivery and activity of therapeutic oligos.

In conclusion, I should mention that researchers who work with DNA will invariably visit The Eagle when in Cambridge, which is a pub only a short walk from Queens’ College. This pub, which dates back to 1667, is quite famous because it is known with certainty to be the place where Francis Crick interrupted patrons’ lunchtime on February 28, 1953 to announce that he and James Watson had ‘discovered the secret of life’ after they had come up with their proposal for the structure of DNA. Today the pub serves a special ale dubbed “Eagle’s DNA” to commemorate the discovery. Trust me when I say that this ale is mighty tasty, because I enjoyed a pint of it, and while standing in the que for that brew, was inspired to capture this image to share here.

As usual, your comments are welcomed.

Personal photo using a Samsung Galaxy S8

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Sniffing Out Prostate Cancer

  • Current Prostate-Specific Antigen (PSA) Tests Are Less than 25% Accurate
  • Trained “Sniffer” Dogs Detect Prostate Cancer-Specific VOCs in Urine with 98% Accuracy
  • Researchers Hope to Analyze VOCs using Gas Chromatography and Validate Results with Canine Studies to Develop Highly Accurate, Non-Invasive Tests for Prostate Cancer

We’re all familiar with news events involving dogs specifically trained to sniff out fugitives, explosives, or cocaine (among other things). This remarkable canine ability is due to some fascinating olfactory factoids. A dog’s sense of smell is a thousand times more sensitive than that of humans, and a dog has more than 220 million olfactory receptors in its nose, while humans have only 5 million.

Taken from finearts. com

Interestingly—if not amazingly—canine sniffing sensitivity has been investigated as a novel means of detecting cancer by simply smelling urine, or more accurately, smelling volatile organic compounds (VOCs) emitted from a urine sample. The following sections provide synopses of some notable publications dealing with new and improved methods for detection of prostate cancer. While reading this post, keep in mind that the principle here is analogous to humans being able to smell a distinctive VOC in their urine after eating asparagus, which is due to formation of volatile asparagusic acid. But I digress…

Dogs Sniff Out Prostate Cancer

The Problem: Prostate cancer represents the fifth most frequent cancer in the world, and according to current CDC statistics is still the number one killer of men in the US, followed by lung and colon cancers, as shown in the chart below.

Taken from health.am

Prostate-specific antigen (PSA) testing is currently used for detection of prostate cancer. Details of the testing process can be read elsewhere, but I’ll briefly describe the steps of the exemplary assay depicted below. In the first incubation phase, specific autoantibodies (present in the sample, calibrators or controls) bind to the immobilized antigen. In the second incubation phase, the dimethyl acridinium ester (DMAE) conjugate reacts with the coated magnetic particle-autoantibody complexes. Non-bound material is washed away after every incubation step, and chemiluminescence is activated by the addition of “trigger” solutions (hydrogen peroxide and an alkali) resulting in oxidation of the ester to a photo-excited form. Return to a stable state is accompanied by the emission of light, which is measured and expressed in Relative Light Units (RLU). A direct relationship exists between the amount of total PSA in the sample and the RLUs detected.

Taken from en.menarinidiagnostics.fr

Since PSA testing is a great tool for the detection of prostate cancer, there is a strong need for more accurate tests. According to an NIH fact sheet, approximately 75% of men who have prostate biopsies due to elevated PSA levels DO NOT have prostate cancer. In fact, over 1 million unnecessary prostate biopsies will be performed in the US this year alone. Reported costs for this biopsy procedure range from $1,500-$6,000, resulting in billions of wasted dollars each year. Moreover, this huge false positive PSA rate exposes millions of men worldwide to an invasive procedure that has risks including sepsis and death.

Taken from pinterest. com

A Canine Solution? In 1989, Williams & Pembroke provided the first evidence for sniffer dogs that could detect VOCs from melanoma cancer in human urine samples. Fast forwarding to 2014, Taverna et al. reported that the olfactory system of highly trained dogs detects prostate cancer in urine samples. Two 3-year-old female German Shepherd explosion-detection dogs were trained to identify prostate cancer-specific VOCs in urine samples from 362 patients with prostate cancer (low-risk to metastatic) and on 540 healthy controls free of any kind of cancer.

Amazingly, dog 1 sensitivity was 100% and specificity was 98.7%, while for dog 2 sensitivity was 98.6% and specificity was 97.6%. Analysis of the few false-positive cases revealed no consistent pattern in participant demographics or tumor characteristics. It was concluded that “[a] trained canine olfactory system can detect prostate cancer specific VOCs in urine samples with high estimated sensitivity and specificity. Further studies are needed to investigate the potential predictive value of this procedure to identify prostate cancer.” While I did not find any such confirmatory follow-up studies with trained dogs, I did find the following investigations of non-canine alternatives to PSA. Interestingly, one of these studies is based on the knowledge that dogs can accurately detect VOCs in urine and the study plans to validate its results with canine studies.

Taken from chromedia.org

A Chromatographic Solution? As a chemist, its seemed reasonable to me to assume that state-of-the-art separation technology could be applied to VOC analysis to develop a more practical and reproducible replacement of PSA tests. I was gratified to find one such report in 2016 by a British research team that used gas chromatography (GC), depicted below, which is commonly employed for separation and detection of smallish, volatile molecules such as VOCs.

Dubbed “Odoreader,” the GC system was developed by a team led by Chris Probert from the University of Liverpool’s Institute of Translational Medicine and Norman Ratcliffe from the University of the West of England in Bristol. The researchers tested the Odoreader on 155 men presenting to urology clinics, of which 58 were diagnosed with prostate cancer, 24 with bladder cancer and 73 with hematuria or weak urine stream without cancer.

For prostate cancer diagnosis, this GC equipped with an automated data analysis system classified samples with 95% sensitivity and 96% specificity, while for bladder cancer diagnosis, the system had 96% sensitivity and 100% specificity. It was concluded that the results of this pilot study “indicate that the GC system is able to successfully identify patterns that allow classification of urine samples from patients with urological cancers,” adding that “larger cohort studies are planned to investigate the potential of this system.”

Not surprisingly, these very promising results have prompted others to investigate analogous GC methods capable of elucidating the structures of key molecules in the mixture of VOCs associated with prostate cancer. Mangilal Agrawal at Indiana University, together with his postdoc, Amanda Siegel, are doing so by coupling the power of GC to separate molecules and the power of mass spectrometry to identify molecules. Then they plan to validate these biomarkers with canine studies much like the one discussed above. Once validated, they will use the biomarkers to develop a non-invasive ‘strip sensor’ or dipstick test that can be used at doctors’ offices to detect the presence of prostate-specific VOCs. They presented preliminary findings using this GC/MS technology at the 2017 National American Chemical Society Meeting in a 15-minute press release video session with Q&A that I watched with interest.

In conclusion, my hope is that these GC based methods, in combination with continued canine studies, will soon lead to much more accurate strip sensor tests to replace PSA testing. These more accurate tests will benefit millions of men around the world by avoiding unnecessary prostate biopsies, and reduce health care costs.

As usual, your comments are welcomed.

Addendum

Taken from Deng et al.

Aptamers (which I’ve blogged about previously and can be prepared from randomized oligonucleotide libraries from TriLink), are also being extensively investigated as potentially more specific prostate cancer detection tests than antibody-based immunoassays. One recent example reported by Deng et al. is depicted below. Basically, a three-layer core–shell nanostructure consisting of a silver core, a silica spacer, and a fluorescent dye RuBpy-doped outer silica layer was fabricated, and allows metal-enhanced fluorescence (MEF). A target-triggered MEF ‘turn-on’ strategy based on the optimized composite nanoparticles was successfully constructed for quantitative detection of prostate specific antigen (PSA), by using RuBpy as the energy donor and BHQ-2 as the acceptor. The hybridization of the complementary DNA of PSA-aptamer immobilized on the surface of the MEF nanoparticles with PSA-aptamer modified with BHQ-2, brought BHQ-2 in close proximity to RuBpy-doped silica shell and resulted in the decrease of fluorescence. In the presence of target PSA molecules, the BHQ-PSA aptamer is dissociated from the surface of the nanoparticles with the fluorescence switched on.

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