Xeno Nucleic Acids (XNAs) Revisited

  • X (‘Xeno’) in XNAs Are Structurally Strange Congeners of DNA or RNA
  • Liu et al. Report X = 3→2Phosphonomethylthreosyl
  • Xenobiology Is an Emerging and Intriguing New Frontier

In scientific nomenclature, ‘xeno‘ is used to indicate something strange or different relative to what exists in nature, and is derived from the Greek word xenos for strange. Xeno nucleic acids (XNAs), reviewed here, are intriguingly “strange” synthetic polymers. They have natural nucleobases (A/G/C/T or U) for encoding genetic information like DNA or RNA, but their backbones are different from the naturally occurring ribose and phosphodiester moieties.  A/G/C/T- or U-bearing XNAs can therefore be thought of as building blocks for non-natural nucleic acid polymers.

Several years ago, I blogged about XNAs in the context of the evolution of DNA/RNA-based life and genetics within the prebiotic universe, which is generally assumed to have existed ~4 billion years ago. All studies of XNAs have previously been limited to model systems in vitro, which begs the question of whether any type of XNA molecule can genetically function in vivo. By reading on, you’ll learn that the answer is yes, as recently reported by Liu et al., and I think you’ll be very surprised by the “strangeness” of the particular XNA found to perform this remarkable feat.

Backstory

Adapted from Anosova et al. Nucleic Acids Res.44, 1007–1021 (2016)

To put the fascinating findings of Liu et al. into perspective, here’s a brief synopsis of the backstory involving the earlier work of others on (L)-α-threofuranosyl (aka threose)-based nucleic acid (TNA), which is shown below and represents the most advanced example of an XNA. The seminal work on TNAs containing opposingly connected (3’→2’) phosphodiester bridges was published in Sciencemagazine in 2000 by a team led by Albert Eschenmoser, who was already famous for his part in the prodigious total synthesis of vitamin B12, which took 12 years!

These researchers reported that TNAs undergo “informational base pairing in antiparallel strand orientation” (i.e. Watson-Crick hybridization), and are capable of cross-pairing with RNA and DNA. Derived from a sugar containing only four carbons vs. five carbons for ribose, TNA was posited as the structurally simplest of all naturally derived XNAs.

Follow-on investigations by Chaput and coworkers led to a publication in 2013 that detailed the surprising ability of TNA to be transcribed from DNA by an available polymerase and then reverse transcribed back into DNA using another available polymerase. The transcription step required chemical synthesis of the four TNA triphosphates (tNTPs) for incorporation by a DNA primer-template complex (black), as depicted here.

Adapted with permission from Chaput and coworkers J. Am. Chem. Soc. 135, 3583−3591 (2013)

More specifically, the DNA primer was annealed to the DNA template in buffer by heating it at 95 °C for 5 min and then cooling it on ice. Primer extension reactions to produce TNA (red) were performed for 1 h at 55 °C using 100 μM tNTPs, 500 nM primer−template complex, 1.25 mM MnCl2, and 0.1 U/μL Therminator™ DNA polymerase. Product analysis used denaturing polyacrylamide gel electrophoresis (PAGE).

Repetition of the above primer extension reaction was followed by strand separation using gel electrophoresis, as depicted below. Extraction of the gel afforded single-stranded DNA (black)-TNA (red) product to which a reverse complementary primer was annealed. SuperScript II™ (SSII)-mediated TNA reverse transcription (RT) was performed for 24 h at 42 °C and analyzed by denaturing PAGE. Mn2+is required to convert TNA into full-length cDNA.

Adapted with permission from Chaput and coworkers J. Am. Chem. Soc. 135, 3583−3591 (2013)

Fidelity of TNA Replication

Chaput and coworkers measured the fidelity of TNA replication by sequencing the cDNA product of the RT reaction after amplification by PCR. This fidelity assay measures the aggregate fidelity of a complete replication cycle (DNA → TNA → DNA), which is operationally different than the accuracy of a single-nucleotide incorporation event. The fidelity determined by this assay is the actual accuracy with which full-length TNA is synthesized and reverse transcribed. Therefore, it reflects the combined effects of nucleotide misincorporation, insertions and deletions (indel), and any mutations that occur during PCR amplification and cloning.

The researchers began by measuring the fidelity of TNA replication for a template (4NT.3G) used in the RT assay with SSII. While TNA replication on 4NT.3G resulted in an overall fidelity that was comparable with other XNA replication systems (~96%), detailed analysis of the mutation profile indicated that G → C transversions account for 90% of the genetic changes. While the precise molecular details of this transversion remain unknown, other results were said to suggest that base stacking plays an important role in the misincorporation of tGTP opposite dG in the template.

Functional XNA In Vivo

As mentioned in the introduction, Liu et al. have recently reported results that demonstrate the first-ever XNA to functionin vivo, thus paving the way for future developments in this intriguing unexplored area. The other novel aspect of their work is the use of a backboned-modified XNA, namely, 3′→2′phosphonomethylthreosyl nucleic acid (tPhoNA). This XNA can be seen below, and is quite different from DNA and TNA. I will now discuss both the in vitroand in vivostudies in this landmark publication.

Adapted with permission from Liu et al. J. Amer. Chem. Soc.140, 6690−6699 (2018)

The initial part of this work deals with the synthesis of the four “triphosphate”-like monomers [PMTApp etc. (PMTNpp) shown here] required for polymerase-mediated transcription reactions analogous to those described above for TNA. Preliminary experiments with Therminator™ led to inadequate transcription and thus to extensive site-directed engineering in order to find an alternative mutant polymerase with acceptable kinetics and processivity. Starting with TgoT, which is a Thermococcus gorgonarius DNA polymerase variant, researchers obtained a TgoT mutant (TgoT EPFLH), an efficient tPhoNA synthetase capable of synthesizing a 57-mer from a DNA template in less than 5 minutes.

Next, they screened the available reverse transcriptases (RTs) that could synthesize DNA against TNA templates and found two (mutants of TgoT and KOD DNA polymerase) that were efficient tPhoNA RTs capable of DNA synthesis from both DNA and RNA primers. Together, the TgoT EPFLH and mutant RTs tested enabled Liu et al. to transfer genetic information from DNA into tPhoNA and recover that information back to DNA with an approximate aggregate error rate of ~20 × 10−3per incorporation, which is a degree of fidelity compatible with the development of aptamers and aptazymes based on this new chemistry.

To evaluate the ability of tPhoNAs as templates for DNA synthesis in E. coli, six 18-mer 5′-phosphorylated DNA−PhoNA−DNA chimera oligonucleotides were chemically synthesized and tested using the established gapped-vector assay, based on the restoration of the active site of thymidylate synthase (thyA) depicted here.

Adapted with permission from Liu et al. J. Amer. Chem. Soc.140, 6690−6699 (2018)

This enzyme catalyzes the conversion of deoxyuridine monophosphate to thymidine monophosphate and is essential for E. coli growth in a medium lacking either thymine or thymidine. The six 18-mer 5′-phosphorylated DNA−PhoNA−DNA chimera oligonucleotides were ligated into a gapped heteroduplex plasmid, where 6 codons surrounding the catalytic Cys146 of the thyA gene had been deleted. The resulting ligation products were then transformed into an E. colistrain lacking thyA. Transformants are able to survive in thymidine-free media only when a PhoNA oligonucleotide chimera is recognized by the bacterial replication machinery and utilized as a template for DNA synthesis, restoring the thymidylate synthase active site. The ratio between bacterial colony numbers in media with or without thymidine indicates the extent of successful templating.

The replacement of a DNA unit by a tPhoNA building block yielded a 2.5-fold decrease in the number of prototrophic transformants compared to the positive controls. Further 2- and 6.5-fold drops in the yield of prototrophic transformants resulted from an extension of the tPhoNA stretch from one to two and three oligonucleotides, respectively. Thus, the successive addition of tPhoNA nucleotides significantly diminished DNA propagation in vivo.

Conclusions by Liu et al.

For in vivo applications, it is desirable that XNAs are both chemically and biologically orthogonal: neither the polymers nor the building blocks interact with natural nucleic acids or proteins, and XNA-synthesizing and -binding proteins do not synthesize or bind natural nucleic acids. The tPhoNAs showed significant levels of orthogonality at both chemical (oligonucleotide properties) and biological (recognition by natural protein) levels. The melting analyses (see Liu et al.) demonstrated that heavily modified tPhoNA did not exhibit detectable hybridization to complementary DNA or RNA, but did retain some potential to form homoduplexes, at least for AT-rich sequences.

tPhoNA also showed signs of biological orthogonality. The four PMTNpp’s were demonstrably poor substrates for natural polymerases, yet tPhoNA could be efficiently synthesized by the engineered polymerase TgoT_EPFLH. Importantly, as Liu et al. engineered TgoT for better tPhoNA synthesis, they observed a noticeable drop in its DNA synthetase function. Given the orthogonality demonstrated both chemically (oligonucleotide annealing) and in vivo(transliteration), Liu et al. suggested that it is likely that TgoT_EPFLH’s broadened substrate specificity can be further engineered to develop an orthogonal polymerase.

Liu et al. concluded that, collectively, the data further suggest that a fully orthogonal genetic system based on tPhoNA and specialist tPhoNA polymerases with minimal interaction with natural dNTPs, nucleic acids, or polymerases, is very much achievable.

Xenobiology: Quo Vadis?

Xenobiology (XB) has been defined as a subfield of synthetic biology involving the study of synthesizing and manipulating biological devices and systems using XNAs. In my opinion, XB is as an emerging branch of non-natural biology in search of utility. Consequently, the application of XB as the “ultimate biosafety tool,” as proposed by iGEM students, is quite intriguing. I should note here that iGEM, which stands for International Genetically Engineered Machine Competition, is intriguing in its own right, and well worth reading about later at this link.

The stated iGEM students’ premise for this ultimate biosafety tool is that the wide use of genetically modified organisms (GMOs) has caused serious concerns on how GMOs will interact with the natural environment. In particular, could a genetically modified microbe escape its constraints, and outcompete organisms found in the natural ecosystem? Since the early days of genetic engineering, biosafety strategies have been employed in order to control risks, and the advent of high-throughput synthetic biology is bringing these concerns to another level: the more we tinker with biology, the more our biosafety needs to be bullet-proof.

The stated aim of the iGEM students is to create a synthetic “man-made” version of biology, that respects the definition of life, but is based on entirely different mechanisms to function. The biochemistry of a xeno-organism uses new XNAs, genetic codes, and cofactors different from those explored in biology, and is therefore incompatible with other forms of life. This allows a much higher level of control: a xeno-organism will not be able to find the xenocompounds in the natural environment nor will it be able to use bacterial communication systems.

Only time will tell whether this technically challenging but valuable goal is achieved. I for one hope so, and, after writing and reflecting on this blog, will add that I’m amazed by the increasing breadth and depth of modified nucleic acid chemistry reflected in XNAs. It seems that the diversity of XNA chemical structures, which has been recently reviewedhere, can still be expanded further. Indeed, the findings of Liu et al. for tPhoNAs point the way to orthogonal genetic systems, and perhaps to the iGEM application mentioned here.

As usual, your comments are welcomed.

An Antisense Oligonucleotide Is the First Drug to Demonstrate Reduction of Mutant Huntingtin Protein in Humans

  • Mutant Huntingtin Protein Causes Huntington Disease (HD), Which Afflicts 30,000 People in the U.S.
  • More Than 200,000 People at Risk of Inheriting HD in the U.S.
  • Developed by Ionis Pharmaceuticals, This Antisense Drug Will Undergo Pivotal Clinical Trials Conducted by Roche

Credit: A Luna Blue

My blog from August 7th, 2018, heralded the clinical efficacy of two oligonucleotide drugs for transthyretin-related amyloidosis. One is an antisense oligonucleotide (ASO) drug, and the other is a short-interfering RNA drug. Five days before this notable achievement, Ionis Pharmaceuticals announced equally important news, stating that the European Medicines Agency granted accelerated review timelines for an ASO (IONIS-HTTRx) for the treatment of people with Huntington’s disease (HD), a neurodegenerative illness.

IONIS-HTTRx is the first drug to demonstrate reduction of mutant huntingtin protein, the underlying cause of HD, which is the focus of the present blog. It should be noted that Ionis and Roche have a long-standing alliance when it comes to HD, under which IONIS-HTTRx (designated RG6042 by Roche) will be evaluated in a pivotal study of a larger patient population to further characterize its safety and efficacy profile in adults with HD.

Basic Facts About HD

Description: According to the NIH, HD (aka Huntington’s disease or Huntington’s chorea) is a progressive brain disorder that causes uncontrolled movements, emotional problems, and loss of thinking ability (cognition). Adult-onset HD, the most common form of this disorder, usually appears in a person’s 30s or 40s. Early signs and symptoms can include irritability, depression, small involuntary movements, poor coordination, and trouble learning new information or making decisions. Affected individuals may have trouble walking, speaking, and swallowing. People with HD also experience changes in personality and a decline in thinking and reasoning abilities. Individuals with the adult-onset form of HD usually live about 15 to 20 years after signs and symptoms begin.

A less common form of HD known as the juvenile form begins in childhood or adolescence. It also involves movement problems and mental and emotional changes. Additional signs of the juvenile form include slow movements, clumsiness, frequent falling, rigidity, slurred speech, and drooling. Juvenile HD tends to progress more quickly than the adult-onset form; affected individuals usually live 10 to 15 years after signs and symptoms appear.

Frequency: HD affects an estimated 3 to 7 per 100,000 people of European ancestry. The disorder appears to be less common in some other populations, including people of Japanese, Chinese, and African descent.

Causes: As depicted here, mutations in the HD gene/HTT gene on chromosome 4 cause Huntington disease. The HTT gene provides instructions for making a protein called huntingtin. Although the function of this protein is unknown, it appears to play an important role in nerve cells (neurons) in the brain, primarily a group of nerve cells at the base of the brain known collectively as the basal ganglia.

Credit: Meletios Verras

The HTT mutation that causes HD involves a DNA segment known as a CAG trinucleotide repeat. Normally, the CAG segment is repeated 10 to 35 times within the gene. In people with HD, the CAG segment is repeated 36 to more than 120 times. People with 36 to 39 CAG repeats may or may not develop the signs and symptoms of Huntington disease, while people with 40 or more repeats almost always develop the disorder.

Neuron structure

CAG codes for glutamine, therefore an increase in the size of the CAG segment leads to the production of an abnormally long version of the huntingtin protein. The elongated protein is cut into smaller, toxic fragments that bind together and accumulate in neurons, disrupting the normal functions of these cells. The dysfunction and eventual death of neurons in certain areas of the brain underlie the signs and symptoms of HD.

Inheritance: As depicted here, HD is inherited in an autosomal dominant pattern wherein one copy of the altered gene in each cell is sufficient to cause the disorder. An affected person usually inherits the altered gene from one affected parent. In rare cases, an individual with HD does not have a parent with the disorder.

As the altered HTT gene is passed from one generation to the next, the CAG trinucleotide repeat often increases in size. A larger number of repeats is usually associated with an earlier onset of signs and symptoms. People with the adult-onset form of HD typically have 40 to 50 CAG repeats in the HTT gene, while people with the juvenile form of the disorder tend to have more than 60 CAG repeats.

Individuals who have 27 to 35 CAG repeats in the HTT gene do not develop Huntington disease, but they are at risk of having children who will develop the disorder. As the gene is passed from parent to child, the size of the CAG trinucleotide repeat may lengthen into the range associated with HD (36 repeats or more).

Genetic Testing: There are 64 available genetic tests for HD listed at a NIH website that you can access here. Among these 64, I am most interested in the 7 tests described as “sequence analysis of the entire coding region,” which to me seem to be the most definitive approach. Of these 7 tests, I clicked on this link for the test named “HTT,” which was the only one of these tests offered by a U.S.-based company, namely, Fulgent Genetics near Los Angeles, CA. While this HTT test is described as using “massively parallel sequencing,” which Fulgent specifies as an Illumina® test, this test’s clinical validity and clinical utility are described as “not provided.” I assume this means that this HTT test can be ordered by a doctor for informational purposes. In any case, there is a tab given for “How To Order” that interested readers can consult.

PCR aficionados will recognize HHT’s CAG repeats to be GC-rich, making them difficult to faithfully amplify on Illumina platforms or through other types of ensemble sequencing methods. However, this can be mitigated by using TriLink CleanAmp® 7-deaza-dGTP. Amplification-free methodology is also available. For example, a poster abstract by Pacific Biosystems (PacBio) describes how a novel approach using CRISPR/Cas9 for specific targeting of individual human genes, followed by PacBio’s single-molecule long-read sequencing methods, enables sequencing of complex genomic regions that cannot be investigated with other technologies. HTT CAG repeat-regions were successfully sequencing in this manner.

ASO Studies Targeting Huntington

Use of an ASO to interfere with the expression of mutant HTT can be traced back to 20 years ago, through the following series of publications by various research groups:

Of these, interested readers can consult the last item by Kordasiewicz et al. (2012); a lengthy, detailed report by collaborators at four sites, including Ionis (then named Isis Pharmaceuticals). In brief, this study demonstrated that transient infusion of ASOs into the cerebral spinal fluid of symptomatic HD mouse models not only delays disease progression, but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal.

Rhesus monkey eating

Similar ASO infusion into non-human primates (Rhesus monkeys) was shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, these findings established a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.

Finally, Kordasiewicz et al. note that huntingtin is reportedly essential for one or more early developmental steps. However, no evidence to date has demonstrated toxicity following suppression of huntingtin in the adult brain. In fact, their ASO-mediated simultaneous suppression of mutant and normal huntingtin by 60% in the adult rodent striatum, and suppression of normal huntingtin by 45% in the non-human primate striatum, were both well tolerated.

Readers who are practiced in synthetic or medicinal chemistry are likely interested in the structure of IONIS-HTTRx. After considerable research, I found a SlideShare on LinkedIn, shown below. The SlideShare shows a 5-10-5 20-base gapmer comprised of ‘Generation 2+’ ASO with all-phosphorothioate- and 2’ MOE-modifications. It should be noted, however, that this is only an exemplary generic structure, as the actual sequence is proprietary, according to Anne Smith, the Director of Clinical Development at Ionis, who I contacted for permission to show this image.

Exemplary generic structure of IONIS-HTTRx. With permission from Anne Smith, Ionis

Before outlining future clinical studies of this ASO in the next section, I’ll conclude this section by providing a link to more than 100 items listed in Google Scholar for “Huntington and TriLink,” which can be perused later, as there are many interesting finds. One exemplary item that caught my attention was a patent for single-domain antibodies, which can be used in therapeutic methods to inhibit huntingtin protein aggregation.

Roche’s Clinical Trials of IONIS-HTTRx Renamed as RG6042

In December 2017, Roche acquired development and marketing rights to RG6042 from Ionis. According to an article from September 24, 2018 in Huntington’s Disease News, two new clinical studies by Roche of IONIS-HTTRx—now RG6042—for HD are planned to start by the end of 2018, and will begin enrolling participants by early 2019. These studies will help researchers understand progression of HD and the therapeutic effectiveness of RG6042, which may ‘potentially be the biggest breakthrough in neurodegenerative disease in the past 25 years,’ according to an interview with C. Frank Bennett, PhD, Ionis’ Senior Vice President of Research and franchise leader for the neurological programs.

The upcoming HD Natural History study and the Phase 3 GENERATION HD1 trial were announced at the recent 2018 European Huntington’s Disease Network plenary meeting in Vienna. The 15-month HD Natural History study will assess the correlation between mutant huntingtin protein in cerebral spinal fluid and in other clinical measures of HD, and will also evaluate wearable devices to measure disease burden. There is no therapeutic treatment in this study, as the goal is to understand the natural progression of the disease. The study will include up to 100 early-stage symptomatic patients at sites in the U.S., U.K., Canada, and Germany, and its results are expected to provide valuable information for the Phase 3 GENERATION HD1 study.

The two-year, global GENERATION HD1 trial will evaluate the long-term safety and effectiveness of RG6042 in up to 660 patients with symptoms of HD. It will be ‘the world’s first Phase 3 study to measure the effect’ of a therapy that lowers the amount of mutant huntingtin protein, according to Bennett. The trial will be conducted at 80 to 90 sites in 15 countries around the world.

2019 Breakthrough Prize for Bennett

On October 17, 2018, The Breakthrough Prize Foundation and its well-known sponsors—Sergey Brin, Priscilla Chan and Mark Zuckerberg, Ma Huateng, Yuri and Julia Milner, and Anne Wojcicki, announced the recipients of the 2019 Breakthrough Prize, awarding a collective total of $22 million to nine researchers for important achievements in the Life Sciences, and in Fundamental Physics and Mathematics. Considered the world’s most generous science prize, each Breakthrough Prize is for $3 million.

C. Frank Bennett. With his permission

By remarkable coincidence, The Breakthrough Prize in Life Sciences this year was jointly awarded to C. Frank Bennett at Ionis, and Adrian R. Krainer at Cold Spring Harbor Laboratory. The citation reads “for the development of an effective antisense oligonucleotide therapy for children with the neurodegenerative disease spinal muscular atrophy.”

Spinal muscular atrophy (SMA) is a rare but devastating disease, and the leading genetic cause of infant death. Many children with SMA die before their second birthday. Now, it is no longer a death sentence. Frank Bennett, a pharmacologist, and Adrian Krainer, a biochemist, built upon their discoveries on antisense technology and the natural process of RNA splicing to produce the first drug to treat SMA—Nusinersen (marketed by Biogen as Spinraza). It was approved by the FDA in 2016.

Those who are interested can learn more about SMA and Nusinersen by reading my October 2016 blog, which discusses this exciting breakthrough.

The work by Bennett on an ASO for treatment of SMA, and now his principal involvement in the development of IONIS-HTTRx as a promising drug for HD, are extraordinary contributions to science and society.

I’m more than pleased to have had the opportunity to collaborate with Frank in the early days of Isis Pharmaceuticals.

As usual, your comments are welcomed.

Addendum

After writing this blog, I came across some important news regarding the identification of sensitive indicators of HD progression and outcome of therapeutic intervention. Byrne et al. assessed mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations in cerebrospinal fluid (CSF), as well as blood in parallel with clinical evaluation and magnetic resonance imaging in premanifest and manifest HD mutation carriers. The concentration of CSF mHTT accurately distinguished between controls and HD mutation carriers, whereas NfL concentration, in both CSF and plasma, was able to segregate premanifest from manifest HD. These findings were said to provide evidence that biofluid concentrations of mHTT and NfL have potential for early and sensitive detection of alterations in HD, and could be integrated into both clinical trials and the clinic itself.

3’-End Labeling of RNA or DNA by a Polymerase Ribozyme

  • Revisiting a Polymerase Ribozyme for 3’-End Labeling Oligos
  • Using a Wide Variety of Modified Nucleotide Triphosphates from TriLink to Demonstrate Versatility of Labeling

Gerald Joyce. Taken from scripps.edu

In September 2016, I wrote a blog featuring a remarkable publication by the Gerald Joyce lab at Scripps Research Institute in La Jolla, CA. The researchers wrote about the in vitro evolution of an RNA catalyst (i.e. ribozyme) that had RNA polymerase activity and could amplify RNA. This purely RNA-based synthetic chemistry, in the complete absence of any proteins, provided further evidence for the feasibility of “RNA world,” a phenomenon first discussed by Walter Gilbert in 1986, who hypothesized the existence of prebiotic era billions of years ago during which life began without DNA or proteins.

This blog post once again spotlights the Joyce lab, but in the context of applying this novel polymerase ribozyme as a means to carry out 3’-end labeling of RNA or DNA with 50 modified nucleotides. I’m pleased to add that many of the requisite modified nucleotide triphosphates were obtained from TriLink! Interested readers can consult this June 2018 publication in Nucleic Acids Research for more details if they wish to supplement the brief overview that will be given here.

Introduction

RNA polymerase ribozymes are in vitro evolved RNA molecules that extend an RNA primer on a complementary RNA template using NTP substrates. Currently, the most advanced RNA polymerase ribozyme is the ‘24-3’ polymerase, which was reported in 2016 by Horning & Joyce to have an extension rate of ~1 nucleotide (nt) per minute, and can operate on most template sequences. Using specially designed templates, the 24-3 polymerase can generally be limited to the addition of only a single modified nucleotide, thus enabling efficient 3’-end labeling of a target RNA or DNA using various NTP and dNTP analogs shown here in red.

Taken from Joyce and coworkers Nucleic Acids Research 2018

The highly structured 24-3 polymerase ribozyme, which is depicted here in 2D, contains 180 nt. The ribozyme also has a short “tag” sequence (5’ GUCAUUG 3’) at the 5’ end of the polymerase that is complementary to a sequence (3’ CAGUAAC 5’) at the 5’ end of the template. Besides this feature, the template sequence is not constrained. The primer, which corresponds to the template nucleic acid, binds to the template through Watson–Crick pairing and is extended by the polymerase to achieve 3’-end labeling.

Although the 24-3 polymerase ribozyme can add multiple successive NTPs to the 3’ end of a template-bound primer, the reaction can mostly be restricted to the addition of a single residue by choosing an appropriate template and providing only one of the four nucleobase substrates. By way of example and as shown here, four templates were constructed, each with a different templating nucleotide (red) at the first position of primer extension, followed by several non-complementary nucleotides. Together, this set of templates enables the testing of triphosphate analogs containing each of the four nucleobases.

Taken from Joyce and coworkers Nucleic Acids Research 2018

Exemplary Results

Although a great variety of functionalized nucleotides can be prepared by chemical synthesis, this study by Joyce and coworkers focuses on commercially available nucleotide triphosphate analogs, such as sugar, nucleobase, and backbone modifications, in order to demonstrate the general utility of the approach. Fifty different analogs were tested in a reaction employing a 0.8 μM RNA (or DNA) primer with the following sequence: 5’ UUGCUACUACACGAC 3’ (or corresponding DNA sequence), together with 1 μM ribozyme and 1 μM RNA template. The reactions were carried out in the presence of 200 mM MgCl2 and 0.5 mM NTP analog at pH 8.3 at 17C for 1 h. Yields by PAGE ranged from 11% to 89% overall, with 84% to 89% yield for five of the analogs.

The exemplary results tabulated here highlight the versatility of 24-3 polymerase ribozyme toward incorporation of NTP analogs with very diverse molecular structures that provide different types of functionality.

Exemplary NTP Analogs (TriLink) and Incorporation Yield by the 24-3 Polymerase Ribozyme

NTP analog Yield % NTP analog Yield %
N6-methyl-2-amino-ATP 49 2’-amino-dATP 85
7-propargylamino-dGTP 89 2’-amino-dGTP 70
biotin-16-aminoallyl-dUTP 28 2’-amino-dCTP 80
Pseudo-UTP 66 2’-amino-dUTP 50
α-thio-ATP 70 5-aminoallyl-CTP 67
α-thio-GTP 86 Cy5-aminoallyl-CTP 47
α-thio-CTP 84 5-formyl-CTP 85
α-thio-UTP 11 5-formyl-UTP 58
thieno-GTP 50 1-borano-dGTP 37
thieno-UTP 25 1-borano-dCTP 12

For instance, N6-methyl-2-amino-ATP is a member of the diaminopurines that are discussed elsewhere, while pseudo-UTP shown below is an isomer of UTP that is now widely used in modified mRNAs.

Pseudo-UTP. Taken from TriLink BioTechnologies

In a TriLink white paper by Paul and Yee titled PCR incorporation of modified dNTPs: the substrate properties of biotinylated dNTPs, it is noted that the high affinity of streptavidin for the biotin ligand is one of the strongest and most widely utilized interactions in biology. The strength and specificity of this interaction has been exploited in many biological applications, including secondary label introduction and affinity isolation. While there are various length linkers that have been employed for attachment of biotin to the nucleotide, the relatively long biotin-16-aminoallyl-2′-dUTP used for incorporation by 24-3 polymerase ribozyme is often preferred.

Modification of the 3’-end by incorporation of 7-propargylamino-dGTP, 2’-amino dNTPs or 5-aminoallyl-CTP provides a reactive primary amine group as a versatile “chemical handle” to attach virtually any type of moiety that is needed for an application, whether that be a detectable label or synthetic peptide. The α-thio-NTPs (aka 1-thio-NTPs) and 1-borano-dNTPs demonstrate that these phosphate modifications are compatible with the 23-3 polymerase ribozyme.

α-Thio-ATP (1-Thio-ATP). Taken from Trilink BioTechnologies // 1-Borano-dCTP. Taken from TriLink BioTechnologies

Thieno-UTP. Taken from TriLink BioTechnologies

5-Formyl-nucleotides provide a reactive formyl (i.e. -CHO) group for conjugation reactions with, for example, hydroxylamine-functionalized labels of the type reported elsewhere. The incorporation of thieno-NTPs is interesting because of the inherent fluorescent properties of this relatively new class of analogs offered by TriLink, which can be read about at this link.

Concluding Comments

According to the aforementioned Joyce publication, the simple, one-step installation of a fluorophore or affinity probe using the 24-3 polymerase ribozyme is likely to have broad application, offering an attractive alternative to 3’-end labeling using a polymerase protein such as poly(A) polymerase or terminal transferase. These polymerase proteins operate in the template-independent manner, and thus result in multiple successive additions, unless the NTP analog itself is a chain terminator.

As usual, your comments are welcomed.

Highlights from the 2018 International Roundtable (IRT) on Nucleosides, Nucleotides, and Nucleic Acids

  • Over 400 Attendees from Around the World Congregate at UCSD
  • Four Days Full of Topics Spanning Basic Chemistry Through Therapeutics
  • Jerry Comments on His Five Favorites

According to Dr. Yogesh Sanghvi, this IRT 2018 logo was created by Prof. Yitzhak Tor, who artistically used the elements of XXIII to symbolize sunshine and waves typical to the venue of La Jolla, California.

The 23rd (XXIII) International Roundtable (IRT) on Nucleosides, Nucleotides, and Nucleic Acids was held at the University of California, San Diego (UCSD) in La Jolla, California on August 26 – 30, 2018. This 23rd biannual event, which was sponsored by the International Society of Nucleosides, Nucleotides, and Nucleic Acids (IS3NA) was attended by over 400 researchers in all levels of academia and industry from around the world. Award lectures (2), invited lectures (17), oral presentations (26), and posters (198) spanned a plethora of cutting edge scientific topics, ranging from the origins of life to the development of novel therapeutics. Local organizers were Prof. Yitzhak Tor, Chair (UCSD), Dr. Yogesh Sanghvi (Rasayan, Inc.) and Dr. Rick Hogrefe (TriLink BioTechnologies).

I thoroughly enjoyed attending this 2018 IRT, where I had the opportunity to completely immerse myself in diverse aspects of chemistry, biochemistry, molecular biology, medicinal chemistry, and drug development—all related to nucleosides, nucleotides, and nucleic acids. The meeting gave me the opportunity—and challenge—of selecting five noteworthy presentations, shared here in random rather than rank order. There are several other presentors with fantastic work that I apologize for not being able to discuss at this time. For example, Dr. Alexandre Lebedev at TriLink Biotechnologies, gave an excellent oral presentation titled: Efficient initiation of in vitro mRNA transcription with Cap 0, Cap 1 and Cap 2 oligonucleotide primers (CleanCap®). The presentation highlighted TriLink groundbreaking CleanCap Technology, a chemical solution that provides high mRNA capping efficiency, and avoids self/non-self intracellular responses. If you would like a copy of this presentation, please contact TriLink here.

CRISPR Cloaking

An ongoing “hot topic” is sequence-specific cutting of DNA with CRISPR-Cas9 for gene editing as a research tool or therapeutic modality. I have previously blogged about this here. Given the widespread interest on the subject, it’s apropos to start with a poster titled Reversible RNA acylation for CRISPR-Cas9 gene editing control in cells presented by Maryam Habibian, a postdoc in Eric Kool’s group at Stanford University.

As shown here, Kool’s lab has recently published on the reaction of RNA in aqueous buffer with an azide-substituted acylating agent, which yields several 2′-OH acylations per RNA strand in as little as 10 minutes. This poly-acylated (“cloaked”) RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine results in spontaneous loss of acyl groups (“uncloaking”) that fully restores RNA folding and biochemical activity.

Taken from Kadina et al. Angew Chem Int Ed Engl. (2018)

Data in this poster showed that an azide-substituted reagent efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. These cloaked sgRNAs completely inhibit the endonuclease activity of Cas9 in vitro and in living HeLa cells. However, the sgRNA activity is efficiently recovered both in vitro and in cells by treatment with water-soluble phosphines. This study highlights the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function.

Chemically Modified DNAzymes

Prof. David Perrin at the University of British Columbia in Canada gave an oral presentation titled Chemically modified DNAzymes as sequence-specific ribonuclease-A mimics—from potential therapeutics to the origin of life. He noted that the use of a synthetic RNAzyme or DNAzyme to cut a particular sequence target mRNA for use as a possible therapeutic agent has been a concept for ~40 years, but has not yet come to realization. The principal challenge, he added, is to find a suitably structured nucleic acid that catalyzes efficient phosphodiester bond cleavage in RNA in the absence of Mg+2 or at the relatively low Mg+2 concentrations in cells.

As shown here, this presentation described the in vitro selection of novel RNA cleaving DNAzymes that are selected using 8-histaminyl-deoxyadenosine (imidazole-A), 5-guanidinoallyl-deoxyuridine (guanodino-U), and 5-aminoallyl-deoxycytidine (amino-C), along with dGTP. These modified dNTPs provide key functionalities reminiscent of the active sites of ribonucleases, notably RNase A.

Taken from Perrin and coworkers OP9 Abstract IRT 2018

Remarkably, these exceptional catalysts display classic enzymatic properties of Michaelis-Menten kinetics in the absence of Mg+2. Interested readers can access complete details on this exciting work here. Perrin added that, in honor of the late Stanley Miller (UCSD), whose pioneering work on the origin of life included the possibility of a highly-decorated RNA world, this work represents a chemist’s approach to biomimicry for testing hypotheses of the origin of life in an RNA-world that must of co-opted synthetic modifications, and underscores the use of modified dNTPs for the selection of modified aptamers. You can read more about aptamers in several of my previous blogs.

Direct Sequencing of N6-Methyladenosine in RNA

N6-Methyladenosine. Taken from wikipedia.org

Enzyme-mediated post-transcriptional RNA modifications are dynamic, and may have functions beyond fine-tuning the structure and function of RNA. Understanding these epitranscriptomic RNA modification pathways and their functions may allow researchers to identify new layers of gene regulation at the RNA level, according to a “grand challenge” discussed in a previous blog. N6-Methyladenosine (m6A), shown here, is the most abundant modification in eukaryotic mRNA and long noncoding RNA (lncRNA). It is found at 3-5 sites on average in mammalian mRNA, and up to 15 sites in some viral RNA.

In addition to this relatively low density, specific loci in a given mRNA are a mixture of unmodified- and methylated-A residues, thus making it very difficult to detect, locate, and quantify m6A patterns. Importantly, there is now an elegant solution to this problem. In an invited lecture by Prof. Andreas Marx at the University of Konstanz in Germany titled Elucidating the information layer beyond the genome sequence, an engineered polymerase was said to differentiate between unmodified- and methylated-A residues.

Taken from Marx and coworkers Angew Chem Int Ed Engl. (2018)

This novel method, which was recently published, involves in vitro evolution and screening to evolve a reverse-transcription (RT)-active KlenTaq DNA polymerase mutant (RT‐KTQ G668Y Y671A) that delivers prominent RT signatures at m6A sites in different sequence contexts. As shown here, this novel polymerase exhibits increased misincorporation opposite m6A compared to unmodified A. Application of this DNA polymerase in next-generation sequencing allowed for identification of m6 A sites directly from the sequencing data of untreated RNA samples.

Phosphorothioate-Modified Oligo Therapeutics

Pioneering investigations of phosphorthioate (PS)-modified nucleic acids by Prof. Fritz Eckstein, followed by fully automated synthesis of PS-modified oligodeoxynucleotides by Prof. Wojciech Stec and yours truly, enabled many other researchers to develop PS-ODNs as therapeutic agents. Although I have previously blogged about this topic, the utility and prevalence of PS-modifications in ODN-based therapeutics was a common theme throughout many presentations at IRT 2018.

Most prominently, in my opinion, Dr. Punit Seth at Ionis Pharmaceuticals in Carlsbad, California gave an invited lecture titled Engineering selectivity into therapeutic oligonucleotides through chemical design. The talk largely dealt with PS-ODNs and included a slide with the following summary:

  • PS-ODNs interact with several plasma proteins with a range of binding affinities
    • PS content and single-stranded nature are important for binding
    • Binding can be rationalized by an avidity model wherein each PS contributes a fraction to overall binding
  • Interaction with plasma proteins can have functional consequences
    • Binding to α-2-macroglobulin can reduce uptake pathways
    • Strong binding to HRG can reduce activity
    • Lipid conjugation enhances potency in muscle through interactions with albumin and lipoproteins
  • PS-ODNs interact with cell-surface proteins such as Stabilin scavenger receptors
    • Stabilins clear anionic polymers of the extra-cellular matrix suggesting a common pharmacophore with anionic PS-ODNs

The influence of antisense PS-ODN Sp and Rp stereochemistry on such pharmacological factors, including RNase-H activity, has been reported by Wave Life Sciences using stereoselective synthesis methodology introduced by Wada (see also Baran). Extending this approach, Troels Koch at the Roche Innovation Center in Copenhagen gave an invited lecture titled Stereodefined LNA Phosphorothioates: Design, synthesis and properties. In particular, he described investigations of 2′-O, 4′-C methylene bridged moieties commonly referred to as “locked nucleic acids” (LNAs), shown here:

Taken from zon.trilinkbiotech.com  //  2′-O, 4′-C methylene LNA. Taken from wikipedia

Koch stated that LNAs have, over the last 15 years, been intensively used in RNA therapeutics because LNAs offer high affinity that translates into higher potency for RNA targeting. He added that nearly all of these LNA oligonucleotides have PS linkages. His presentation illustrated the diversity of measurable properties of stereodefined PS-LNAs. Importantly, it was shown that identifying the best diastereomers from a large random mixture is not trivial. Several identification tactics were described, including the use of quantum mechanical modelling as a guide towards finding the best use of stereodefined LNA.

In striking contrast to the aforementioned focus on inclusion and improvement of PS linkages in therapeutic oligonucleotides, Prof. Jesper Wengel at the University of Southern Denmark in Odense gave an oral presentation titled Novel DNA-mimicking monomers for gapmer antisense oligonucleotides, wherein the objective is to increase gene knock-down specificity by complete removal or substantial reduction of PS linkages and other strategies. His current design is to use phosphodiester (PO)-linked “3-10-3” LNA-DNA-LNA gapmers with palmitic acid-derived moieties attached, as shown below, and bridging N or O in the LNA residues.

Taken from Wengel et al. OP24 at IRT 2018 (Photo by Jerry Zon)

Discovery of a Nucleotide Analog Drug for Ebola Virus

In my blog on the 2014 outbreak of the deadly Ebola virus, I indicated the need for more resources allocated towards the development of a prophylactic vaccine. While such work continues, I was very pleased to learn of promising results obtained for a new drug against Ebola, which would provide treatment for individuals already infected with the virus.

Dr. William Lee at Gilead Sciences, Inc. in Foster City, California, reported in an invited lecture titled Remdesivir (GS-5734): An Antiviral Nucleotide Analog for the Treatment of Ebola Virus that this nucleotide prodrug (shown here) of a novel nucleoside analog has shown broad spectrum in vitro activity against filoviruses, corona viruses, paramyxoviruses, and flaviviruses. Importantly, Remdesivir has demonstrated potent in vivo efficacy against multiple strains of the Ebola virus in the rhesus monkey infection model. His talk reviewed the data in rhesus, the manufacturing challenges, and the limited exposure in patients exposed to the Ebola viruses.

Taken from Lee IL17 Abstract IRT 2018

Concluding Comments

In my opinion, IRT 2018 was indeed jam-packed with innovative and interesting presentations by a diverse array of researchers from around the world, all united by the common thread of nucleosides, nucleotides, and nucleic acids. In addition to the science, there was ample opportunity to renew friendships and, more importantly, network and exchange contact information with new people for potential collaborations on mutually interesting projects.

Presented by Prof. Roger Stromberg, Karolinska Institutet, Sweden and Secretary of IS3NA (Photo by Jerry Zon)

Every IRT meeting includes an announcement (shown here) of the next venue, which for the 24th (XXIV) IRT in 2020 will be Stockholm, Sweden, locally organized by Prof. Roger Stromberg and held at the Karolinska Institutet.

I hope to see you there!

As usual, your comments are welcomed.

Footnote

While walking through the UCSD campus to the “kick off” Keynote Lecture by Prof. Gerald Joyce (Salk Institute) titled RNA-Targeted drug discovery, to be followed by an all-attendees reception sponsored by TriLink, I came across and photographed the large, brightly colored statue shown here.

UCSD Sun God (Photo by Jerry Zon)

My curiosity about this eye-catching, fanciful figure led me to learn that it is called Sun God, and is an art object created by Niki de Saint Phalle (1930-2002), who is best known for her oversized figures that embrace contradictory qualities such as good and evil. She lived in New York in the 1960s when she was prominent in the development of “happenings” and other artistic efforts involving the integration of art and life. She lived and worked in La Jolla from 1992 until her death in 2002.

De Saint Phalle’s Sun God was the first work commissioned by the Stuart Collection of UCSD and was her first outdoor commission in America. The exuberantly colored, fourteen-foot bird is placed atop a fifteen-foot concrete arch and sits on a grassy area between near the Faculty Club. The students started the Sun God Festival in 1984. It has become one of the largest annual campus events.

The Sun God has become a landmark on the UCSD campus. Students have embellished the statue at various times with giant sunglasses, a cap and gown, a UCSD ID card, and a nest of hay with eggs. Sun God has also been adorned with earphones and a radio/tapeplayer, turning the statue into a “Sony Walkbird,” and has sported a machete and headband for its disguise as “Rambird.” It appears on T-shirts and mugs. The grassy area beneath it is a popular site for rendezvous and celebrations.

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Spotlight on TriLink Product Applications

  • Nearly 500 Publications in 2017 Cite Use of TriLink Products
  • Jerry Spotlights 20 Citing Oligos, Nucleotides, mRNA and Aptamers
  • 10 of These 20 Spotlighted Items Show Global Reach of TriLink Products

While thinking about possible topics to blog about, it occurred to me that researching recent publications on the applications of TriLink products would likely lead to many options. Using Google Scholar to do just that, I was given nearly 500 items, which is indeed plenty. However, choosing which to feature was neither an easy nor objective task. Having said that, and with sincere apologies to publications not spotlighted here, my “faves” and comments are given below, listed arbitrarily (not ranked) in four product categories: oligonucleotides, nucleotides, mRNA, and aptamers.

Taken from depositphotos.com

For convenience, each publication title can be clicked on to access the original article. Links to the cited TriLink products are also provided, alongside links to other adjunct information. Several trending “hot topics” and previous blogs are also noted.

Oligonucleotides

Taken from researchgate.net

Nucleotides

8-oxo-dGTP; taken from TriLink BioTechnologies // dPTP; taken from TriLink BioTechnologies

mRNA

Modified mRNA for new therapeutic approaches continues to be an amazingly hot area of R&D, which I have previous dubbed “modified mRNA mania” in a previous blog. Interested readers can peruse this link to ~300 items found in my Google Scholar search for TriLink and mRNA publications in 2017.

pseudo-UTP; taken from TriLink Biotechnologies // 2-thio-UTP; taken from TriLink Biotechnologies

Aptamers

2’-F-dCTP; taken from TriLink BioTechnologies // 2’-F-dUTP; taken from TriLink BioTechnologies

Global Reach

A pleasantly surprising aspect of the selected-product search results given above is the worldwide distribution of researchers using TriLink products. This global reach, if you will, is evident from the following countries outside of the USA, which I made point of mentioning:

The Netherlands, India, Austria, Switzerland, Turkey, Germany, Italy, Belgium, Republic of Korea, and Denmark.

All of the publications listed above were selected solely on the type of TriLink product used. Given the relatively small “sample size” of these selected publications, which are only 20-of-500, finding investigators in 10 countries outside of the USA is a compelling testimonial for the TriLink global reach.

World Science Day

Truth be told, when I was searching for a fitting image to visually convey the concept of “global science,” I came across the fact that the United Nations Educational, Scientific, and Cultural Organization (UNESCO) has designated November 10 as World Science Day, with an emphasis on peace and development. The stated intention is to highlight “the important role of science in society and the need to engage the wider public in debates on emerging scientific issues. It also underlines the importance and relevance of science in our daily lives.”

Taken from monitor.co.ug

According to UNESCO, “[t]he theme for 2018 is ‘Science, a Human Right’, in celebration of the 70th anniversary of the Universal Declaration of Human Rights (art. 27), and of the Recommendation on Science and Scientific Researchers. Recalling that everyone has a right to participate in and benefit from science, it will serve to spark a global discussion on ways to improve access to science and to the benefits of science for sustainable development.”

To me, this is a long-term objective which is indeed critical for betterment of future generations.

As usual, your comments are welcomed.

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Clinical Efficacy Found: Two Oligonucleotide Drugs for Transthyretin (TTR)-Related Amyloidosis (ATTR)

  • ATTR Is a Class of Life-Threatening and Progressive Genetic Diseases    
  • ATTR Results from Misfolding of TTR, a Blood Protein Carrier of Retinol (Vitamin A), and Thyroxine (T4)
  • TTR mRNA Knockdown by an Antisense Oligonucleotide (ASO) or a Short-Interfering RNA (siRNA) Leads to Clinical Efficacy for ATTR

A recent blog here lauded the beginning of American and European clinical studies of gene-editing by CRISPR, which is enabled by sequence-specific interaction of synthetic guide RNAs with genomic DNA targets. At the end of that post, I mentioned that future clinical successes with CRISPR may encounter the challenge of unforeseen drug development issues, as has been the case for antisense oligonucleotide (ASO) and short-interfering RNA (siRNA) approaches. It took a couple of decades for FDA-approved ASO drugs to progress from “bench to bedside,” and the first siRNA drug has yet to be approved.

After opining the above just a few weeks ago, I am pleased to say that another ASO drug and the first siRNA drug have been reported to both be on the brink of FDA approval, based on back-to-back publications of the results of Phase 3 clinical studies in the highly respected Journal of New England Medicine. Interestingly, both of these mechanistically different oligonucleotide drugs are aimed at the same clinical indication, namely, transthyretin (TTR)-related amyloidosis (ATTR). This blog will serve as an introduction to ATTR, as well as a brief summary of both of these very promising clinical studies.

Introduction to ATTR

As detailed elsewhere, ATTR represents a class of life-threatening and progressing diseases that is associated with the misfolding of TTR, a major blood protein that is a carrier of retinol (vitamin A) and thyroxine (thyroid hormone T4). TTR is primarily (>95%) produced in the liver as a tetramer, as depicted below. Amyloid aggregation is believed to be a result of decreased tetramer stability, resulting in dissociation of TTR into monomers. These monomers are prone to unfolding, and can self-assemble into oligomers and amyloid fibrils found in peripheral neurons, the gastrointestinal tract, and heart.

Taken from link.springer.com

The three major forms of ATTR disease are described in the NIH Genetic Home Reference (GHR), which also provides information about their underlying genetics and diagnosis by, for example, DNA sequencing. In hereditary ATTR with polyneuropathy [aka familial amyloid polyneuropathy (FAP)], the peripheral nerves are primarily affected, while in cardiomyopathy-related ATTR [aka familial amyloid cardiomyopathy (FAC)], neuropathy is usually less prominent.

According to the GHR, ATTR among Americans of European descent is estimated to affect one in 100,000 people, while FAC is far more frequent among people with African ancestry. For example, it is estimated that this form affects between three to four in 100 African Americans. Although the reasons for this disproportionately higher incidence are unknown, suggestions on how to raise awareness on this issue are discussed in a review I found while researching FAC among African Americans.

For the most part, patients develop a severe disease and die within 5 to 15 years after onset. While these forms of ATTR can be ascribed to a dominant expression of the TTR gene variants, (their number is ~100, as depicted here in an artistic rendering), only wild-type TTR is expressed in senile systemic amyloidosis (SSA), a type of amyloidosis frequently found in elderly people.

Taken from pharmaceuticalintelligence.com

The ATTR pathway above depicts the possibility of using oligonucleotides that interfere with mRNA function to block the synthesis of TTR. While blocking synthesis of a specific mutant of TTR is possible, biochemical and clinical evidence suggests that the wild-type TTR can also significantly contribute to the disease. In 2016, results obtained from an in vitro model system were jointly published by Ionis Pharmaceuticals and Alnylam Pharmaceuticals. The study supports the notion that ASOs or siRNAs can block TTR synthesis, and can therefore represent a possible therapy for ATTR. These findings paved the way for each company to independently carry out individual clinical development programs for oligonucleotide drug candidates, as outlined below.

ASO Drug Inotersen

Ionis’ inotersen (formerly IONIS-TTRRx/ISIS 420915), which is a 2′-O-methoxyethyl (MOE)– and phosphorothioate (PS)-modified RNA (orange)-DNA (black) “gapmer” ASO shown here, is an inhibitor of the hepatic production of TTR. Readers interested in ASO chemistry and mechanistic details for gapmers can consult an excellent review by Shen and Corey.

Taken from Shen and Corey Nucleic Acids Research (2018)

Taken from Buxbaum Journal of New England Medicine (2018)

As shown here, the genome of patients with hereditary amyloidotic polyneuropathy contains one copy of wild-type TTR and one copy with a base substitution (mutation), resulting in a change in the amino acid sequence. In hepatocytes, both copies are equally transcribed and translated. Inotersen binds to wild-type and mutant TTR mRNA transcripts, resulting in their degradation by ribonuclease H.

In healthy volunteers, inotersen showed dose-dependent and sustained reductions of circulating TTR levels. Ionis then conducted a randomized, double-blind, placebo-controlled, Phase 3 trial (NEURO-TTR) to determine the efficacy and safety of inotersen treatment in patients with hereditary transthyretin amyloidosis with polyneuropathy, in the presence or absence of cardiomyopathy. According to Benson et al., eligible patients were randomly assigned (in a 2:1 ratio) to receive 300 mg of inotersen or placebo. Patients 18 to 82 years of age received three subcutaneous injections during the first week to achieve near steady-state drug levels, followed by a once-weekly subcutaneous injection for the next 64 weeks.

A total of 172 patients (112 in the inotersen group and 60 in the placebo group) received at least one dose of a trial regimen, and 139 (81%) completed the intervention period. In the inotersen group, reductions in circulating TTR reached steady-state levels by week 13 and were sustained through the end of the intervention period. From week 13 to week 65, decreases in serum TTR from baseline levels in the inotersen group reached a median nadir of 79%.

Primary efficacy assessments favored inotersen. These improvements were independent of disease stage, mutation type, or the presence of cardiomyopathy. The most frequent serious adverse events in the inotersen group were glomerulonephritis [in 3 patients (3%)] and thrombocytopenia [in 3 patients (3%)]. It was concluded that “inotersen improved the course of neurologic disease and quality of life in patients with hereditary transthyretin amyloidosis” and that “[t]hrombocytopenia and glomerulonephritis were managed with enhanced monitoring.”

siRNA Drug Patisiran

According to the above cited review by Shen and Corey, and as shown here, Alnylam’s patisiran is a double-stranded siRNA comprised of 3’-end DNA (black), RNA (pink), and 2’-O-MOE RNA (blue) moieties. Partisiran is delivered in complex with a lipid nanoparticle (LNP), (also depicted here), and is described in detail elsewhere by Acuitas Therapeutics, an LNP drug-delivery company that partnered with Alnylam.

Taken from Shen and Corey Nucleic Acids Research (2018)

Taken from acuitatx.com

The mechanism of action depicted here for the siRNA patisiran involves the formation of an RNA-induced silencing complex (RISC) with both wild-type and mutant TTR mRNA transcripts and subsequent mRNA degradation.

Taken from Buxbaum Journal of New England Medicine (2018)

Adams et al. have now reported results from a Phase 3 clinical trial by Alnylam wherein patients were randomly assigned (in a 2:1 ratio) to receive patisiran (0.3 mg per kilogram of body weight) or placebo intravenously over a period of approximately 80 minutes, once every 3 weeks for 18 months. A total of 225 patients 24 to 83 years of age were randomly assigned to receive patisiran (148 patients) or placebo (77). In the patisiran group, the reduction in serum TTR levels was rapid and sustained over a period of 18 months, with a median reduction of 81% that remained similar across age, sex, or genotype.

Primary efficacy endpoints favored patisiran and approximately 20% of the patients who received patisiran and 10% of those who received placebo had mild or moderate infusion-related reactions; the overall incidence and types of adverse events were similar in the two groups. It was concluded that “patisiran improved multiple clinical manifestations of hereditary transthyretin amyloidosis.”

One Expert’s Opinion

Joel Buxbaum, an MD and Professor Emeritus in the Department of Molecular Medicine at the Scripps Research Institute, accompanied these back-to-back publications about inotersen and patisiran in the Journal of New England Medicine with an Editorial. Based on his publications in this field, Buxbaum is an expert on the molecular basis of acquired and hereditary human disease related to human amyloidosis. After summarizing the results of each clinical trial, Buxbaum posed the following scientifically intriguing questions, which had not occurred to me after reading these two reports.

“Although each of the trials unequivocally shows a therapeutic effect, there remain questions. Would a single patient have a response to each of the therapeutics to the same degree? If so, it is in the patient’s interest to use the least expensive therapy. If not, N-of-1 trials of sequential treatments, although cumbersome, would be required to identify the best treatment for each patient, conditional on the availability of a rapidly responsive, validated surrogate marker of disease.” In regard to the currently trending concept of N-of-1 trials, Buxbaum refers the reader to a commentary in Nature by Nicholas Schork titled Personalized medicine: Time for one-person trials, from which I borrowed this illustration that visually coveys Schork’s point.

Taken from Schork Nature (2015). Illustration by Greg Clarke

Buxbaum continues his expert commentary as follows:

“It is also possible that a combination of interventions would elicit a more pronounced, durable therapeutic effect. Although there remains much work to be done, the trials by Adams et al. and Benson et al. represent a landmark: together, they show that the rate of progression of a peripheral neurologic disease can be slowed, and perhaps ameliorated, through the use of oligonucleotide drugs that are administered systemically.”

In my humble opinion, the promising outcomes of these two trials are indeed great news for people afflicted with ATTR, who can be future recipients of this oligonucleotide therapy. The results are likewise great news for families and friends. The many researchers, who collectively contributed—directly or indirectly—to achieving this milestone, are also undoubtedly pleased by these results.

As usual, your comments are welcomed.

Kool’s Cool Chemistry

  • Eric T. Kool Has a Long-String of Widely Cited Papers on Innovative Nucleic Acid Chemistry   
  • Kool’s Latest Contribution Involves Reversible Chemistry for PhotoCloaking RNA
  • Kool’s Application of This to Aptamers is Very Cool, In My Opinion

Eric T. Kool (taken from Stanford.edu)

Readers of my blog know that my writing style tends toward using alliterations, which is evident in this blog’s title referring to Prof. Eric T. Kool and his chemistry, and that cool is the slang sense, i.e. awesome, swell, nifty, etc.

At the outset, I should say that Stanford University-based Kool has a long string of very ingenious publications in diverse aspects of nucleic acids, which attract many readers evidenced by ~1,400 citations per year according to Google Scholar metrics. Kool has received numerous awards and honors that are listed at The Kool Lab website, together with his lab’s research interests.

An appreciation of the diversity of Kool’s scientific contributions can be gleaned by later perusing his ~270 publications at this PubMed link, but for now I’ll be focusing on his most recent publication titled RNA Control by Photoreversible Acylation. This article requires—unfortunately—a paid subscription to J. Amer. Chem. Soc. or purchasing access to a pdf, so I’ve tried my best to give you herein a synopsis of what Kool has achieved in this study, and why it’s so cool.

Caged Nucleic Acids

One of the ways to externally initiate (i.e. turn on) nucleic acid biochemical or biological function in vitro or in vivo is to use a “caging” strategy. The term caging generally refers to installation of one or more removable groups on a molecule in such manner so as to render the molecule inactive. For nucleic acids (and proteins, etc.) this has typically been achieved with photoremovable groups, such that irradiation with UV light removes the caging group and restores functional activity.

Taken from Deiters and coworkers ChemBioChem 2008

An illustrative example of this, published by Deiters and coworkers in 2008, is shown here for controlling gene silencing in mammalian cells with light-activated antisense agents. In this case, phosphorothioate-modified antisense agents were rendered inactive by installation of NPOM groups on nucleobases to prevent hybridization to a target mRNA. Brief irradiation with light at 365 nm removes the NPOM caging groups, enables sequence-specific binding to mRNA, and thus, blocks translation and/or leads to RNase H-catalyzed mRNA degradation.

A similar approach has been applied to functional studies of RNA. However, obtaining such photocaged DNA or RNA has usually required synthesis of chemically modified phosphoramidite monomer building blocks for solid-phase synthesis of the desired oligonucleotides. This limits utility to investigators who are able to deal with organic synthesis or have budgets to purchase relative costly photocaged monomers or custom synthesized photocaged oligonucleotides. Applying photocaging to long RNA is even more problematic.

Kool’s Cool Chemistry

To address these obstacles, Kool set out to develop a general method that can be applied post-synthetically to synthetic and native RNAs regardless of length, and moreover could be easily carried out by non-chemists. Based on studies (cited earlier) by others showing that 2’-OH groups of RNA can be selectively acylated in aqueous buffers with activated acyl compounds, Kool envisaged a strategy depicted here. An appropriately designed “PhotoCloaking” agent (PCA) is reacted with 2’-OH groups post-synthetically to block structure and interactions, but are rendered photo-responsive by including photocleavable bonds.

In his publication, Kool reasoned that “addition of several such blocking groups to the RNA (‘cloaking’) would in principle cover and protect it from folding and interacting with other molecules. Subsequently, the acylation could be reversed by exposing RNA to light, removing the blocking groups and switching on activity.” The PCA design strategy included combination of a reactive acyl group with an o-nitroveratryl photo-labile group, as shown here for PCAs 1 and 2 having unmethylated and methylated veratryl groups, respectively, to control the efficiency of photocleavage. Also included was a dimethylaminoethyl group to enhance solubility, and (after testing) 2-chloroimidazole as having the ideal level of reactivity.

A short 12-mer synthetic RNA oligo was used to evaluate 2’-OH acylation in water under mild conditions, in conjunction with denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometry (MS), which indicated polyacylation with up to five groups (aka labels). Next, uncloaking of RNA by photoremoval of the PCA labels in water by irradiation with 365 nm light was confirmed by PAGE and MS. A 16-mer synthetic RNA oligo was similarly polyacylated and shown by native gel analysis and UV to have diminished ability to hybridize to complementary DNA. The reactivity of a synthetic hammerhead ribozyme previously reported was similarly disabled by PhotoCloaking and subsequently restored by exposure to UV light.

Kool then hypothesized that this postsynthetic polyacylation strategy would offer a convenient and simple method for preparing a photocontrolled aptamer of any length, which I have touted as a useful class of compounds in previous blogs. Kool’s test for this was especially interesting, in my opinion, because he used a 150-nt RNA transcript called Broccoli. This oddly named RNA is an aptamer that folds into a compact tertiary structure which binds DFHBI dye giving rise to a strong increase in fluorescence, and thus serves as an RNA mimic of well-known green fluorescent protein.

Kool had to use the more reactive PCA 2 to prepare a suitably cloaked version of Broccoli, which exhibited very dim fluorescence compared to uncloaked (i.e. untreated) Broccoli. When the cloaked aptamer was exposed to UV light and incubated with DFHBI, fluorescence was completely restored to the level of the untreated sample. The capability to use this PhotoCloaking strategy to switch on RNA function in cells was assessed by transfecting a 237-nt cloaked Broccoli-dimer RNA construct into a human (HeLa) cell line and then exposing these cells to light. As can be seen from the results shown here, the cloaking and uncloaking worked just as intended.

Epifluorescence microscopy of HeLa cells transfected with untreated or cloaked 237-nt Broccoli. Taken from Kool and coworkers J Am Chem Soc 2018

Kool concluded that “a great advantage of this postsynthetic labeling is that it allows for one-step photoprotection of long, biologically relevant RNAs that are difficult or impossible to synthesize via solid-state oligonucleotide synthesis. Since PhotoCloaking can be achieved without specialized equipment, the new method could potentially find widespread use for study of the biology of RNAs.”

Very cool indeed!

As usual, your comments are welcomed.

Top 10 Genes in the Human Genome! (by Number of Citations)

  • The ‘Guardian of The Genome’ p53 Tops the List of Top 10 Genes
  • Cancer-Related Genes Are Prevalent Among the Top 10
  • TriLink Is Proud of Its Products Mentioned in Studies of the Top 10

People seem to be fascinated with “Top 10” lists, which currently exceed 134,000 according to The Top Tens® website. Later, you can search this mind-boggling list for whatever strikes your fancy; for now, let’s focus on what’s trending in nucleic acids which is the intent of this blog. In that regard, and linked to the notion of “Top 10” lists, venerable Nature magazine recently published a news piece titled The Greatest Hits of the Human Genome. This caught my attention as definitely a blog-worthy topic, as it had a tag line offering readers “a tour through the most studied genes in biology,” which sounded very intriguing to me.

Interested readers can peruse the entire news item in Nature, but for the moment here’s my synopsis of Nature’s list of Top 10 Genes, along with my findings for representative examples of how TriLink products have been used for each of these Top 10 Genes.

The Top 10 Genes

According to Nature, software engineer pre-doctoral student Peter Kerpedjiev became intrigued to rank the world’s most studied human genes, and eventually collaborated with Nature in analyzing data extracted from the NCBI/NLM PubMed database. Following is the rank-order list of the Top 10 most studied genes, along with a brief functional definition of each gene, and then a graphical depiction of the number of citations for each gene. Incidentally, you’ll see that naming genes is not systematic, and simply adopts letter and number abbreviations used in the early literature as descriptors.

  1. TP53 Tumor-suppressor protein p53 is mutated in up to half of all cancers.
  2. TNF Tumor necrosis factor has been a drug target for cancers and inflammatory diseases.
  3. EGFR Epidermal growth factor receptor is a membrane-bound protein often mutated in drug-resistant cancers.
  4. VEGFA Vascular endothelial growth factor A promotes growth of blood vessels.
  5. APOE Apolipoprotein E has important roles in cholesterol and lipoprotein metabolism.
  6. IL6 Interleukin 6 has several important roles in immunity.
  7. TGFB1 Transforming growth factor beta 1 controls cell proliferation and differentiation.
  8. MTHFR Methylenetetrahydrofolate reductase helps to process amino acids.
  9. ESR1 Estrogen receptor 1 has been a focus in breast, ovarian and endometrial cancers.
  10. AKT1 Encodes a signaling protein that phosphorylates other proteins to activate them.

Adapted from Nature Vol 551, 2017; Source: Peter Kerpedjiev/NCBI-NLM

Topping the list is a gene called TP53, initial recognition of which goes back to publications in the early 1980s. The popularity of TP53 shouldn’t come as news to most biologists, as it is a tumor suppressor widely known as the ‘guardian of the genome’. The p53 gene is reported to be the most frequently mutated gene in human cancer: about 50% of all human cancers have lost p53 or express an inactive, mutant p53.

Core domain of p53 (green) bound to DNA (blue). Six most frequently mutated amino acids in yellow; red ball is zinc ion. Taken from Cho et al. Science, Vol 265, 1994

Given its preeminent position among all human genes studied, I had intended to provide a short synopsis of p53 functions identified to date. However, reading reviews quickly revealed that these functions are far too complex for me to adequately and succinctly summarize. Instead, I recommend consulting one such recent review by Haupt & Haupt, with the attention-grabbing title p53 at the start of the 21st century: lessons from elephants. According to these authors,

“[H]ow p53 defends against DNA damage to preserve the genome and fight cancer is still being elucidated, despite more than 35 years of intense molecular and cellular study. Until very recently, the field predominantly focused on the role of p53 as a transcriptional regulator, particularly on its transactivation targets that drive arrest and apoptosis. The capacity of p53 to suppress tumors independent of key mediators of these processes has challenged accepted knowledge. The complexity of the p53 response continues to emerge along with new understanding of the contribution of p53 to transcriptional repression and also revisitation of the concept of p53 transactivation-independent function, which was first reported more than 20 years ago.”

Research into human cancer also brought scientists to TNF, the runner-up to TP53 as the most-referenced human gene of all time, with more than 5,300 citations in the NLM data. TNF encodes a protein—tumor necrosis factor—named in a 1975 publication because of its ability to kill cancer cells. But attempts to harness TNF’s anticancer action proved unsuccessful, as would-be therapeutic forms of the TNF protein were highly toxic when tested in people.

Like other popular genes, APOE is well studied because it’s central to cardiovascular disease, which is still one of the biggest unsolved health problems. First described in the mid-1970s as a transporter involved in clearing cholesterol from the blood, initial use of APOE protein as a lipid-lowering treatment was eventually supplanted by the statins. Now there is interest in an allele (i.e. genetic variant) of APOE, APOE4 that is associated with amyloid plaques and greatly increased risk of Alzheimer’s disease, about which I’ve blogged previously.

Amyloid plaques. Taken from slideshare.net

Due to space limitations, I won’t comment on the remaining seven Top 10 genes, but will include this quote from the Nature news piece that comments on what influences a gene’s popularity, so to speak:

“[I]t takes a certain confluence of biology, societal pressure, business opportunity and medical need for any gene to become more studied than any other. But once it has made it to the upper echelons, there’s a ‘level of conservatism’, says Gregory Radick, a science historian at the University of Leeds, UK, ‘with certain genes emerging as safe bets and then persisting until conditions change’. The question now is how conditions might change. What new discoveries might send a new gene up the chart—and knock today’s top genes off their pedestal?”

TriLink’s Connections to The Top 10 Genes

In a previous blog, I highlighted selected applications of TriLink’s products in publications during a given year. Extending this to the present Top 10 human genes, I searched Google Scholar for each of these genes cross-indexed with TriLink, and then selected a publication for each case. The results of each search can be perused later using the link embedded in each gene name, but for now here are snippets of each selected publication along with the TriLink product used and a link to the article.

  1. TP53 Immunization with p53 for rejecting established tumors; phosphorothioate CpG oligos; Daftarian et al.
  2. TNF Antisense inhibition of TNF synthesis; phosphorothioate oligos; Zhaowei et al.
  3. EGFR Aptamer-siRNA inhibition of EGFR; 2’-fluoro CTP; Giangrande & coworkers.
  4. VEGFA Aptamer for VEGF; 2’-O-methyl NTPs; Burmeister et al.
  5. APOE Modified mRNA delivery; 1methylpseudouridine-5-triphosphate; Pardi et al.
  6. IL6 Biosensor for IL6 protein and DNA; biotinylated oligo; Wang et al.
  7. TGFB1 RNA-seq of leukemia cells; Cy3 and Cy5 labeled random 9-mers; Wilhelm et al.
  8. MTHFR Genotyping MTHFR mutants in blood; Molecular Beacon probes; Shi et al.
  9. 9. ESR1 SNP genotyping ESR1 associated with breast cancer; custom synthesized oligos as probes; Gold et al.
  10. AKT1 Antisense inhibition of AKT1; phosphorothioate oligos; Yoon et al.

Concluding Comments

When researching and writing this blog, several thoughts crossed my mind. Firstly, investigations of genes related to cancer in one way or another comprise a significant portion of The Top 10 literature. Given the prevalence and seriousness of cancer worldwide, this is not surprising, and perhaps expected.

Secondly, the news article in Nature that I cited in the introduction states that out of the 20,000 or so protein-coding genes in the human genome, only 100, which is only 0.5% of the 20,000 protein-coding genes, account for more than 25% of all the papers tagged in the NCBI/NLM PubMed database. The news item added that thousands of genes go unstudied in any given year, and quoted Helen Anne Curry, a science historian at the University of Cambridge, UK as saying ‘It’s revealing how much we don’t know about because we just don’t bother to research it.’ Although this reminds me a bit of the currently popular, self-evident saying “we don’t know about what we don’t know”, the implication is that we don’t know much about an extensive portion of the human protein-coding genome.

Thirdly, from a company perspective, it was evident that TriLink’s long-time expertise in modified nucleic acids has enabled, over the years, many investigations of all types related to these Top 10 genes, which is good thing.

Finally, from a personal perspective, it was gratifying to find that my early development of automated synthesis of phosphorothioate oligos evolved to provide ready access to this remarkably unique and useful class of modified nucleic acids pioneered by my long-time friend Fritz Eckstein.

As usual, your comments are welcomed.

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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.

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.