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.

Frightening Fungus Among Us

  • Clinical Alert for Candida auris (C. auris) Issued by CDC
  • US Concerned About C. auris Misidentification and Drug Resistance
  • Sequencing C. auris DNA in Clinical Samples is Preferred for Identification
Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

When I was a kid and didn’t know better, there was a supposedly funny rhyme that “there’s fungus among us.” While this saying is thankfully passé nowadays, the growing number of infections by a formerly obscure but deadly fungus is frightening. This so-called “superbug” is an antibiotic-resistant fungus called Candida auris (C. auris) that’s worth knowing about, and is the fungal focus of this blog.

First, Some Fungus Facts

Fungi are so distinct from plants and animals that they were allotted a biological ‘kingdom’ of their own in classification of life on earth, although that was only relatively recently, i.e. 1969. There are 99,000 know fungi, which exist in a wide diversity of sizes, shapes and complexity that extends from relatively simple unicellular microorganisms, such as yeasts and molds, to much more complex multicellular fungi, such as mushrooms and truffles.

It was previously thought that genomes of all fungi are derived from the genome of the model fungus Saccharomyces cerevisae, which has been used in winemaking, baking and brewing since ancient times. However, genome sequencing of more than 170 fungal species has revealed that, while the genome size of S. cerevisae is only ~12 Mb, seven species of fungus have genome sizes larger than 100 Mb. This is attributed to various evolutionary pressure-factors generating transposable elements, short sequence repeats, microsatellites, and genome duplication, and noncoding DNA.

Fungal cell walls are made up of intertwined fibers mostly comprised of long chains of chitosan, the same tough compound found in the exoskeletons of animals such as spiders, beetles and lobsters. The chitin in fungal cells is entangled with glucans and other wall components, such as proteins, forming a mass that protects the cell membrane behind it—and posing a formidable barrier against antifungal drugs.

Taken from Wikipedia.org

Taken from Wikipedia.org

In researching whether there are any nucleic acid drugs against fungi, I found one early patent by Isis (now Ionis) Pharmaceuticals for use of antisense phosphorothioate-modified oligonucleotides for the treatment of Candida infections, but virtually no other reports. I suspect that will change in the future as pathogenic fungi and other disease-causing microbes become more resistant to conventional drugs.

Fungal infections of the skin are very common and include athlete’s foot, jock itch, ringworm, and yeast infections. While these can usually be readily treated, infections caused by pathogenic fungi have reportedly risen drastically over the past few decades. Moreover, with the increase in the number of immunocompromised (burn, organ transplant, chemotherapy, HIV) patients, fungal infections have led to alarming mortality rates due to ever increasing phenomenon of multidrug resistance.

Segue to a Serious Situation

Emergence of drug-resistant fungi is, in part, the segue to the serious story of the present blog. The other part being incorrect identification of a certain fungus as being a common candida yeast, which is not only scary but seemingly inexcusable in today’s era of highly accurate PCR-based assays to accurately identify microorganisms. Here’s the situation in a nutshell.

  1. auris infection, which is associated with high mortality and is often resistant to multiple antifungal drugs, was first described in 2009 in Japan but has since been reported in countries throughout the world. Unlike many Candida infections, C auris is a hospital-acquired infection that is contracted from the environment or staff of a healthcare facility, and it can spread very quickly.

To determine whether C. auris is present in the United States and to prepare for the possibility of transmission, the Centers for Disease Control (CDC) and Prevention issued a clinical alert in June 2016 requesting that C. auris cases be reported.

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

This official alarm bell, if you will, was triggered by the following facts:

  • Many isolates are resistant to all three major classes of antifungal medications, a feature not found in other clinically relevant Candida
  • auris identification requires specialized methods such as a MALDI-TOF mass spectrometry or sequencing the 28s ribosomal DNA, as pictured below.
  • Using common methods, auris is often misidentified as other yeasts, which could lead to inappropriate treatments.

The CDC subsequently found that seven cases were identified in Illinois, Maryland, New York and New Jersey. Five of seven isolates were either misidentified initially as C. haemulonii or not identified beyond being Candida. Five of seven isolates were resistant to fluconazole; one of these isolates was resistant to amphotericin B, and another isolate was resistant to echinocandins. While no isolate was resistant to all three classes of antifungal medications, emergence of a new strain of C. auris that is would pose a serious public health issue.

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Based on currently available information, the CDC concluded that these cases of C. auris were acquired in the U.S., and several findings suggest that transmission occurred:

  • First, whole-genome sequencing results demonstrate that isolates from patients admitted to the same hospital in New Jersey were nearly identical, as were isolates from patients admitted to the same Illinois hospital.
  • Second, patients were colonized with auris on their skin and other body sites weeks to months after their initial infection, which could present opportunities for contamination of the health care environment.
  • Third, auris was isolated from samples taken from multiple surfaces in one patient’s health care environment, which further suggests that spread within health care settings is possible.

A related Fox News story adds that C. auris was found on a patient’s mattress, bedside table, bed rail, chair, and windowsill. Yikes!

While the above situation in the U.S. might not seem particularly worrisome to you, the potential for emergence of more infectious C. auris strains with higher lethality should be of concern. That has already reportedly occurred in several Asian countries and South Africa. Obviously, deployment of the best available methods for pathogen identification can, in principle, lessen the likelihood of the emergence and/or spread of C. auris in the U.S. and other countries.

Case for Point-of-Care C. auris Nanopore Sequencing?

Taken from extremtech.com 

Taken from extremtech.com

Regular readers of my previous blogs know that I’m an enthusiastic fan of the Oxford Nanopore Technologies minION sequencer, which is proving to be quite useful for characterizing pathogens in very remote regions on Earth—and even on the International Space Station to diagnose astronaut infections! Notwithstanding various current limitations for minION sequencing of microbes, it seems to me that it would be relatively straightforward to generate minION data for many available samples of pathogenic fungi and genetically related microbes to assess the feasibility using minION for faster, cheaper, better unambiguous identification of C. auris minION in centralized or Point-of-Care applications.

Taken from rnaseq.com

Taken from rnaseq.com

If you think this suggestion is farfetched, think again, after checking out these 2016 publications using minION:

The 51.4-Mb genome sequence of Calonectria pseudonaviculata for fungal plant pathogen diagnosis was obtain using minION.

The first report of the ~54 Mb eukaryotic genome sequence of Rhizoctonia solani, an important pathogenic fungal species of maize, was derived using minION.

Sequence data is generated in ~3.5 hours, and bacteria, viruses and fungi present in the sample of marijuana are classified to subspecies and strain level in a quantitative manner, without prior knowledge of the sample composition.

CDC on C. auris Status and FAQs

In the interest of concluding this blog with the most up-to-date and authoritative information, I consulted the CDC website and found statements and replies to FAQs that are well worth reading at this link.

As a scientist, my overriding question concerns the lack of adoption of improved microbiological methods by hospitals and clinics. The above noted misidentifications of C. auris infections resulting from use of flawed lab analyses seems unacceptable. Although I don’t know all the facts or statistics to generalize, I suspect that there are other incorrect lab analyses due to use of outdated methods. On the other hand, I’m hopeful that, with the FDA’s widely touted Strategic Plan for Moving Regulatory Science into the 21st Century, the section entitled Ensure FDA Readiness to Evaluate Innovative Emerging Technologies—think nanopore sequencing—becomes actionable, sooner rather than later.

Changing established—dare I say entrenched—clinical lab tests is not simple or easy, but if it doesn’t begin it won’t happen, about which I’m quite certain. I can only wonder why development of infectious disease analytical methods and treatments seem to require a crisis. Sadly, I think it boils down to the complexities and socio-political dynamics of who pays.

Frankly, it’s my personal opinion that maybe it’s time Thomas Jefferson’s philosophy about hammering guns into plows is directed to health care.

Postscript

After writing this blog, I learned that T2 Biosystems has received FDA approval to market in the U.S. the first direct blood test for detection of five yeast pathogens that cause bloodstream infections: Candida albicans and/or Candida tropicalis, Candida parapsilosis, Candida glabrata and/or Candida krusei.

Yeast bloodstream infections are a type of fungal infection that can lead to severe complications and even death if not treated rapidly. Traditional methods of detecting yeast pathogens in the bloodstream can require up to six days, and even more time to identify the specific type of yeast present. The T2Candida Panel and T2Dx Instrument (T2Candida) can identify these five common yeast pathogens from a single blood specimen within 3-5 hours.

T2Candida incorporates technologies that break the yeast cells apart, releasing the DNA for PCR amplification for detection by greatly simplified, miniaturized nuclear magnetic resonance (NMR) technology, as can be seen in this video.

In my opinion, this fascinating new technology is another example of what could be rapidly deployed toward detecting C. auris.

Jerry’s Favs from the Recent OTS Meeting

  • 12th Annual OTS Meeting in Montreal Très Excitant!
  • RNAi Approach to Treating Preeclampsia Spurred by Researchers’ Personal Experiences
  • Ionis Video for Spinal Muscular Atrophy Amazes the Audience
  • 30 years later, PS-Modified Oligonucleotides Continue to be Enabling!
montreal

City line of Montreal. Taken from oligotherapeutics.org

The 12th Annual Meeting of the Oligonucleotide Therapeutics Society (OTS) held on September 25-28, 2016 in French-speaking Montreal, Quebec, Canada brought together hundreds of enthusiastic investigators from around the world. All attendees share a common interest in oligo-based therapeutics, and we gratefully say merci beaucoup to the organizers of this very well run event.

Commenting here on my “favs” is limited by space, and highly subjective—by intent—focusing on only several impressions that struck me as worth sharing. Readers interested in perusing all of the lecture titles and speaker biosketches can do so at this link, which also lists corporate sponsors—including TriLink—and connects with the regularly updated OTS website.

Jerry’s balcony view of OTS 2016 presentations at the Centre Mont-Royal venue

Jerry’s balcony view of OTS 2016 presentations at the Centre Mont-Royal venue

Before getting to my selected topics, I wish to congratulate the OTS Board of Directors and Scientific Advisory Council for their ongoing valuable contributions to this society, and continuing efforts to include participation by the new generation of young investigators, who I’m sure will collectively make exciting advances in the field of oligonucleotide therapeutics. It was pleasure for me to me meet some of these “next gen” scientists, and learn about their current work, which is quite sophisticated by comparison to what I and others did in the early—dare I say primitive—era of antisense oligonucleotide drug discovery.

Toward Treating Preeclampsia: Personalized Drug Developers’ Stories

Preeclampsia (PE) is a disorder that occurs during pregnancy, affects both the mother and the fetus, and is characterized by elevated blood pressure, swelling and protein in the urine. According to a Preeclampsia Foundation fact sheet, every minute somewhere in the world a woman dies in pregnancy or childbirth, which amounts to more than 500,000 deaths each year. In addition, PE causes ~15% of premature births in industrialized countries and is the number one reason doctors decide to deliver a baby early.

Dr. Melissa J. Moore is a professor in the RNA Therapeutics Institute and the Department of Biochemistry and Molecular Pharmacology at the University of Massachusetts Medical School. Taken from hhmi.org

Dr. Melissa J. Moore is a professor in the RNA Therapeutics Institute and the Department of Biochemistry and Molecular Pharmacology at the University of Massachusetts Medical School. Taken from hhmi.org

In her OTS lecture on clinical development of an RNA interference (RNAi) approach to treat PE, Dr. Melissa J. Moore (pictured below) introduced an attention-grabbing personal element when she revealed her bout with PE. Coincidentally, she discovered that her then attending physician, Dr. S. Ananth Karumanchi, had begun his quest for identifying PE-causality at the molecular level prompted by his daughter’s dangerously premature birth due to PE. This turned out to be a truncated kinase receptor abbreviated sFlt1, which Moore described as Karumanchi’s break-through discovery for possible development of PE therapeutics.

The condensed version of these two remarkably interwoven, PE-related personal stories—that you can hear first-hand from Moore on YouTube—was an agreement between Moore and Karumanchi to collaborate on discovering whether double-stranded short-interfering RNA (siRNA) targeting sFlt1 could be useful as an RNAi-based PE therapeutic agent.

Melissa Moore’s ultrasound sonogram. Taken from her YouTube video

Melissa Moore’s ultrasound sonogram. Taken from her YouTube video

Moore went on to gratefully acknowledge her colleague at the RNA Therapeutics Institute, Prof. Anastasia Khvorova, for setting up “by hook or by crook” the first non-profit academic facility for large-scale production of siRNA suitable for clinical development. This led to designing and producing a nuclease-resistant siRNA comprised of 2’-OMe/2’-F ribonuceosides and phosphorothioates at the ends, with an attached hydrophobic cholesterol moiety for improved delivery.

Even more impressive—at least to me—was Moore’s ability to access pregnant baboons in a non-human primate model of PE. This is inherently difficult due to issues involving non-human primates for any research, and is technically much more challenging compared to, for example, infectious disease models. Dr. Moore showed lots of compelling results from her model studies, and concluded her talk by saying that clinical studies were planned.

In closing this section, it’s worth noting that Moderna—a leading mRNA therapeutics company—has recently announced its appointment of Dr. Moore as Chief Scientific Officer of Moderna’s mRNA Research Platform.

Ionis Pharmaceuticals Drug Video for Spinal Muscular Atrophy Amazes the Audience

Dr. Stanley T. Crooke. Taken from ionispharma.com

Dr. Stanley T. Crooke. Taken from ionispharma.com

This year’s OTS Lifetime Achievement Award address by Dr. Stanley T. Crooke, founder and CEO of Ionis Phamaceuticals (formerly Isis Pharmaceuticals), was presented to an auditorium packed with attendees who, like me, greatly admire Crooke’s many scientific and commercial contributions to the field over the past decades. These currently include more than 450 (!) scientific publications and 38 drugs in the pipeline, with 3 finishing Phase III—very impressive and promising indeed!

Ionis’ impact on providing efficacious oligonucleotide-based therapies to patients was conveyed most powerfully—in my opinion—by a video about Cameron, an infant SMA patient who was born with Spinal Muscular Atrophy (SMA). According to an NIH fact sheet, SMA is a genetic disease that causes weakness and wasting of the voluntary muscles in the arms and legs of infants and children. These disorders are linked to an abnormal or missing gene known as the survival motor neuron gene 1 (SMN1), without which motor neurons in the spinal cord degenerate and die.

As detailed elsewhere by Chiriboga et al., Nusinersen (aka ISIS-SMNRx or ISIS 396443) is a 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) designed to alter splicing of SMN2 mRNA and increase the amount of functional SMN protein produced, thus compensating for the genetic defect in the SMN1 gene. The cartoon shown below depicts how this ASO targets an hnRNP-A1/A2–dependent splicing silencer in intron 7 of the SMN pre-mRNA. Nusinersen displaces hnRNP proteins from this silencer site on the SMN2 pre-mRNA, facilitating accurate splicing of SMN2 transcripts (e.g., increasing the synthesis of transcripts containing exon 7) and resulting in increased production of full-length SMN protein.

Taken from researchgate.org after Chiriboga et al.

Taken from researchgate.org after Chiriboga et al.

Ionis and its commercial partner Biogen announced in August 2016 that Nusinersen met the primary endpoint pre-specified for the interim analysis of ENDEAR, the Phase 3 trial evaluating Nusinersen in infantile-onset (Type 1) SMA. The analysis found that infants receiving Nusinersen experienced a statistically significant improvement in the achievement of motor milestones compared to those who did not receive treatment. In September, Biogen announced that it applied for Priority Review by the FDA that, if granted, would shorten the review period of Nusinersen following the Agency’s acceptance of the NDA filing by Ionis and Biogen.

Cameron at 2+ years enjoying the moment with his mom. Taken from YouTube

Cameron at 2+ years enjoying the moment with his mom. Taken from YouTube

The amazing effect of Nusinersen warranting this Priority Review is best appreciated—in my opinion—by watching a YouTube video showing the progress of Cameron during his treatment. When viewing this video, keep in mind that Cameron’s Type I SMA is typically evident at birth or within the first few months, and that symptoms include floppy limbs and trunk, feeble movements of the arms and legs, swallowing and feeding difficulties, and impaired breathing. Sadly, the prognosis is poor for babies with SMA Type I. Most die within the first two years. But not so for Cameron, shown below at 2+ years, thanks to Nusinersen!

Phosphorothioate-Modified Oligonucleotides Continue to be Enabling—30 Years On!

men

Left: Prof. Fritz Eckstein. Taken from oligotherapeutics.org Right: Prof. Wojciech J. Stec. Taken from cbmm.lodz.pl

That nuclease-resistant phosphorothioate (PS)-modified internucleotide linkages have had an enabling influence on all manner of oligo-based drug development is evident from the first-ever OTS Lifetime Achievement Award in 2015 being given to Prof. Fritz Eckstein for his pioneering and life-long work on PS linkages in DNA and RNA. Following Fritz’s lead, and aided by the availability of ABI’s DNA synthesizer, Prof. Wojciech J. Stec and yours truly published the first completely automated method for synthesis of fully or partially PS-modified DNA oligos in 1984. This opened the door for early antisense experiments with PS-modified oligos, which have continued now for 30 years!

Perhaps surprisingly, the ever evolving field of oligonucleotide therapeutics continues to rely on PS-modifications in a myriad of mechanistically distinct strategies, such as “classic” antisense to mRNA, siRNA, anti-miRNAs, modulators of RNA splicing, etc. In this regard, Dr. Crooke’s concluding remarks on his goal of deciphering the “code” for protein binding to chemically modified ASO, led me to muse about the negatively charged P-Smoiety in such binding of PS-containing ASO.

Taken from zon.trilinkbiotech.com

Taken from zon.trilinkbiotech.com

My initial thought was that P-S likely enhances binding to positively charged amino acid moieties in proteins, due to greater polarizability vs. P-O, and that Sp or Rp P-Sstereochemistry may likely influence binding. Moreover, this spatial aspect of a binding “code” can now be studied using stereospecific synthesis of PS-ASO by either Stec’s OTP method or Wada’s oxazaphospholidine method. Time will tell—stay tuned!
As usual, your comments here are welcomed.

Zon on Zon’s Zebrafish

  • Leonard Zon Uses Zebrafish to “Fish” for Candidate Drug Compounds
  • Two Candidate Drugs are in Clinical Trials for Cancer Treatments
  • Zon Interviews L. Zon
Leonard I. Zon, M.D. Taken from pediatrics.mc.vanderbilt.edu.

Leonard I. Zon, M.D. Taken from pediatrics.mc.vanderbilt.edu.

The first time I was asked if I was related to the scientist Leonard Zon, I honestly had to reply that I didn’t know, and out of curiosity later looked up his publications, which were quite numerous for a then newish investigator. His current biosketch expertise includes pioneering research in the new fields of stem cell biology and cancer genetics. Dr. Leonard I. Zon is the Grousbeck Professor of Pediatric Medicine at Harvard Medical School, an Investigator with the Howard Hughes Medical Institute, and Director of the Stem Cell Program at Children’s Hospital Boston—that’s impressive!

I found Leonard Zon’s unusual zebrafish-based research and accomplishments therefrom definitely blogworthy, and the coincidences of both our surnames and involvement in science are kind of an unusual “double-doppelgänger,” if you will. In any case, it’s always a surprise to meet your double, even if only in name and profession.

A few regular readers of my blog will likely smile and think that Zon on Zon’s Zebrafish is yet another instance of my penchant for alliteration. However, reading the following snippets about Leonard Zon’s clever—dare I say zany—use of zebrafish for his research will illustrate why they are unusual, interesting and commercially viable.

On the other hand, I must admit that I too smiled at the thought of this unique opportunity to post an interview of one Zon by another Zon, which reminded me a bit of Zappa Plays Zappa. But I digress…

Zebrafish as a Model for Organogenesis

Leonard Zon currently has nearly 250 research publications listed in PubMed, which is a large number by any measure, but that’s even more impressive when you take into account that his first was in 2002—only 14 years ago. That translates to an average of about 3 publications every 2 months each year!

Zon’s inaugural publication in 2002 was an in-depth review in venerable Science in which the abstract presciently reads in part as follows:

Organs are specialized tissues used for enhanced physiology and environmental adaptation. The cells of the embryo are genetically programmed to establish organ form and function through conserved developmental modules. The zebrafish is a powerful model system that is poised to contribute to our basic understanding of vertebrate organogenesis. This review develops the theme of modules and illustrates how zebrafish have been particularly useful for understanding heart and blood formation.

As will be elaborated below, Zon’s most recent publication in 2016—also in Science—has extended the zebrafish model to now include melanoma. If you’re asking yourself, why use zebrafish, the answer is partly due to convenience derived from the unique features and accelerated life cycle of zebrafish.

Zebrafish life cycle. Taken from en.wikipedia.com.

Zebrafish life cycle. Taken from en.wikipedia.com.

Seen right, these advantages include its small size, easy care, and rapid generation time. In addition—and very importantly—the embryos and growing zebrafish are transparent, allowing for continuous observation of developing organs under the light microscope.

Mutagenesis screens allow examination of defects in early organogenesis and late organ function. These many advantages of investigating zebrafish—and Zon’s huge facility comprising thousands of tanks—are nicely explained and shown in a video, which I found well worth viewing. The video also includes Zon’s specially bred, virtually transparent species of zebrafish named—humorously—Casper, after Casper the Friendly Ghost. Pictures below are (a) the transparent Casper zebrafish; (b) the non-transparent wild-type zebrafish; and transparent Casper the Friendly Ghost, which brings back my childhood memories. But again I digress…let’s get back to science!

(a) Casper, (b) wild type zebrafish. Taken from nature.com. Transparent Casper the Friendly Ghost. Taken from enchantedamerica.wordpress.com

(a) Casper, (b) wild type zebrafish. Taken from nature.com. Transparent Casper the Friendly Ghost. Taken from enchantedamerica.wordpress.com

Aided by the availability of DNA sequence information for the zebrafish genome, researchers have published ~2,000 (!) reports dealing with antisense gene “knockdown” using phosphorodiamidate oligonucleotides. By numerical coincidence, the first such report appeared in 2000, and was prophetically entitled Effective targeted gene ‘knockdown’ in zebrafish.

Taken from igtrcn.org.

Taken from igtrcn.org.

As shown below, these rather unusual oligonucleotides—dubbed “morpholinos” by resemblance of the 6-membered ring to morpholine—can be injected directly into zebrafish embryos. Interested readers can consult a detailed “how to” guide on use of morpholinos in zebrafish.

Going forward, however, I expect that uber-hot CRSPR/Cas9 gene editing will be widely adopted, based on the titles of these two pioneering publications in 2016:

Stem Cells and Beyond

Now that we know a bit about Zon’s zebrafish and how to knockdown or edit a gene for functional genomics (aka “gene functionation”), let’s zero in on what Zon studies and how he does that. In a nutshell, the overarching science in Zon’s lab deals with stem cells, which are undifferentiated cells that can differentiate into specialized cells, as well as divide to produce more stem cells, as depicted below.

Taken from nas-sites.org.

Taken from nas-sites.org.

According to Zon’s website, the hematopoietic system that forms various types of blood cells is an excellent model for understanding tissue stem cells. This conceptual relationship is very important because it provides insight to cell differentiation regulation, and involvement in aging, disease, and oncogenesis. In addition, better understandings of the regulation of hematopoietic stem cell biology and lineage differentiation improves diagnosis and treatment of human hematopoietic disorders (aka “blood cancers”) and bone marrow transplantation therapies.

Differentiation of different blood cells from hematopoietic stem cell to mature cells. Taken from wikipwedia.com.

Differentiation of different blood cells from hematopoietic stem cell to mature cells. Taken from wikipwedia.com.

From Zebrafish to Clinic

Scientific theory is nice, but success is best, and Leonard Zon’s theory of using zebrafish to manipulate human stem cells for discovering therapies seems indeed to be headed for success, according to an article in the Harvard Gazette.

Zon and others at the Harvard Stem Cell Institute (HSCI) have published initial results of a Phase Ib safety study wherein 12 adult patients undergoing umbilical cord blood transplantation received two umbilical cord blood units, one untreated and the other treated with the small molecule 16,16-dimethyl prostaglandin E2 (dmPGE2). This molecule had been found in Zon’s zebrafish screen, which I’ll outline below.

Fate Therapeutics, a San Diego-based biopharmaceutical company of which Zon is a co-founder, sponsored the investigational new drug (IND) application, under which the aforementioned clinical program was conducted, thus translating his research findings from the laboratory—dare I say tank—into the clinic.

Zon’s Zebrafish Yield New Approaches to Treat Muscular Dystrophies

According to NIH’s National Institute of Neurological Disorders and Stroke website, muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. It adds that there is no specific treatment to stop or reverse any form of MD. Consequently, MD is a compelling target for new drug discovery, and Zon’s zebrafish are being used for such discovery in the following way.

Each zebrafish produces about 200 (!) eggs per week, so Zon’s lab collects and deposits a single egg into each well of a multi-well plate for individual but parallelized treatment with individual chemicals to screen for effects on differentiation, thus screening many compounds in a speedy manner.

Zon and coworkers concluded that “these studies reveal functionally conserved pathways regulating myogenesis across species [zebrafish, mouse, and human] and identify chemical compounds that…differentiate human iPSCs into engraftable muscle.”

Let’s hope that human clinical trials using this novel therapeutic approach enabled by Zon’s zebrafish soon prove successful for treatment of MD.

Zon’s Zebrafish Also Enable Elucidation of Melanoma Development

According to a fact page by the American Cancer Society, skin cancer is the most common of all cancers. About 3.5 million cases of basal and squamous cell skin cancer are diagnosed in this country each year. Melanoma, a more dangerous type of skin cancer, will account for more than 73,000 cases of skin cancer in 2015.

Zon and a large group of 18 coworkers have recently reported in venerable Science work that has been heralded in a New York Times article. This study is very comprehensive and involves lots of “heavy duty” molecular and cellular biology, which you can read in detail in the aforementioned linked article. Snippets of the key findings are as follows.

  • Benign melanocytic skin cells carry oncogenic BRAF-V600E mutations and can be considered a “cancerized” field of melanocytes, but they rarely convert to melanoma.
  • In an effort to define events that initiate cancer, they used a melanoma model in the zebrafish in which the human BRAF-V600E oncogene is driven by the melanocyte-specific mitfa
  • When bred into a p53 mutant background, these fish develop melanoma tumors over the course of many months.
  • The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and is specifically reexpressed only in melanoma tumors, making it an ideal candidate for tracking melanoma from initiation onward.
  • As show below, they developed a crestin:EGFP reporter that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors.
  • They show through live imaging of transgenic zebrafish crestin reporters that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model.
Taken from Zon and coworkers Science 2016.

Taken from Zon and coworkers Science 2016.

Zon Interviews L. Zon

Dr. Zon and some of his 4,000 zebrafish tanks. Taken from bizjournals.com.

Dr. Zon and some of his 4,000 zebrafish tanks. Taken from bizjournals.com.

After researching Leonard Zon’s aforementioned unique—and promising—use of zebrafish to advance basic science and discover new therapies, I contacted him by email to “interview” him, regarding several points. My questions (JZ) and his answers (LZ) are as follows.

JZ: Aside from the cord blood clinical trial mentioned in the Harvard Gazette, are any other human clinical trials being carried out based on your zebrafish findings?

LZ: We have had two chemicals discovered in zebrafish, and ultimately went to a clinical trial. The first was a di-methyl form of PGE2 for cord blood transplantation for leukemia. The second was leflunomide, an arthritis drug that paused transcription in neural crest cells and is being evaluated for metastatic melanoma.

JZ: Is Fate Therapeutics your only startup company?

LZ: I started Scholar Rock about 3 years ago. This company is targeting the TGF-B family of ligands. Has about 25 employees, and is in Cambridge. I am about to start a third company.

JZ: Is your zebrafish method for screening chemicals patented?

LZ: We have patents on several screening methods, but in general we patent the chemicals we find.

JZ: Zebrafish offer many reported advantages for your kind of research, but what is a primary disadvantage?

LZ: The major disadvantage of the zebrafish is that the system occasionally lacks definition. For instance, in the blood system, we have one monoclonal antibody against one epitope. We really need to create reagents for the field that brings it in line with other systems such as mice and humans.

JZ: How many tanks and zebrafish are maintained in your two labs?

LZ: We have 4000 tanks and about 300,000 fish.

JZ: Did you name your transparent Casper zebrafish after Casper the Friendly Ghost?

LZ: Absolutely.

In closing, I should add that the huge amount of information on zebrafish as a model organism for human disease and drug discovery from many labs has been centralized and organized in a database that is available through The Zebrafish Information Network (ZFIN) for researchers to share at the ZFIN Community Wiki.

I hope that you found this blog interesting, and I welcome your comments.

Joe Zon with some of his famous guitars at NAMM Show 2015. Taken from ilan.me.

Joe Zon with some of his famous guitars at NAMM Show 2015. Taken from ilan.me.

Postscript

Truth be told, compared to being asked if I’m related to Leonard Zon, I’m more frequently asked if I’m related to Zon guitars, which apparently are quite well known, and are produced in Redwood City, CA by Joe Zon, who is pictured below. My reply to that frequent question is that I don’t know if I’m related, but will someday look into that, as well as whether I’m distantly related to Leonard Zon.

Anti-Antisense Makes Sense

  • Antisense Oligos Blocking Antisense RNAs Makes Proteins for Therapeutics
  • New Antisense Approach is Analogous to (-1) x (-1) = 1
  • Drug Company OPKO-CURNA is Taking This Approach to the Clinic

I apologize for the somewhat cryptic headline and mathematical byline for this blog, but they really do encapsulate the following: the underlying molecular biology involves a novel antisense oligo approach to “turn on” a protein, in contrast to all previous use of an antisense oligo to “turn off” a protein. I’ll rationalize the mathematical analogy later.

Taken from nature.com

Taken from nature.com

Recognizing the potential therapeutic value of this “turn-on” mechanism, a startup company with the quirky name cuRNA—a contraction for “cure” and “RNA”—was founded, and was acquired by OPKO—a health care conglomerate—and renamed OPKO-CURNA.

What follows is a condensed version of the full story of yet another example of how basic discoveries in nucleic acid research have “morphed” into new therapeutic strategies, which in turn lead to the genesis of small start-ups that oftentimes get acquired by bigger pharma companies. All of these kinds of stories include variations on a theme that are both informative and interesting—in my opinion.

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Reflections on Advances in Medicinal Oligonucleotides

  • Oligos Are Not “Magic Bullets”
  • Oligos Have, Nevertheless, Enabled New Drug Paradigms
  • Oligos Continue to Attract Significant Corporate Investments

Reflections

The 10th Annual Meeting of the Oligonucelotide Therapeutics Society (OTS) is in full swing today in San Diego, CA where it began on Oct 12 and concludes on Oct 15. Having worked on antisense oligos since the early days (~30 years ago) participating in this meeting led me to several thoughts that I wish to share with you in this post.

First of all, contrary to many sceptics in those early days, the concept of using synthetic oligonucleotides as an entirely new class of medicinal agents has not only survived but also greatly expanded in terms of the biological target/mechanism of action and types of oligo constructs used—each with a seemingly endless array of chemical modifications to evaluate. In approximate chronological order of discovery, these targets and the types of oligos they have now come to include are listed below coincidentally, most of these are represented in the 2014 OTS agenda.

Target
mRNA
dsDNA
transcription factors
immunostimulation
proteins
splice junctions
RNA interference
Oligo Type
antisense
triplex
dsDNA decoys
CpG
aptamers
blockers
siRNA

 

 

 

 

 

Secondly, while it has been possible for oligo chemists to design and synthesize a plethora of modified oligos to achieve optimized nuclease stability, binding to target, etc., efficient delivery has remained the single most challenging problem to deal with. In talks on medicinal oligos, this situation is oftentimes eluded to as something to the effect of “there are only three remaining problems to solve: delivery, delivery, and delivery.”

Lastly, contrary to early hopes of being Dr. Ehrlich’s “magic bullets” (see caption below), oligos didn’t quite prove to be the new paradigm for a speedy concept-to-clinic solution. As all of us in the oligo world know, oligo-based therapeutics have encountered long and costly R&D timelines and clinical development paths typical of all other classes of therapeutic compounds.

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