CRISPR in the Clinic…Coming Soon

  • Trio of CRISPR Discoverers Awarded a $1 Million Kavli Prize
  • CRISPR Therapeutics, a Startup Company, Will Soon Start Clinical Trials
  • New Issue: Concerns for Cancer

Over the past few years, I have periodically blogged about CRISPR-based gene editing, which has been arguably the hottest trending topic in nucleic acid-targeted therapy for about the past five years or so. The catalyst for this burst of publications was a 2012 report in Science on a study led by Doudna and Charpentier (see below). The study focused on the potential utility of CRISPR-Cas9 for genome editing, and it currently has over 5000 citations in Google Scholar. There are ~7400 articles in PubMed indexed to CRISPR, and it is evident from my chart shown here that there is strong growth in the annual number of CRISPR publications in the PubMed database.

Number of CRISPR publications in PubMed

In May 2018, three pioneers in CRISPR technology—Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin, Virginijus Šikšnys (see Footnote) of Vilnius University in Lithuania, and Jennifer Doudna of the University of California, Berkeley—were awarded the $1 million Kavli Prize in Nanomedicine. This highly prestigious prize from The Norwegian Academy of Science and Letters was awarded “for the invention of CRISPR-Cas9, a precise nanotool for editing DNA, causing a revolution in biology, agriculture, and medicine.”

Emmanuelle Charpentier, Virginijus Šikšnys and Jennifer Doudna (left to right). Taken from quantamagazine.org

As far as invention goes, there has been continued litigation, recently summarized in a GEN interview with law professor Jacob Sherkow titled CRISPR in the Courthouse. The University of California, Berkeley (UC) and the Broad Institute of MIT and Harvard are at odds over foundational patents covering CRISPR-Cas9. Interested readers should consult “late breaking news,” which covers the recent issuance of a US patent to UC and its partners.

Notwithstanding unresolved intellectual property matters, so-called “surrogate companies” for the holders of these key patents include Editas Medicine (MIT/Harvard), Caribou Biosciences/Intellia Therapeutics (UC and University of Vienna), and CRISPR Therapeutics (Emmanuelle Charpentier), the latter of which is the focus of the present blog. As you’ll read below, CRISPR Therapeutics is developing a “CTX001” approach for the treatment of Sickle cell disease and β-thalassemia for clinical trials this year, which is a highly anticipated milestone—both scientifically and commercially.

CRISPR Therapeutics CTX001

Sickle cell disease and β-thalassemia are caused by genetic mutations in the β-globin gene, which codes for the β subunit of hemoglobin that, as depicted below, is the oxygen carrying component of red blood cells. In these diseases, hemoglobin is missing or defective, which results in devastating medical problems. The approach developed by CRISPR Therapeutics is designed to mimic the presence of fetal hemoglobin (HbF; aka γ-globin) that is present in newborn babies. HbF is a form of hemoglobin that is quickly replaced by adult hemoglobin. However, in rare cases where HbF persists in adults, it provides a protective effect for those who have Sickle cell disease and β-thalassemia.

Taken from socratic.org

CTX001 is an ex vivo therapy in which autologous (i.e. self-donated) cells are harvested directly from the patient. CRISPR Therapeutics then applies its gene-editing technology to the cells outside of the body, making a single genetic change designed to increase HbF levels in a patient’s own blood cells. The edited cells are then reinfused and are expected to produce red blood cells that contain HbF in the patient’s body, thus overcoming the hemoglobin deficiencies caused by these diseases.

The gene-editing mechanism for CTX001 presented by CRISPR Therapeutics at the American Society of Hematology (ASH) in December 2017 is depicted below. In researching this CRISPR-based mechanism, I found a publication by Bjurström et al. that helps to better understand this depiction. In brief, the zinc-finger transcriptional factor BCL11A has been shown to silence HbF genes in human cells during development, and thus directly regulates HbF switching.

Taken from CRISPR Therapeutics

BCL11A silences HbF by associating with other known γ-globin transcriptional repressors. The gene binds to the locus control region as well as other intergenic sites, which prevents the interaction between the locus control region and the HbF globin gene required for fetal globin expression. Using a guide RNA and Cas9 to enable permanent site-specific genome engineering through a DNA repair pathway, knockdown of the BCL11A gene can be an effective strategy for reactivating HbF and restoring functional erythrocytes.

The aforementioned ASH presentation by CRISPR Therapeutics also includes an overview of Sickle cell disease and β-thalassemia, as shown here. According to an informative historical article that I found, Sickle cell disease and β-thalassemia are related genetic disorders that can cause fatigue, jaundice, and episodes of pain ranging from mild to very severe. They are inherited, and usually both parents must pass on an abnormal gene in order for a child to have the disease. Much more genetic information on these two disorders is available on the NIH Genetics Home Reference.

Taken from CRISPR Therapeutics

CRISPR Therapeutics Clinical Studies Status

The December 2017 ASH presentation by CRISPR Therapeutics received widespread media coverage that heralded the highly anticipated “bench-to-bedside” transition for CRISPR technology. CTX001 was able to efficiently edit the target gene in more than 90 percent of hematopoietic stem cells to achieve about 40 percent of HbF production, which investigators believe is sufficient to improve a patient’s symptoms. Study results also showed that CTX001 affects only cells at the target site and that it has no off-target effects on hematopoietic stem cells, thereby appearing to be a safe potential treatment.

These positive results prompted CRISPR Therapeutics to start a collaboration with Vertex Pharmaceuticals to develop and commercialize CTX001 treatment of Sickle cell disease and β-thalassemia. It was also announced that CRISPR Therapeutics and Vertex are planning to submit an investigational new drug (IND) application to the Food and Drug Administration (FDA) to start a Phase 1/2 clinical trial in Sickle cell disease in the United States in 2018. In addition, CRISPR Therapeutics also submitted a clinical trial application (CTA) for CTX001 to advance into a Phase 1/2 clinical trial in patients with β-thalassemia in Europe in 2018. This trial will evaluate the safety and effectiveness of CTX001 in adult patients with transfusion-dependent β-thalassemia.

After the above announcement, News Atlas reported that the FDA placed a clinical hold on this Phase1/2 trial of CTX001 pending, according to CRISPR Therapeutics, ‘the resolution of certain questions that will be provided by the FDA as part of its review of the IND.’

Concerns for Cancer

In studies published in June 2018 in venerable Nature Medicine, researchers from Sweden’s Karolinska Institute and, separately, Novartis, found that cells whose genomes are successfully edited by CRISPR-Cas9 have the potential to seed tumors inside a patient. CRISPR-Cas9 works by cutting both strands of the DNA double helix. That “injury” causes a cell to activate a gene called p53, which has been called the “Guardian Angel of the Genome” and is the most studied of all human genes, which you can read about in one of my previous blogs.

Whichever action p53 takes, the consequence is the same: CRISPR doesn’t work as intended because the genome edit is mended, or the cell dies. The flip-side of p53 repairing CRISPR edits, or killing cells that accept the edits, is that cells that survive with the edits do so because they have a dysfunctional p53. The reason why that could be a problem is that p53 dysfunction can cause cancer. 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.

As a result, the Novartis paper concludes that “it will be critical to ensure that [genome-edited cells] have a functional p53 before and after [genome] engineering.” The Karolinska team warns that p53 and related genes “should be monitored when developing cell-based therapies utilizing CRISPR-Cas9.”

An article in statnews.com quotes the CEO of CRISPR Therapeutics, Sam Kulkarni, as saying that these p53 findings are “something we need to pay attention to, especially as CRISPR expands to more diseases. We need to do the work and make sure edited cells returned to patients don’t become cancerous.”

Closing Comments

Taken from highwaysupply.net

Many years ago, I was among the early investigators of antisense therapeutics, which at the time was viewed as a new paradigm that would enable faster bench-to bedside, compared to traditional small molecule drug development. In reality, the antisense approach encountered unforeseen complications and required ~30 years of development to reach demonstrable clinical utility, which I previously wrote about in another blog. Short-interfering RNA (siRNA)-based therapeutics also encountered similar struggles.

While past history is not a predictor of the future, in my humble opinion, CRISPR-based clinical strategies will continue to have to deal with unexpected issues, such as the above p53 situation. While I remain hopefully optimistic about future clinical successes for CRISPR, I won’t be surprised if some of these achievements come slower than currently anticipated.

As usual, your comments are welcomed.

Footnote

According to June 8, 2018 Science News at a Glance, Virginijus Šikšnys, whose role in the invention of the revolutionary genome editor CRISPR has often been overlooked, received some vindication when he was named a co-winner of the prestigious Kavli Prize in Nanoscience. Šikšnys will share the $1 million award with Doudna and Charpentier, who have received far more attention. Šikšnys first showed that the CRISPR system could be transferred from one bacterium to another. And like Doudna and Charpentier, he independently designed a way to steer the CRISPR complex to specific targets on a genome, which he called “directed DNA surgery.”

SaveSave

SaveSave

SaveSave

Nature’s Number One

  • David Liu is Nature Magazine’s Number One Person Who Mattered in 2017
  • Liu’s Team Has Modified CRISPR to Achieve Single-Base Editing of the Human Genome
  • This Opens the Door for Developing Therapies for Tens of Thousands of Human Genetic Diseases

Nature magazine is considered by many—including yours truly—to be among the best sources of scientifically related news, publications, and editorials. Consequently, when I read the cover of a recent issue of Nature featuring its picks for the “ten people who mattered” in 2017, I was immediately intrigued, and felt compelled to read about who and why these persons were selected—especially for the uber-prestigious number one pick. You can read about all these folks later by clicking here, but for now I’ll focus only on David Liu, who was chosen by Nature as the number one person who mattered in 2017, which is a very special accolade.

The “Gene Corrector”

Referring to Liu as the “Gene Corrector” was Nature magazine’s way of concisely encapsulating the fact that Liu’s laboratory was able to take the already well-known CRISPR system, which I’ve blogged about extensively, to its highest possible pinnacle of performance. Namely, editing only a single nucleotide in the entire human genome comprised of six billion nucleotides, which is the ultimate in specificity for human genome editing. How this was achieved is briefly outlined in the next section, but before getting to that I think it’s nice to put a face with a name, and also include key contributors to this remarkable feat.

According to news from the Broad Institute, which along with Harvard and Howard Hughes Medical Institute are Liu’s multi-academic affiliations, this recent landmark achievement involved contributions by Nicole Gaudelli, currently a postdoctoral fellow in Liu’s lab; Alexis Komor, a former postdoctoral fellow in Liu’s lab who is now an assistant professor at the University of California San Diego; current graduate student Holly Rees; former graduate students Michael Packer and Ahmed Badran, and former postdoctoral fellow David Bryson.

Holly Rees, David Liu, and Nicole Gaudelli (Credit: Casey Atkins). Taken from broadinstitute.org

Evolution of Base Editing

Evolution of a “base editor” by David Liu has involved a progression of investigations that began in 2013 when Liu joined a host of other luminaries in founding a company now called Editas Medicine to develop treatments based on CRISPR technology. It became evident that despite the great potential for gene editing using CRISPR technology, clinical applications could be limited by the unpredictability of CRISPR/Cas9. Although the Cas9 enzyme cuts DNA where directed by guide RNA, researchers must rely on the cells’ own DNA-repair systems to fix the break, which can create a variety of different edits to the genome.

Liu’s lab looked for ways to improve on that. In 2016, then postdoc Alexis Komor and others on Liu’s team reported its first base editor. They used engineered fusions of CRISPR/dCas9 (a catalytically “dead” Cas9 mutant) and a naturally occurring cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA. These fusion constructs do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, which is copied by polymerases into DNA as T, thereby thus converting a C•G base pair into a T•A within a window of approximately five nucleotides.

Taken from Komor et al. Nature 2016.

The approach has since been deployed in a range of organisms, from wheat to zebrafish and mice. And in September 2017, researchers in China reported that they had used Liu’s base editor to correct a single-letter mutation, or point mutation, for a blood disorder (β-thalassemia) in human embryos, although the edited embryos were not allowed to develop further.

According to the Nature article, Liu postdoc Nicole Gaudelli was eager to build on that work and create an analogous system that could instead deaminate adenine to produce inosine (I). Within the constraints of a polymerase active site, inosine pairs with C and therefore is read or replicated as G. The original A•T base pair is thus replaced with a G•C base pair at the target site. However, no naturally occurring enzymes are known to deaminate adenine in DNA. Nature adds that Gaudelli was therefore proposing to “break a cardinal rule in the Liu lab: no one takes on a project if the first step is to create a new enzyme. The risk of lost time and failure is too high.” Fortunately, Liu nevertheless encouraged her to pursue this risky challenge.

Taken from Gaudelli et al. Nature 2017.

The Broad news article quotes Gaudelli as saying the main challenge for her while developing an adenine base editor (ABE) was “overcoming the psychological hurdle of whether or not ABE could go from concept to reality, since the key component of the editor did not exist naturally and had to be evolved in our lab. It was important to keep the faith that we could not only dream of such a molecular machine, but also build it.”

Building the required hypothetical deoxyadenosine deaminase turned out to be a difficult task that necessitated considerable, sophisticated molecular biological engineering that is more than I can succinctly summarize here. Interested readers will have to consult details in the publication by Guadelli et al. that describes the strategy and execution of numerous rounds of this enzyme evolution and engineering.

During this process they characterized the most promising ABEs from later rounds in depth by choosing a set of 17 human genomic targets that place a target A at position 5 or 7 of the protospacer (see above scheme) and collectively included all possible NAN sequence contexts, wherein N = A, G, C or T. The base editing efficiency of the most active editor (ABE7.10) overall averaged 53 ± 4% at the 17 sites tested, exceeded 50% at 11 of these sites, and ranged from 34–68%. These results were said to compare favorably to the typical C•G to T•A editing efficiency reported by Komor et al.

Guadelli et al. determined that the activity windows of late-stage variants are approximately 4–6 nucleotides wide, from approximately protospacer positions 4 to 7 for ABE7.10, and positions 4 to 9 for several other ABEs. Importantly, they concluded that “the precise editing window boundaries can vary in a target-dependent manner,” which to me implies that application of this methodology will require case-by-case genetic disease-specific optimization with potentially varying degrees of success.

Guadelli et al. did not detect any apparent ABE-induced A•T to G•C DNA editing outside on-target or off-target protospacers following ABE treatment. Although additional studies were said to be needed to examine possible untoward RNA editing by ABEs, they observed no elevated adenine mutation rate among four abundant mRNAs in ABE7.10-treated HEK293T cells compared to untreated cells, nor any apparent ABE toxicity in bacterial or human cells under the conditions investigated.

Editing Disease-Relevant Mutations

Taken from ironitout.org

Finally, Guadelli et al. tested the potential of ABEs to introduce disease-suppressing mutations to correct pathogenic mutations in human cells. In one example, the genetic iron storage disorder hereditary hemochromatosis (HHC) was tested. HHC is commonly caused by a G to A mutation at nucleotide 845 in the human HFE gene, resulting in a C282Y substitution in the HFE protein that leads to excessive iron absorption and potentially life-threatening elevation of serum ferritin.

They transfected DNA encoding ABE7.10 and a guide RNA that places the target adenine at protospacer position 5 into an immortalized lymphoblastoid cell line harboring the HFE C282Y genomic mutation. Editing efficiency was measured by high-throughput DNA sequencing of genomic DNA, which showed clean conversion of the Tyr282 codon to Cys282 in 28% of sequencing reads from transfected cells, with no evidence of undesired editing or indels at the on-target locus.

Concluding Remarks

While the development of ABE is an exciting step forward in base editing, more work remains before base editing can be used to treat patients with genetic diseases, including tests of safety, efficacy, and side effects. “Creating a machine that makes the genetic change you need to treat a disease is an important step forward, but it’s only one part of what’s needed to treat a patient,” said Liu in the Broad news item. “We still have to deliver that machine, we have to test its safety, we have to assess its beneficial effects in animals and patients and weigh them against any side effects—we need to do many more things.”

“But having the machine is a good start.”

I fully agree, and hope that you will too.

As usual, your comments are welcomed.

Addendum

After writing this blog, it was announced that there is now a specialty journal dedicated to CRISPR-related investigations. The February 2018 inaugural issue of this new journal—aptly named The CRISPR Journal—is shown below.

SaveSave

Aptamers and Clinical Applications

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

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

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

Overview of Aptamer Functional Versatility

Taken from genelink.com

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

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

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

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

Aptamer Drugs Listed in ClinicalTrials.Gov

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

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

Taken from aidsinfo.nih.gov

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

Status of Aptamer Studies in ClinicalTrials.Gov

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

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

Taken from doctorabidi.com

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

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

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

Table of Active Studies Listed in ClinicalTrials.Gov in 2017

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

 

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

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

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

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

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

Conclusions and Prospects

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

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

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

As usual, your comments are welcomed.

Footnote

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

Taken from iomers.net

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

SaveSave

SaveSave

SaveSave

SaveSave

SaveSave

Jerry’s Favs from the Recent 7th Cambridge Symposium

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

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

Taken from na-cb.co.uk

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

Taken from worldachitecture.org

Overview of the Symposium

Mike Gait. Taken from histmodbiomed.org

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

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

Subject areas this year included:

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

Marv Caruthers. Taken from colorado.edu

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

Jerry’s Favs from the Symposium

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

DNA Can Function as an Enzyme!

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

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

Taken from Silverman Acc Chem Res (2015)

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

RNA Polymerase Activity Without Proteins!

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

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

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

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

Systemic Brain Delivery of Therapeutic Oligos!

Taken from igtrcn.org

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

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

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

Parting Thoughts and The Eagle

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

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

As usual, your comments are welcomed.

Personal photo using a Samsung Galaxy S8

SaveSave

SaveSave

SaveSave

Advances in Aptamer Applications – Part 2

  • Top Cited Aptamer Publications Over the Past Three Years
  • Jerry’s Picks for Top 3 Aptamer Publications So Far This Year
  • TriLink Products Cited in Numerous Aptamer Publications

Aptamers are highly structured nucleic acids that bind to a specific target molecule. RNA or DNA aptamers are usually selected from a very large pool (aka library) of random sequences, and can be comprised of either natural and/or chemically modified nucleotides. My first blog on aptamers was titled Aptamers: Chemistry Bests Mother Nature’s Antibodies. This purposefully provocative claim was intended to emphasize the growing body of evidence that collectively indicates aptamers can perform better than antibodies in many applications.

NMR-derived structures of aptamers binding to either a large protein or small molecule. Taken from genelink .com

Because it has been nearly four years since that boastful blog in 2013, I thought it was time to survey aptamer applications published since then to comment on what has been trending or is otherwise notable. I found more than 1,500 articles in PubMed for 2014 through 2017 (estimate) that have the search term “aptamer” in the title or abstract. Given this huge number of publications, I used Google Scholar citation frequency as a numerical indicator of interest, importance and/or impact for these publications in each year. I also decided to focus on original publications that, by definition, excludes review articles. 

Top 3 Cited Publications in 2014

  1. Activatable fluorescence/MRI bimodal platform for tumor cell imaging via MnO2 nanosheet–aptamer nanoprobe (109 citations)

This Chinese team of researchers led by uber-prolific Weihong Tan, about whom I’ve previously blogged, designed a novel methodology for imaging tumor cells using quenched-fluorescent aptamers. In the presence of target cells, the binding of these “dark” aptamers to cell surface markers weakens the adsorption of aptamers on MnO2 nanosheets causing partial fluorescence recovery (i.e., unquenching), thus illuminating the target cells, as well as facilitating endocytosis into target cells. After endocytosis, reduction of MnO2 nanosheets by glutathione further activates the fluorescence signals and generates large amounts of Mn2+ ions as a contrast agent for magnetic resonance imaging (MRI).

Taken from pubs.rsc.org

  1. A phase II trial of the nucleolin-targeted DNA aptamer AS1411 in metastatic refractory renal cell carcinoma (88 citations)

Taken from mct.aacr.org

The anticancer mechanism of action for DNA aptamer AS1411, which has multiple G-quadruplex moieties that disrupt cancer cell replication following nucleolin-mediated uptake, is depicted below and detailed elsewhere. In this clinical study, it was found that AS1411 appears to have limited activity in patients with metastatic renal cell carcinoma. However, rare, dramatic and durable responses can be observed and toxicity is low. Further studies with AS1411 and other nucleolin-targeted compounds may benefit from efforts to discover predictive biomarkers for response.

  1. An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1 (61 citations)

Aflatoxin B₁ structure. Taken from wikipedia.org

Aflatoxin B₁ (AFB1) produced by Aspergillus flavus and A. parasiticus is considered the most toxic aflatoxin and it is highly implicated in hepatocellular carcinoma in humans. In this work by Korean researchers, a rapid and simple dipstick assay based on an aptamer has been developed for determination of AFB1 contamination in food. The dipstick assay format was based on a competitive reaction of a biotin-modified aptamer specific to AFB1 between target and Cy5-modified DNA probes. Streptavidin and anti-Cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min.

Top 3 Cited Publications in 2015

  1. Aptamer-conjugated silver nanoparticles for electrochemical dual-aptamer-based sandwich detection of staphylococcus aureus (63 citations)

Taken from sciencedirect .com

Staphylococcus aureus (S. aureus) is one of the most important human pathogens and causes numerous illnesses. This report by Iranian researchers describes a sensitive and highly selective dual-aptamer-based sandwich immunosensor for the detection of S. aureus. As depicted below, a biotinylated primary anti-S.aureus aptamer was immobilized on streptavidin coated magnetic beads (MB), which serves as a capture probe. A secondary anti-S.aureus aptamer was conjugated to silver (Ag) nanoparticles such that, in the presence of target bacterium, a sandwich complex is formed on the MB surface and the electrochemical signal of Ag is measured by anodic stripping voltammetry.

  1. Aptamer-based fluorescence biosensor for chloramphenicol determination using upconversion nanoparticles (59 citations)

Chloramphenicol. Taken from Wikipedia .com

Chloramphenicol (CAP) shown below is a naturally occurring antibiotic that is artificially manufactured for use in veterinary and human medicine. Due to its adverse effects in humans, use of the antibiotic is restricted and, in Europe, ‘zero tolerance’ for CAP in food products has been legislated. In this report by Chinese researchers, detection of CAP uses aptamer-conjugated magnetic nanoparticles for both recognition and concentration, together with upconversion nanoparticles for detection. The method was validated for measurement of CAP in milk vs. a commercially available enzyme-linked immunosorbent assay (ELISA) method.

  1. A new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of Staphylococcus aureus (48 citations)

Taken from mdpi .com

This work by Chinese investigators describes a novel aptamer/graphene interdigitated gold electrode piezoelectric sensor for detecting S. aureus by binding to the aptamer, which is immobilized on the graphene via the π–π stacking of DNA bases, as depicted below. When S. aureus is present, aptamer dissociates from the graphene and thus leads to change of oscillator frequency of the piezoelectric sensor.

Top 3 Cited Publications in 2016

  1. Aptamer–MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen (38 citations)

This study in the UK uses a thiolated DNA aptamer for prostate specific antigen (PSA) immobilized on the surface of a gold electrode. Controlled electropolymerization of dopamine around the complex served to create an imprint of the complex following removal of PSA. This molecularly imprinted polymer (MIP) cavity was found to act synergistically with the embedded aptamer to provide recognition properties superior to that of aptamer alone. A generalized depiction for producing a MIP is shown below.

Taken from sigmaaldrich .com

  1. Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device (34 citations)

Taken from wikipedia.org

Monitoring the periodic diurnal variations in cortisol (aka hydrocortisone, show below) from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. This publication from China reports use of aptamer-functionalized gold nanoparticles pre-bound with electro-active triamcinolone for detection of cortisol based on its competitive binding to the aptamer by monitoring a signal from the displaced triamcinolone using square wave voltammetry at graphene-modified electrodes. The assay was benchmarked vs. ELISA and radioimmunoassays.

  1. Multifunctional aptamer-based nanoparticles for targeted drug delivery to circumvent cancer resistance (32 citations)

Taken from Liu et al. Biomaterials (2016)

In yet another publication from China, Liu et al. report use of a G-quadruplex nanostructure to target cancer cells by binding with nucleolin, in a manner analogous to that mentioned above. A second component is double-stranded DNA (dsDNA), which is rich in GC base pairs that can be applied for self-assembly with doxorubicin (Dox) for delivery to resistant cancer cells. These nanoparticles were found to effectively inhibit tumor growth with less cardiotoxicity.

Jerry’s Top 3 Publication Picks for 2017-to-Date

Here are my Top 3 “fav” aptamer articles published during the first half of 2017, and my reasons for these aptamer selections—pun intended. Interested readers can consult the original publication for technical details.

  1. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome

CRISPR/Cas9 is unquestionably—in my opinion—the hottest topic in nucleic acid-based R&D these days, as I have previously blogged about. Off-target effects of CRISPR/Cas9 can be problematic, so using targeted delivery to cells of interest is an important approach for mitigating this problem. In this study, an aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery with adaptability to other situations. The chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand, and the approach “provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.”

  1. A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva

This report claims the first ever development of a split aptamer that achieves enhanced target-binding affinity through cooperative binding. In this instance, a split cocaine-binding aptamer incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. This system afforded specific, ultra-sensitive, one-step fluorescence detection of cocaine in saliva without signal amplification. This limit of detection meets the standards recommended by the European Union’s Driving under the Influence of Drugs, Alcohol and Medicines program.

  1. Detection of organophosphorus pesticide–Malathion in environmental samples using peptide and aptamer based nanoprobes

Environmental contamination with pesticide residues has necessitated the development of rapid, easy and highly sensitive approaches for the detection of pesticides such as malathion, a toxic organophosphorus pesticide, widely used in agricultural fields. These Indian investigators employed an aptamer, cationic peptide and unmodified gold nanoparticles. The peptide, when linked to the aptamer renders the gold nanoparticles free and therefore, red in color. When the aptamer is associated with malathion, however, the peptide remains available to cause the aggregation of the nanoparticles and turn the suspension blue. The sensitivity was tested in real samples and the results implied the high practicability of the method.

Aptamer Publications in 2014-Present Citing TriLink Products

I was pleasantly surprised to find more than 250 publications on aptamers in Google Scholar citing the use of TriLink products since 2014. This volume of literature is way too large to summarize succinctly, so I decided to do a quick scan to select the following items that provide an indication of the broad diversity of applications partially enabled by TriLink products:

2’-F-UTP. Taken from trilinkbiotech .com

In closing, I should first mention that, while scanning the aptamer/TriLink publications mentioned above, it was evident that the most frequently cited TriLink products were 2’-F-CTP and 2’-F-UTP, which are incorporated into aptamers to impart nuclease resistance, as discussed on a TriLink webpage.

My second and last comment is that, as you may have noticed, there seems to be a high proportion of aptamer publications coming out of China and/or coauthored by Chinese investigators collaborating with researchers in other countries. This despite the fact that Chinese publications in Life Sciences are ~6-times fewer that those from the US, according to reliable statistics. I have no idea why this is so, but thought it’s an intriguing factoid.

As usual, your comments are welcomed.

SaveSave

SaveSave

SaveSave

SaveSaveSaveSave

SaveSave

SaveSave

SaveSave

National ALS Advocacy Day

  • Tuesday May 16, 2017 is National ALS Advocacy Day
  • Mark Your Calendar and Get Involved!
  • Read on to Find Out Why Involvement Matters

Lou Gehrig (1903-1941). Taken from Wikipedia.org

Amyotrophic lateral sclerosis (ALS) is more commonly referred to in North America as “Lou Gehrig disease.” Henry Louis “Lou” Gehrig was a record-setting baseball All-Star for the New York Yankees from 1923 through 1939, when he voluntarily took himself out of the lineup to stunned fans after his play was hampered by ALS. Sadly, Mr. Gehrig died only two years later, which indicates the rapidity of ALS disease progression.

ALS was first found in 1869 by French neurologist Jean-Martin Charcot, but it wasn’t until Lou Gehrig’s affliction that national and international attention was brought to the disease. ALS is now known to be a group of neurological diseases that mainly involve the degeneration of nerve cells (neurons) responsible for controlling voluntary muscle movement, like chewing, walking, breathing and talking. Motor neurons are nerve cells that extend from the brain to the spinal cord and to muscles throughout the body.

Taken from irelandms.com

The disease is relentlessly progressive, meaning the symptoms get continuously worse over time. Both the upper motor neurons in the brain and the lower motor neurons in the spine degenerate or die, and stop sending messages to the muscles. Unable to function, the muscles gradually weaken, start to twitch, and waste away (atrophy). Eventually, the brain loses its ability to initiate and control voluntary movements.

Currently, there is no cure for ALS and no effective treatment to halt, or reverse, the progression of the disease. Most people with ALS die from respiratory failure, usually within 3 to 5 years from when the symptoms first appear. However, about 10 percent of people with ALS survive for 10 or more years.

It is generally estimated there are over 30,000 people living with ALS in the United States at any given time, and that the number worldwide is around 450,000. Someone is diagnosed with ALS every 90 minutes. The life-time incident rate for an average person is often estimated at between 1-in-400 to 1-in-600 people—an incidence rate comparable to that for multiple sclerosis.

Who Gets ALS and Why?

Following are some reliable facts that I selected from an authoritative NIH website for ALS:

  • ALS affects people of all races and ethnic backgrounds.
  • Caucasians and non-Hispanics are most likely to develop the disease.
  • Although the disease can strike at any age, symptoms most commonly develop between the ages of 55 and 75.
  • Men are slightly more likely than women to develop ALS. However, the difference between men and women disappears with aging.
  • Military veterans are about 1.5 to 2 times more likely to develop ALS, and is recognized as a service-connected disease by the U.S. Department of Veterans Affairs.
  • 90% of ALS cases are considered sporadic, i.e. the disease seems to occur at random.
  • 10% of ALS cases are familial, i.e. an individual inherits the disease from his or her parents.
  • Mutations in more than a dozen genes have been found to cause familial ALS

How is ALS treated?

Riluzole. Taken from healthtap.com

No cure has yet been found for ALS. However, there are treatments available that can help control symptoms, prevent unnecessary complications, and make living with the disease easier. In 1995, the FDA approved riluzole (Rilutek), the only disease-modifying drug to date for ALS. Riluzole has multiple neural mechanisms of action, and is believed to reduce damage to motor neurons by decreasing levels of glutamate, which transports messages between nerve cells and motor neurons. Unfortunately, clinical trials in people with ALS showed that riluzole prolongs survival by only a few months.

BrainStorm Cell Therapeutics & NurOwn®

In researching clinical trials for hopefully much more effective therapies for ALS, I came across a company in Israel named BrainStorm Cell Therapeutics Inc. (BCTI) that offers a form of stem cell therapy for ALS that appears to be quite promising. Following is a short synopsis of what I found.

Space-filling model of BDNF. Taken from Wikipedia.org

BCLI has developed a patented stem cell-based technology that delivers a growth factor that can help neurons live longer at or near the site of injury or damage. More specifically, a mesenchymal stem cell isolated from an ALS patient is grown in a cell culture under certain conditions to produce a differentiated phenotype that secretes brain-derived neurotrophic factor (BDNF) at a level at least five-times greater than normal. The term “factor” is generic in biomedical parlance, and in the case of BDNF refers to a protein pictured to the right.

After these “super secreting” cells are obtained ex vivo, they are reintroduced (aka implanted) into the same ALS patient (i.e. an autologous transplant; see schematic diagram) wherein BDNF acts on neurons of the central nervous system and the peripheral nervous system, helping to support the survival of existing neurons, and encourage the growth and differentiation of new neurons and synapses.

Taken from Byrne JA. Overcoming clinical hurdles for autologous pluripotent stem cell-based therapies. OA Stem Cells 2013 Sep 01;1(1):3.

Details for this cell-harvesting, ex vivo differentiation, selection, and implantation can be read in this 2014 article by BCTI in Clinical Translation Medicine. BCTI has registered these differentiated BDNF-secreting cells as NurOwn®, which I assume is intended to sound bit like “your own”—to reflect the autologous nature of the transplant—and be a linguistic blend of neuron and own. In any case, the important point is that safety and efficacy have been reported in a recently published Phase 1/2 and 2a open-label, proof-of-concept studies of patients with ALS at the Hadassah Medical Center in Jerusalem, Israel.

In the Phase 1/2 part of the trial, 6 patients with early-stage ALS were injected intramuscularly (IM) and 6 patients with more advanced disease were transplanted intrathecally (IT), i.e. via the spinal cord. In the Phase 2a dose-escalating study, 14 patients with early-stage ALS received a combined IM and IT transplantation of autologous BDNF-secreting cells. Interested readers can consult this publication for details, but the bottom line, if you will, was that the possible clinical benefits would be hopefully confirmed in an upcoming clinical trial.

My further research on this led to a February 2017 press release by BCTI announcing that City of Hope’s Center for Biomedicine and Genetics (CBG) in Duarte, California will produce clinical supplies of NurOwn® adult stem cells for the BCTI’s planned randomized, double-blind, multi-dose Phase 3 clinical study in patients with ALS. It added that CBG is expected to support all U.S. medical centers that will be participating in the Phase 3 trial.

A second February 2017 press release by BCTI announced an agreement with Centre for Commercialization of Regenerative Medicine (CCRM) in Toronto, Canada to support the market authorization request for NurOwn® and explore the opportunity to access Health Canada’s early access pathway for treatment of patients with ALS as early as 2018.

Other ALS Clinical Studies

My “go to” authoritative source of reliable information about clinical trials is NIH’s ClinicalTrials.gov website that has an updated database that can be searched and filtered in many ways. When I searched for ALS clinical trials that were recruiting patients, I was heartened to find over 180 studies that can be perused via this link. For each study, there is information about purpose, study design and measures, eligibility, contacts and locations.

Incidentally, as regular readers of my blog will know, modified mRNA (mod mRNA) therapeutics is a relatively new and very promising modality for treating diseases that respond to providing or supplementing proteins. Given that DNA vectors encoding BDNF mRNA are readily available, I’m hoping that a mod mRNA for BDNF will soon be investigated as yet another avenue of treatment for ALS.

Advocate for ALS!

Taken from alsa.org

The ALS Association (ALSA) has the stated mission “to discover treatments and a cure for ALS, and to serve, advocate for, and empower people affected by ALS to live their lives to the fullest.” ALSA’s website offers various ways for any individual to become an advocate for ALS, such as becoming informed and donating much needed money or time, participating in the Walk to Defeat ALS® that draws people of all ages and athletic abilities together (see picture) to honor the courageous souls who are affected by ALS, to remember those who have passed, and to show support for the cause.

One of my long-time friends has recently been diagnosed with ALS, which in part led me to research this blog to help inform him, and led me to find a Walk to Defeat ALS® in which to participate. I encourage you to do advocate for ALS in whatever way you wish and are able.

As usual, your comments are welcomed.

Nanobombs for Light-Activated MicroRNA Drug Delivery

  • Nanoparticles Designed to “Explode” Upon Irradiation with Light
  • Light Used Can Penetrate Skin and Tissue
  • Nanoparticle “Cargo” of MicroRNA Released Locally, Where Needed 

Every once in a while the title of a publication grabs my attention so much that I just have to read it, and that’s what happened when I read this one A Near-Infrared Laser-Activated “Nanobomb” for Breaking the Barriers to MicroRNA Delivery.

Taken from ajamtafireworks.com

My interest was primarily piqued by the word ‘nanobomb,’ which I had never heard of even though I consider myself quite well-read scientifically. Moreover, microRNA (miRNA) and drug delivery are both currently trending hot topics. I couldn’t find an eye catchy image of a nanobomb other than fireworks by this name sold in India, which are oddly touted as being “pollution free”! But I digress…

Delivery of miRNA and other types of nucleic acid-based drugs continues to be perhaps the biggest challenge facing successful clinical development of this broad new class of drugs. In that regard, an expert review titled Nucleic acid delivery: the missing pieces of the puzzle?, written by my good friend and long-time collaborator Prof. Frank Szoka (UCSF), is a “must read” for those of you who are interested in further details and perspectives by an expert.

Prof. Xiaoming He. Taken from bme.osu.edu

Another “hook” in the nanobomb article’s title was its use of lasers, which are also trending in biomedical or biotechnological applications. So, all of this taken together, led me to request a pdf from the corresponding coauthor, Prof. Xiaoming He at Ohio State University, who promptly complied. Since it’s a pay-to-read publication, I’ve tried my best to convey the essence of this work in what follows.

Symphony of Science

Like a symphony, which is an elaborate musical composition for full orchestra, I learned that Prof. His nanobombs for laser-induced drug delivery are also quite elaborate and composed of a full array of scientific principles. To my surprise, a Google Scholar search of the term “nanobomb” gave a large number—more than one hundred—of items that included a wide variety of ways to use nano-size, i.e. sub-micron materials and light to somehow “blow up” or otherwise kill diseased cells, and thus fall under the umbrella-term photodynamic therapy.

Carbon nanotubes (~100 nm diameter) seen through an electron microscope. Taken from item.fraunhofer.de

For example, there’s a report in 2005 coauthored by Eric Wickstrom—who coincidentally is a friend of mine from early antisense oligo days—on the first application of single-wall carbon nanotubes (SWCNT) as potent therapeutic nanobombs for killing breast cancer cells in vitro. This is accomplished by adding water molecules into SWCNTs, which then get adsorbed on target cells for 800-nm (red) laser light irradiation to vaporize the entrained water.

A more elaborate rendition of nanobomb drug delivery was published in 2013 by Lu et al., who reported that intracellular gold nanoparticles under laser irradiation generate “nanobubbles” that can kill cells. To formulate a targeting gold nanoparticle as a cancer cell-specific “nanobomb,” a 31-nt DNA aptamer was conjugated to the gold surface. While biological results from this approach have yet to be published, there’s a good tutorial on the properties and applications of gold nanoparticles that are commercially available in discrete sizes, as seen here:

Taken from simaaldrich.com

Symphonic Variations of Science

This section heading extending my musical metaphor for nanobomb drug delivery is meant to convey the fact that the study by Prof. He and his coworkers featured herein is a symphony-like variation of photodynamic therapy composed of many parts. This work stands out—in my opinion—as being a remarkably comprehensive proof-of-concept involving lots of in vitro data and, more importantly, compelling in vivo animal results. I think you’ll “tune in”—musical pun intended—on this as I now summarize the complex scientific symphony by He and coworkers pictured below.

(A) Three agents are encapsulated inside nanoparticles: microRNA-34a (miR-34a) anti-mRNA drug for gene therapy of prostate cancer stem cells, indocyanine green (ICG) for absorbing laser light, and ammonium bicarbonate for gas generation under heating. (B) Laser light causes the nanoparticles to expand, penetrating the cancer cell’s endosomal/lysosomal barrier (green circles), blowing up cancer cells (yellow), and releasing miR-34a to inhibit protein CD44, which is crucial for cancer stem cell survival. Credit: Hai Wang et al./Advanced Materials. Taken from kurzweilai.net

What’s the Nanobomb?

The top part of the above depiction labeled (A) doesn’t do justice to the elegant methodology used to prepare the water-in-oil-in-water (W-in-O-in-W) double-emulsion nanobomb, so interested readers will have to consult the publication by He and coworkers for details. However, as shown in A, the nanobomb’s active components are indocyanine green (ICG), ammonium bicarbonate (NH4HCO3) and microRNA-34a (miR-34a), the latter of which I found is a single-stranded 22-nt RNA rather than the misleading drawn double-stranded species. Chitosan’s amino groups form a polyplex with miRs like miR-34a.

I also found many references to miR-34a as an anticancer agent, and in this instance it serves to negatively regulate CD44 overexpression, which is associated with survival and growth of prostate cancer stems cells (CSCs) as previously studied by a number of other groups.

Indocyanine green (ICG). Taken from Wikipedia

Having said this, the real novelties of this nanobomb are—in my opinion—its “ignition” and “explosive” materials, namely ICG and NH4HCO3, respectively. Some among you may recognize from the name or structure of ICG that it’s a “chemical cousin,” so to speak, of cyanine-3 and cyanine-5 dyes popular for labeling, which BTW are now available from TriLink as NHS esters upon inquiry.

The ICG dye is used to absorb laser light and convert the light’s energy into heat, which then causes NH4HCO3 to very rapidly generate CO2 and NH3 gases and thus release miR-34a (as depicted in part B of the above cartoon from He and coworkers). This “release” aspect deserves the following further comments.

Why’s a Nanobomb Needed?

This rhetorical question relates to the “D-word,” i.e. delivery of miR (or other classes of oligo agents) into a cell’s cytoplasmic compartment where it is intended to block target mRNA expression after its release from the endosome/lysosome compartment, as depicted in B above. The following electron micrograph shows what these sub-compartments look like.

Electron micrograph of endosomes in human HeLa cells. Early endosomes (E), 5 minutes after internalization; late endosomes/ multivesicular bodies (M) and lysosomes (L) are visible. Bar, 500 nm. Taken from Wikipedia.com

As depicted by He and coworkers, laser light via ICG is used to “ignite” or trigger “explosive” release of miR-34a from these compartments, rather than rely on natural phenomenon or use of pH-sensitive triggering mechanisms. For those of you who are not familiar with the endosome/lysosome compartment, details can be found in the aforementioned review by Prof. Frank Szoka, which was coauthored by Juliane Nguyen.

Is the Nanobomb Effective In Vivo?

Saline (control); HLPP = formulation; miR-34a (R) unformulated plus laser light (L); HLPP with ICG (I) and NH4HCO3 (A) plus L; HLPP with A and R plus L; HLPP with I and A and R plus L. Taken from He and coworkers.

Prof. He and his coworkers carried out extensive controlled experiments in vitro and in vivo to assess efficacy (as well as biodistribution and safety) of their nanobombs, and to me the most compelling data are shown below. Briefly, mice capable of harboring tumors derived from subcutaneously placed human prostate CSCs were treated with various formulations or control, and afterwards the extent of tumor growth was accessed in various ways including by tumor weight. It’s obvious in (C), shown below, that the only statistically significant (*) effect was obtained by the nanobomb formulation.

But is This Nanobomb Practical?

Here’s what He and coworkers state with regard to the scope and practicality of their nanobomb approach:

“Although the tissue penetration of the NIR [near infrared laser] is limited to less than ~1 cm and multiple polymers are needed for preparing the nanosystem, NIR could be delivered into deep tissue using minimally invasive approaches [e.g. endoscopy] and the preparation of the nanosystem using the double-emulsion method is quite straightforward. Therefore, the present study demonstrates the great potential of the NIR laser-activated “nanobomb” for microRNA delivery to achieve augmented cancer therapy.”

Time will tell what aspects of this approach will ultimately be reduced to practice in the clinic and/or lead to an approved photodynamic therapy for cancer. However, in the meantime, I’m betting that it will also “ignite” others to explore variations on this symphony of science.

As usual, your comments are welcome.

Postscript

After this blog was written, Jalani et al. published a paper titled Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery. This work is conceptually related—in my opinion—to the above nanobombs in that low energy red-light-absorbing nanoparticles emit higher energy UV light (i.e. photon upconversion) to simulataneously image and release drugs specially absorbed on these nanoparticles. This NIR light penetration, UV imaging, and drug release are said therein to be effective down to a depth of ~2 cm of tissue, which is twice that reported above by He and coworkers.

Chinese Scientists to Pioneer First Human CRISPR Clinical Trial

  • Chinese Team Begins First CRISPR-Based Anticancer Immunotherapy Clinical Study
  • US Consortium’s CRISPR Clinical Study OK’d by NIH Panel
  • Survey Indicates More Worry than Enthusiasm for Gene-Editing in Babies
Taken from jeantet.ch

Taken from jeantet.ch

Regular readers of my blog will recall a number of previous postings on gene editing using CRISPR-Cas9 (aka CRISPR), which has rapidly become the hottest trend in nucleic acid-based biotechnology and medical research. That opinion is partly based on the fact that this relatively new technology has already amassed thousands of publications, including 1,250 in 2015 alone! Additional evidence follows from the numerous start-up and established biopharma companies pursuing CRISPR-based therapeutics, which is “the biggest biotech discovery of the century” according to one report.

Now, even more exciting developments for CRISPR are on the horizon. Chinese researchers are poised for the first human clinical trial using CRISPR, and an analogous study in the US awaiting FDA approval. Both of these trials involve forms of cancer immunotherapy, which is a very hot field in itself, and is briefly introduced in the next section.

Cancer Immunotherapy

Cancer immunotherapy is the use of the immune system to treat cancer either actively, passively, or by a combination of these approaches, which exploit the fact that cancer cells often have macromolecules on their surface that can be detected by the immune system (aka tumor-associated antigens, TAAs). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs, whereas passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.

Numerous antibody therapies have been approved by the FDA and other regulatory authorities worldwide. In contrast, active immunotherapies, which usually involve the removal of immune cells from the blood or from a tumor for reintroduction to the patient, have lagged in such approvals. In fact, the only US-approved cell-based therapy is Dendreon’s Provenge®, for the treatment of prostate cancer. A recent series of NY Times articles highlights some powerful examples of cancer immunotherapy, especially from the perspective of interviews with patients and their physicians.

Taken from howitworksdaily.com

Taken from howitworksdaily.com

For the purpose of this blog, I’ll add that adoptive T-cell therapy is a form of passive immunization by the transfusion of T-cells, which are found in blood and tissue and usually activate when they find “foreign” pathogens. These pathogens can be either infected cells, or antigen presenting cells present in tumor tissue, where they are known as tumor infiltrating lymphocytes. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

As depicted below, T-cells specific to a tumor antigen can be removed from a tumor sample or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the resultant cells being reinfused. Importantly, activation can take place through genetic engineering, such as gene editing by CRISPR.

Cancer specific T-cells can be obtained by fragmentation and isolation of tumor infiltrating lymphocytes, or by genetically engineering cells from peripheral blood. The cells are activated and grown prior to transfusion into the recipient (tumor bearer). Taken from Wikipedia.org

Cancer specific T-cells can be obtained by fragmentation and isolation of tumor infiltrating lymphocytes, or by genetically engineering cells from peripheral blood. The cells are activated and grown prior to transfusion into the recipient (tumor bearer). Taken from Wikipedia.org

CRISPR Goes Clinical in China

As I write this post, Chinese scientists at Sichuan University’s West China Hospital in Chengdu will reportedly become the first in the world to inject people with cells modified using the CRISPR gene-editing method. This landmark trial will involve patients with metastatic non-small cell lung cancer who have not responded to chemotherapy, radiation therapy and/or other treatments.

The team will extract T cells from the blood of the trial participants and then use CRISPR to knock out a gene encoding a protein called PD-1 that normally regulates the T cell’s immune response to prevent it from attacking healthy cells. As depicted below, this so-called checkpoint has been targeted by small molecule inhibitors (anti PD-1), but with somewhat limited success so far—notably including failure of a would-be billion-dollar drug (Opdivo®) recently reported by Bristol-Myers Squibb. Nevertheless, the presently envisaged PD-1 gene-edited cells will be amplified in the lab and re-introduced into the patient’s bloodstream to circulate and, hopefully, attack the cancer.

Taken from smartpatients.com

Taken from smartpatients.com

Potential Concerns of CRISPR Clinical Trials

While the Chinese trial may be groundbreaking, it is not without risk. There is concern that the edited T-cells could attack normal tissue as it’s well documented that CRISPR can result in gene edits at the wrong place in the genome. To help mediate this risk, the T-cells will be validated by biotechnology company MedGenCell—a collaborator on the trial—to ensure that the correct gene is knocked out before the cells are re-introduced into patients.  Since the primary purpose of this phase one trial is to determine if the approach is safe, participants will be closely monitored for side effects, and researchers will pay close attention to biological markers in the blood.

Some may say that this type of trial in humans is premature, but China has always tried to be a leader in CRISPR research. Carl June, a clinical researcher in immunotherapy at the University of Pennsylvania in Philadelphia explains, ‘China places a high priority on biomedical research.’ And Tetsuya Ishii, a bioethicist in Japan, is quoted in Nature magazine as saying that ‘When it comes to gene editing, China goes first.”

Initial CRISPR Clinical Trial in USA Awaiting Review Board and FDA Approvals

While the Chinese team seems positioned to be the first to conduct a CRISPR clinical trial, the US isn’t far behind. As reported earlier this summer, the Recombinant DNA Research Advisory Committee (RAC) at the NIH has approved an analogous—but genetically more complex—proposal to use CRISPR-edited T cells for cancer immunotherapy. The RAC reviews all proposals for human trials involving modified DNA that are conducted in the United States, and the investigators now have to convince review boards at their own institutions, and the FDA, to allow the trial. This will hopefully be done by the end of 2016.

The proposed trial is a collaborative effort involving the University of Pennsylvania, M.D. Anderson Cancer Center in Texas, and the University of California, San Francisco. This trial will be funded by a $250-million immunotherapy foundation formed in April 2016 by former Facebook president Sean Parker. According to a news item in Nature, The University of Pennsylvania will produce the edited cells, and will recruit and treat patients alongside the collaborating medical centers in Texas and California.

In this study, researchers will perform three CRISPR edits with T cells from patients with several types of cancers: 1. a gene for a protein engineered to detect and target cancer cells will be inserted, 2. a natural T-cell protein that could interfere with this process will be removed and 3. the gene for a protein that identifies the T cells as immune cells and prevents the cancer cells from disabling them will be removed. The researchers will then infuse the edited cells back into the patient.

Both of the studies being performed by the Chinese and US teams are very exciting to me. I will be paying close attention to the progress of these studies over the next few months and hope to report back to you with some very interesting and ground breaking results.

Survey Indicates Public Concern in the US About Gene Editing in Babies

A Pew Research Center pole focusing on concerns about gene editing was recently reported in Nature. The poll surveyed more than 4,700 people in the United States and the results indicate that more people are concerned than enthusiastic about gene editing.

The quadrants below (from 0-100%) show responses to the question, “How worried or enthusiastic do you feel about gene editing to reduce disease risk in babies?”

Taken from nature.com

Taken from nature.com

While these findings speak for themselves, I hasten to add that this same pole also asked “Have you heard or read about this topic?” The following replies (in quadrants from 0-100%) indicate that a substantial percentage of respondents who have not at all read about gene editing nevertheless expressed either worry or enthusiasm for gene editing. Ditto for those who read a little about gene editing.

babies

It would seem, then, that we should take this poll with a grain (or two) of salt as it apparently includes a substantial amount of uninformed opinion. This is not too surprising given the complex nature of gene editing and it’s relatively new position in research. We can see from the results of this poll that a significant amount of education may be needed to garner increased public support for gene editing. Hopefully, the results of the studies highlighted above will begin to pave this road.

Hope for the Best but Expect Complications

This section heading, which summarizes my parting comments about the advent of CRISPR-enabled clinical trials, is based upon my having participated in several decades of research on nucleic acid-based concepts for radically different approaches to traditional small-molecule therapeutics. First was the idea of using chemically modified antisense oligonucleotides to block gene expression, which encountered non-specificity issues, low potency, delivery, and a host of other issues that took two decades to sort through. Then there was the idea of using chemically modified short-interfering RNA (siRNA) for modulating gene expression via RNA interference (RNAi). This, too, encountered similar problems in reaching the clinic. Ditto for anti-microRNAs (antagomirs).

I’m hoping that CRISPR-based therapies meet with success far faster—and prove to be affordable to society. I won’t, however, be surprised if progress is slow—and quite expensive.

Are you excited about potential CRISPR-based therapies? Do you have concerns about their safety and efficacy? Do you believe the general public is ready to accept gene editing therapies? Please share your thoughts as your comments are always welcome.

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.

Continue reading

Point-of-Care PCR 2.0

  • Ubiquitome Quickens Pace of POC Apps for Its Freedom4
  • Cepheid Unveils its POC Diagnostics System
  • Hopkins Crew Brews “Coffee Mug-Sized” Gizmo for Fully Automated Chlamydia Testing
Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Regular readers of this blog will recall a recent byline exclaiming “Honey I Shrunk the qPCR Machine”, which spotlighted the unveiling of startup company Ubiquitome’s first point-of-care (POC) product—Freedom4—developed in New Zealand. Up until then, this far away—for me—exotic island country brought to mind folks fondly nicknamed Kiwi—after the native flightless bird, not Chinese fruit. Mightily impressed by this tiny but powerful qPCR device, I vowed to thereafter keep an eye on these Kiwis’ democratized POC apps enabled by its nifty handheld 4-sample high-performance qPCR device.

Continue reading