Daily Archives: September 17, 2022

Scientists have developed a new technique to reveal the hidden human genome.

Numerous short RNA sequences that code for microproteins and peptides have been identified, providing new opportunities for the study of diseases and the development of drugs.

Researchers from Duke-NUS Medical School and their collaborators have discovered thousands of previously unknown DNA sequences in the human genome that code for microproteins and peptides that could be critical for human health and disease.

Much of what we understand about the known two per cent of the genome that codes for proteins comes from looking for long strands of protein-coding nucleotide sequences, or long open reading frames, explained computational biologist Dr Sonia Chothani, a research fellow with Duke-NUS Cardiovascular and Metabolic Disorders (CVMD) Programme and first author of the study. Recently, however, scientists have discovered small open reading frames (smORFs) that can also be translated from RNA into small peptides, which have roles in DNA repair, muscle formation and genetic regulation.

Scientists have been seeking to identify smORFs and the tiny peptides they code for since smORF disruption can cause disease. However, the currently available techniques are quite limited.

Much of the current datasets do not provide information that is detailed enough to identify smORFs in RNA, added Dr Chothani. The majority also comes from analyses of immortalised human cells that are propagatedsometimes for decadesto study cell physiology, function and disease. However, these cell lines arent always accurate representations of human physiology.

Chothani and her colleagues from Singapore, Germany, the United Kingdom, and Australia present an approach they created to address these challenges in a recentstudy published in Molecular Cell. They scoured existing ribosome profiling datasets for short strands of RNA with periodic three-base sections that covered more than 60% of the RNAs length. They then performed their own RNA sequencing and Ribosome profiling to establish a combined data set of six kinds of cells and five types of tissue derived from hundreds of patients.

Analyses of these data identified nearly 8,000 smORFs. Interestingly, they were highly specific to the tissues that they were found in, meaning that these smORFs may perform a function specific to their environment. The team also identified 603 microproteins coded by some of these smORFs.

The genome is littered with smORFs, said Assistant Professor Owen Rackham, senior author of the study from the CVMD Programme. Our comprehensive and spatially resolved map of human smORFs highlights overlooked functional components of the genome, pinpoints new players in health and disease and provides a resource for the scientific community as a platform to accelerate discoveries.

Professor Patrick Casey, Senior Vice-Dean of Research at Duke-NUS, said, With the healthcare system evolving to not only treat diseases but also prevent them, identifying potential new targets for disease research and drug development could open avenues to new solutions. This research by Dr Chothani and her team, published as a resource for the scientific community, brings important insights to the field.

Reference: A high-resolution map of human RNA translation by Sonia P. Chothani, Eleonora Adami, Anissa A. Widjaja, Sarah R. Langley, Sivakumar Viswanathan, Chee Jian Pua, Nevin Tham Zhihao, Nathan Harmston, Giuseppe DAgostino, Nicola Whiffin, Wang Mao, John F. Ouyang, Wei Wen Lim, Shiqi Lim, Cheryl Q.E. Lee, Alexandra Grubman, Joseph Chen, J.P. Kovalik, Karl Tryggvason, Jose M. Polo, Lena Ho, Stuart A. Cook, Owen J.L. Rackham and Sebastian Schafer, 15 July 2022, Molecular Cell.DOI: 10.1016/j.molcel.2022.06.023

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Revealing the Hidden Genome: Unknown DNA Sequences Identified That May Be Critical to Human Health - SciTechDaily

Mental illness is a growing public health problem. In 2019, an estimated 1 in 8 people around the world were affected by mental disorders like depression, schizophrenia or bipolar disorder. While scientists have long known that many of these disorders run in families, their genetic basis isnt entirely clear. One reason why is that the majority of existing genetic data used in research is overwhelmingly from white people.

In 2003, the Human Genome Project generated the first reference genome of human DNA from a combination of samples donated by upstate New Yorkers, all of whom were of European ancestry. Researchers across many biomedical fields still use this reference genome in their work. But it doesnt provide a complete picture of human genetics. Someone with a different genetic ancestry will have a number of variations in their DNA that arent captured by the reference sequence.

When most of the worlds ancestries are not represented in genomic data sets, studies wont be able to provide a true representation of how diseases manifest across all of humanity. Despite this, ancestral diversity in genetic analyses hasnt improved in the two decades since the Human Genome Project announced its first results. As of June 2021, over 80% of genetic studies have been conducted on people of European descent. Less than 2% have included people of African descent, even though these individuals have the most genetic variation of all human populations.

To uncover the genetic factors driving mental illness, I, Sinad Chapman and our colleagues at the Broad Institute of MIT and Harvard have partnered with collaborators around the world to launch Stanley Global, an initiative that seeks to collect a more diverse range of genetic samples from beyond the U.S. and Northern Europe, and train the next generation of researchers around the world. Not only does the genetic data lack diversity, but so do the tools and techniques scientists use to sequence and analyze human genomes. So we are implementing a new sequencing technology that addresses the inadequacies of previous approaches that dont account for the genetic diversity of global populations.

To study the genetics of psychiatric conditions, researchers use data from genome-wide association studies that compare the genetic variations between people with and without a particular disease. However, these data sets are mostly based on people of European ancestry, largely because research infrastructure and funding for large-scale genetics studies, and the scientists conducting these studies, have historically been concentrated in Europe and the United States.

One way to close this gap is to sequence genetic data from diverse populations. My colleagues and I are working in close partnership with geneticists, statisticians and epidemiologists in 14 countries across four continents to study the DNA of tens of thousands of people of African, Asian and Latino ancestries who are affected by mental illness. We work together to recruit participants and collect DNA samples that are sequenced at the Broad Institute in Massachusetts and shared with all partners for analysis.

Prioritizing the voices and priorities of local communities and scientists is foundational to our work. All partners have joint ownership of the project, including decision-making and sample and data ownership and control. To do this, we build relationships and trust with the local communities we are studying and the local university leaders and scientists with whom we are partnering. We work to understand local cultures and practices, and adapt our collection methods to ensure study participants are comfortable. For example, because there are different cultural sensitivities around providing saliva and blood samples, we have adapted our practices by location to ensure study participants are comfortable.

We also freely share knowledge and materials with our partners. There is a two-way exchange of information between the Broad Institute and local teams on study progress and results, enabling continual learning, teaching and unity between teams. We strive to meet each other where we are by exchanging practices and training scientists to support the development of locally grown and locally led research programs.

Our collaboration with African research groups provides a prime example of our model. For example, our African research colleagues are co-leaders on the grants that fund the lab equipment, scientists and other staff for projects based at their study sites. And we help to support the next generation of African geneticists and bioinformaticians through a dedicated training program.

Collecting samples from more diverse populations is only half of the challenge.

Existing genomic sequencing and analysis technologies do not adequately capture genetic variation across populations from around the world. Thats because these technologies were designed to detect genetic variations based on reference DNA from people of European ancestry, and they reduce accuracy when analyzing sequences that arent derived from the reference genome. When these tools are applied to genetic data from other populations, they fail to detect much of the rich variation in their genomes. This can lead researchers to miss out on important biomedical discoveries.

To address this issue, we developed an approach to genome sequencing that can detect more genetic variation from populations around the world. It works by sequencing the exome the less than 2% of the genome that codes for proteins in high detail, as well as sequencing the 98% of the genome that does not code for proteins in less detail.

This combined approach reduces the trade-offs geneticists often have to make in sequencing projects. High-depth whole genome sequencing, which reads through the entire genome multiple times to get detailed data, is too costly to do on a large number of DNA samples. While low-coverage sequencing reduces costs by reading smaller segments of the genome, it may miss some important genetic variation. With our new technology, geneticists can get the best of both worlds: sequencing the exome in depth maximizes the likelihood of pinpointing specific genes that play a role in mental illness, while sequencing the whole genome less in depth allows researchers to process large numbers of whole genomes more cost-effectively.

Our hope is that this new technology will allow researchers to sequence large sample sizes from a diverse range of ancestries to capture the full breadth of genetic variation. With a better understanding of the genetics of mental illness, clinicians and researchers will be better equipped to develop new treatments that work for everyone.

Genomic sequencing opened a new era of personalized medicine, which promises to deliver treatments tailored to each individual person. This can be done only if the genetic variations of all ancestries are represented in the data sets that researchers use to make new discoveries about disease and develop treatments.

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Uncovering the genetic basis of mental illness requires data and tools that aren't just based on white people this international team is collecting...

A Newfound Link Between Cancer and Aging?

A new study in 2022 reveals a thought-provoking relationship between how long animals live and how quickly their genetic codes mutate.

Cancer is a product of time and mutations, and so researchers investigated its onset and impact within 16 unique mammals. A new perspective on DNA mutation broadens our understanding of aging and cancer developmentand how we might be able to control it.

Cancer is the uncontrolled growth of cells. It is not a pathogen that infects the body, but a normal body process gone wrong.

Cells divide and multiply in our bodies all the time. Sometimes, during DNA replication, tiny mistakes (called mutations) appear randomly within the genetic code. Our bodies have mechanisms to correct these errors, and for much of our youth we remain strong and healthy as a result of these corrective measures.

However, these protections weaken as we age. Developing cancer becomes more likely as mutations slip past our defenses and continue to multiply. The longer we live, the more mutations we carry, and the likelihood of them manifesting into cancer increases.

Since mutations can occur randomly, biologists expect larger lifeforms (those with more cells) to have greater chances of developing cancer than smaller lifeforms.

Strangely, no association exists.

It is one of biologys biggest mysteries as to why massive creatures like whales or elephants rarely seem to experience cancer. This is called Petos Paradox. Even stranger: some smaller creatures, like the naked mole rat, are completely resistant to cancer.

This phenomenon motivates researchers to look into the genetics of naked mole rats and whales. And while weve discovered that special genetic bonuses (like extra tumor-suppressing genes) benefit these creatures, a pattern for cancer rates across all other species is still poorly understood.

Researchers at the Wellcome Sanger Institute report the first study to look at how mutation rates compare with animal lifespans.

Mutation rates are simply the speed at which species beget mutations. Mammals with shorter lifespans have average mutation rates that are very fast. A mouse undergoes nearly 800 mutations in each of its four short years on Earth. Mammals with longer lifespans have average mutation rates that are much slower. In humans (average lifespan of roughly 84 years), it comes to fewer than 50 mutations per year.

The study also compares the number of mutations at time of death with other traits, like body mass and lifespan. For example, a giraffe has roughly 40,000 times more cells than a mouse. Or a human lives 90 times longer than a mouse. What surprised researchers was that the number of mutations at time of death differed only by a factor of three.

Such small differentiation suggests there may be a total number of mutations a species can collect before it dies. Since the mammals reached this number at different speeds, finding ways to control the rate of mutations may help stall cancer development, set back aging, and prolong life.

The findings in this study ignite new questions for understanding cancer.

Confirming that mutation rate and lifespan are strongly correlated needs comparison to lifeforms beyond mammals, like fishes, birds, and even plants.

It will also be necessary to understand what factors control mutation rates. The answer to this likely lies within the complexities of DNA. Geneticists and oncologists are continuing to investigate genetic curiosities like tumor-suppressing genes and how they might impact mutation rates.

Aging is likely to be a confluence of many issues, like epigenetic changes or telomere shortening, but if mutations are involved then there may be hopes of slowing genetic damageor even reversing it.

While just a first step, linking mutation rates to lifespan is a reframing of our understanding of cancer development, and it may open doors to new strategies and therapies for treating cancer or taming the number of health-related concerns that come with aging.

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Explainer: The Basics of DNA and Genetic Systems - Visual Capitalist

DetailsCategory: DNA RNA and CellsPublished on Saturday, 17 September 2022 10:29Hits: 269

CAMBRIDGE, MA, USA I September 16, 2022 I Intellia Therapeutics, Inc. (NASDAQ:NTLA), a leading clinical-stage genome editing company focused on developing potentially curative therapeutics leveraging CRISPR-based technologies, today announced positive interim results from an ongoing Phase 1/2 clinical study of NTLA-2002, its second in vivo genome editing candidate. NTLA-2002 is a systemically administered CRISPR candidate being developed for hereditary angioedema (HAE) and is designed to knock out the KLKB1 gene in liver cells, thereby reducing the production of kallikrein protein. Uncontrolled activity of kallikrein is responsible for the overproduction of bradykinin, which leads to the recurring, debilitating and potentially fatal swelling attacks that occur in people living with HAE. The interim data were shared today in an oral presentation at the 2022 Bradykinin Symposium held in Berlin, Germany.

The data presented are from the initial six adult patients with HAE in the ongoing dose-escalation study with a data cut-off date of July 27, 2022. Single doses of 25 mg (n=3) and 75 mg (n=3) of NTLA-2002 were administered via intravenous infusion, and changes from baseline values of plasma kallikrein protein were measured for each patient. Administration of NTLA-2002 led to dose-dependent reductions in plasma kallikrein and achieved maximal reductions by week eight, with mean reductions of 65% and 92% in the 25 mg and 75 mg dose cohorts, respectively. Furthermore, these reductions were sustained through at least 16 weeks in the 25 mg cohort and eight weeks in the 75 mg cohort for which complete cohort biomarker data were available.

In addition to plasma kallikrein levels, HAE attack rates are also being measured in the study, with the first analysis occurring at the end of the pre-specified 16-week primary observation period. To date, all three patients in the 25 mg dose cohort have reached the end of this initial observation period. Patients in this group had a baseline HAE attack rate ranging from 1.1 to 7.2 attacks per month, as confirmed by the investigator. Treatment with a single dose of 25 mg of NTLA-2002 resulted in a mean reduction in HAE attacks of 91% throughout the 16-week observation period. Additionally, two of the three patients have not had a single HAE attack since treatment, and all three patients have been attack free since week 10 (follow-up through weeks 24 - 32). Patients in the 75 mg cohort have not completed the primary 16-week observation period. Attack-rate data for this cohort will be presented at the American College of Allergy, Asthma & Immunology (ACAAI) Annual Scientific Meeting, November 10 14 in Louisville, Kentucky.

Prophylaxis medications are permitted in the Phase 1 part of the study. Two of the three patients in the 25 mg cohort were actively receiving prophylaxis therapy prior to administration of NTLA-2002. For these two patients, the study protocol permitted investigators to withdraw the patients prophylaxis therapy after completion of the 16-week primary observation period. This treatment approach was implemented for the two applicable patients in this cohort, and neither patient has had an HAE attack since discontinuing their prophylaxis therapy through the latest follow-up.

These initial data represent a significant milestone for both Intellia and people around the world suffering from genetic diseases, such as HAE, said Intellia President and Chief Executive Officer John Leonard, M.D. We are strongly encouraged by the greater than 90% reduction in HAE attacks observed in the 25 mg dose cohort, as these interim results support our belief that a single dose of NTLA-2002 has the potential to permanently prevent the debilitating swelling attacks associated with HAE. Additionally, todays announcement continues to validate our genome editing approach and the modular platform we have built. This is now the second time in history clinical data have been generated suggesting we can precisely edit target cells within the human body to potentially treat genetic diseases with a single, systemic administration of a CRISPR-based therapy. We plan to move as quickly and judiciously as possible on behalf of people living with HAE and a number of additional genetic diseases in the months and years ahead.

At both dose levels, NTLA-2002 was generally well-tolerated, and the majority of adverse events were mild in severity. The most frequent adverse events were infusion-related reactions, which were mostly Grade 1 and resolved within one day. There have been no dose-limiting toxicities, no serious adverse events and no adverse events of Grade 3 or higher observed to date. No clinically significant laboratory abnormalities were observed, including any significant elevation in liver enzymes.

Many people living with HAE continue to experience breakthrough attacks despite currently available treatments and often find the burden of untreated attacks, frequent infusions or injections to be tremendously disruptive to their lives, said Hilary Longhurst, M.D., Ph.D., Faculty of Medical and Health Sciences, University of Auckland, New Zealand, and the trials principal investigator in New Zealand. These early data support NTLA-2002 as a potential one-time treatment capable of producing profound reductions in HAE attacks. While the clinical data are still emerging, I am highly optimistic that NTLA-2002 could become a new treatment option for the HAE community.

Based on the interim data presented today, Intellia selected a third dose of 50 mg to be evaluated in the ongoing dose-escalation portion of the Phase 1/2 study. Dosing at this level has recently completed, and Intellia expects to select up to two doses to further evaluate in the Phase 2, placebo-controlled, dose-expansion portion of the study, which is expected to begin in the first half of 2023. Intellia anticipates expanding country and site participation, including U.S. clinical sites, as part of the Phase 2 study.

Intellia Therapeutics Investor Event and Webcast Information Intellia will host a live webcast today, Friday, September 16, 2022, at 8:00 a.m. ET, to provide a clinical update from its in vivo portfolio, during which the company will review the presented clinical data at the 2022 Bradykinin Symposium alongside interim results from NTLA-2001. To join the webcast, please visit this link, or the Events and Presentations page of the Investors & Media section of the companys website at http://www.intelliatx.com. A replay of the webcast will be available on Intellias website for at least 30 days following the call.

About the NTLA-2002 Clinical ProgramIntellias multi-national Phase 1/2 study is evaluating the safety, tolerability, pharmacokinetics and pharmacodynamics of NTLA-2002 in adults with Type I or Type II hereditary angioedema (HAE). This includes the measurement of plasma kallikrein protein levels and activity as determined by HAE attack rate measures. The Phase 1 portion of the study is an open-label, single-ascending dose design used to identify up to two dose levels of NTLA-2002 that will be further evaluated in the randomized, placebo-controlled Phase 2 portion of the study. This Phase 1/2 study will identify the dose of NTLA-2002 for use in future studies.Visit clinicaltrials.gov (NCT05120830) for more details.

About NTLA-2002

Based on Nobel Prize-winning CRISPR/Cas9 technology, NTLA-2002 is the first single-dose investigational treatment being explored in clinical trials for the potential to continuously reduce kallikrein activity and prevent attacks in people living with hereditary angioedema (HAE). NTLA-2002 is a wholly owned investigational CRISPR therapeutic candidate designed to inactivate thekallikrein B1 (KLKB1) gene, which encodes for prekallikrein, the kallikrein precursor protein. NTLA-2002 is Intelliassecond investigational CRISPR therapeutic candidate to be administered systemically, by intravenous infusion, to edit disease-causing genes inside the human body with a single dose of treatment. Intellias proprietary non-viral platform deploys lipid nanoparticles to deliver to the liver a two-partgenome editingsystem: guide RNAspecific to the disease-causing gene and messenger RNAthat encodes the Cas9 enzyme, which together carry out the precision editing.

About Hereditary Angioedema

Hereditary angioedema (HAE) is a rare, genetic disorder characterized by severe, recurring and unpredictable inflammatory attacks in various organs and tissues of the body, which can be painful, debilitating and life-threatening. It is estimated that one in 50,000 people are affected by HAE, and current treatment options often include life-long therapies, which may require chronic intravenous (IV) or subcutaneous (SC) administration as often as twice per week, or daily oral administration to ensure constant pathway suppression for disease control. Despite chronic administration, breakthrough attacks still occur. Kallikrein inhibition is a clinically validated strategy for the preventive treatment of HAE attacks.

About Intellia TherapeuticsIntellia Therapeutics, a leading clinical-stage genome editing company, is developing novel, potentially curative therapeutics leveraging CRISPR-based technologies. To fully realize the transformative potential of CRISPR-based technologies, Intellia is pursuing two primary approaches. The companys in vivo programs use intravenously administered CRISPR as the therapy, in which proprietary delivery technology enables highly precise editing of disease-causing genes directly within specific target tissues. Intellias ex vivo programs use CRISPR to create the therapy by using engineered human cells to treat cancer and autoimmune diseases. Intellias deep scientific, technical and clinical development experience, along with its robust intellectual property portfolio, have enabled the company to take a leadership role in harnessing the full potential of genome editing to create new classes of genetic medicine. Learn more at intelliatx.com. Follow us on Twitter@intelliatx.

SOURCE: Intellia Therapeutics

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Intellia Therapeutics Announces Positive Interim Clinical Data for its Second Systemically Delivered Investigational CRISPR Candidate, NTLA-2002 for...

WALTHAM, Mass.--(BUSINESS WIRE)--Pretzel Therapeutics, a biotechnology company harnessing the intricacies of mitochondrial biology to develop groundbreaking therapies, launched today with a $72.5 million Series A financing to pioneer novel therapies to modulate mitochondrial function. The financing was led by ARCH Venture Partners and Mubadala Capital with participating investors HealthCap, Cambridge Innovation Capital, Cambridge Enterprise, Angelini Ventures, GV, Invus, Eir Ventures, GU Ventures, and Karolinska Institutet Holding.

We are excited to pioneer a new era in the treatment of diseases related to mitochondrial dysfunction. The expertise we have assembled and the platform technologies we have created will allow new inroads into treating both rare genetic diseases as well as common diseases of aging, said Jay Parrish, Ph.D., Chairman of the Board and Chief Executive Officer of Pretzel. Were proud to be backed by an outstanding investor syndicate, with a Series A financing that will allow us to prosecute preclinical development across our pipeline and continue to build out our talented team.

Pretzel is advancing a first-of-its-kind platform to modulate mitochondrial biology, with a vast range of potential applications across rare and common disorders, said Alaa Halawa, MBA, Partner and Head of the U.S. Ventures business at Mubadala Capital. As investors focused on partnering early with companies that will positively impact patients lives, were proud to co-lead the companys Series A financing and to partner with Pretzel on their journey to build the worlds leading center of excellence addressing diseases of mitochondrial dysfunction.

Dysfunctional mitochondria are involved in more than 50 diseases. The most severe of these are broadly termed mitochondrial diseases, a group of rare genetic conditions which affect individuals of all ages. Mitochondrial dysfunction also plays an important role in more common diseases, including aging-related disorders such as Alzheimers and Parkinsons diseases. In addition, modulating mitochondrial biology presents a potential approach to the treatment of diseases not directly caused by mitochondrial dysfunction, for instance cancer and metabolic diseases.

Pretzels platform encompasses three primary technologies to modulate mitochondrial function: Genome correction, genome expression modulation, and mitochondrial quality control. The companys genome correction therapeutics will utilize specialized gene-editing tools to reduce mutated mitochondrial DNA and increase the levels of healthy mitochondrial DNA. Genome expression modulation will be accomplished using small molecules that act on the enzymes involved in mitochondrial DNA replication, transcription, and translation. Finally, mitochondrial quality control will be targeted using small molecules that modulate mitochondrias built-in quality control system.

Founders and Team

Pretzels founders include three leading academics in the field of mitochondrial biology. Claes Gustafsson, M.D., Ph.D., is professor of medical biochemistry at the University of Gothenburg and an expert in mitochondrial gene expression. Michal Minczuk, Ph.D., is a Group Leader and MRC Investigator at the MRC Mitochondrial Biology Unit, University of Cambridge and an expert in mitochondrial genome engineering. Nils-Gran Larsson, M.D., Ph.D., is professor of mitochondrial genetics at the Department of Medical Biochemistry and Biophysics at Karolinska Institutet who has published over 150 articles on mitochondrial biology.

In addition to Drs. Gustafsson, Minczuk, and Larsson, founders Gabriel Martinez, Ph.D. and Paul Thurk, Ph.D., played a formative role in the companys creation based on their deep biotechnology industry expertise. Finally, the company recognizes the contributions of Gunther Kern, Ph.D., MBA; Jeremy Green, Ph.D.; and Christina Trojel-Hansen, Ph.D., to the formation of Pretzel.

Mitochondria have historically been a challenging cellular organelle to target therapeutically, in part because mitochondrial diseases are extremely diverse, both genetically and phenotypically, but also due to the distinctive characteristics of mitochondrial genome function. However, scientific understanding of mitochondrial biology has greatly advanced in recent years, allowing new insights into their role in many prevalent diseases, as well as how they can be therapeutically targeted, said Claes Gustafsson. Its gratifying to form Pretzel to translate these insights into therapies that could meaningfully improve peoples lives.

Pretzel is led by accomplished experts in drug discovery, drug development, and company foundation, and is advised by a Board of Directors and Scientific Advisory Board with deep scientific and industry expertise.

Pretzels leadership team is comprised of:

The companys Board of Directors is comprised of:

The companys Scientific Advisory Board is comprised of:

About Pretzel Therapeutics

Pretzel Therapeutics is a biotechnology company harnessing the intricacies of mitochondrial biology to develop groundbreaking therapies. Pretzel was founded by leading academic experts in mitochondrial biology and is backed by a world-class investor syndicate. The company is headquartered in Waltham, MA and has research facilities in Gothenburg, Sweden. For more information, visit http://www.pretzeltx.com.

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Pretzel Therapeutics Launches With $72.5 Million Series A Financing to Pioneer Mitochondrial Therapies - Business Wire

The actinomycete DEM30355 was isolated from a soil sample, collected from the El Tatio geyser field within an arid part of the Atacama Desert in Chile17. Strain DEM30355 was recovered in the genus Amycolatopsis, based on 16S rRNA analysis, forming a subgroup with Amycolatopsis vancoresmycina DSM 44592T and Amycolatopsis bullii SF27T (see ESI). The genus Amycolatopsis contains 94 species and four subspecies encompassing both extremophiles and producers of bioactive secondary metabolites, including the clinically used vancomycin and rifamycin antibiotics19,20. Preliminary bioactivity screening showed that extracts of Amycolatopsis sp. DEM30355 displayed promising antibiotic activity against B. subtilis, thus we decided to examine the genome of Amycolatopsis sp. DEM30355 for novel biosynthetic potential. Purified genomic DNA from Amycolatopsis sp. DEM30355 was analysed using both PacBio and Illumina sequencing technologies and genome assembly was performed using the combined datasets to give a 9.6Mb draft genome, in 13 contigs. The draft genome of Amycolatopsis sp. DEM30355 was examined using the secondary metabolite analysis software AntiSMASH 6.0.121. Of the 31 biosynthetic gene clusters (BGCs) detected, a PKS cluster was identified showing moderate overall similarity (81%) to that which encodes for rishirilides A and B22,23,24,25,26. These compounds are anthracenone polyketides, originally isolated from Streptomyces rishiriensis OFR-1056, with no reported antibiotic activity. Rishirilide B has been shown to be a moderately potent inhibitor of 2-macroglobulin, glutathione S-transferase and asparaginyl-tRNA synthetase, whilst little is known about the biological role of rishirilide A (Fig.1)18,27,28.

Top ()-Rishirilide A (1) and (+)-rishirilide B (2). Relative stereochemistry of ()-1 and absolute stereochemistry of (+)-2 shown. Bottom. Structure of ()-tatiomicin (3) as derived from NMR and SCXRD experiments. Key COSY (red) and HMBC (blue) correlations shown. Absolute stereochemistry as shown by both vibrational circular dichroism (VCD) and single-crystal X-ray diffraction (SCXRD) resonant scattering experiments. Structural differences of rishirilide A and tatiomicin are highlighted (magenta).

Further inspection of the BGC from Amycolatopsis sp. DEM30355 revealed a highly altered gene synteny (see ESI), compared to the rishirilide BGC, along with the presence of several new genes: one postulated to be involved in PKS biosynthesis (tatS1), two encoding methyltransferases (tatM1 and tatM2), two encoding cyclases (tatC4 and tatC5) and one cytochrome p450 oxidoreductase (tatO11) (Fig.2). Due to the significant variation in the genetic make-up of the BGC, we postulated that it may code for the production of an as yet undiscovered polyketide and as such we set about attempting to identify this molecule from the metabolome of Amycolatopsis sp. DEM30355.

Organization of the tatiomicin BGC. Genes coding for polyketide biosynthesis (red; tatS=starter unit biosynthesis, tatK=chain biosynthesis), polyketide modification (blue; tatO=oxidoreductases, tatC=cyclases, tatM=methyltransferases), regulation (yellow; tatR), transport (green, tatT) and others (grey; tatP=phosphorylase, black; genes not assigned to the tatiomicin BGC based on homology to the rishirilide BGC and proposed biosynthetic pathway)).

Preliminary analysis of the fermentation supernatant of Amycolatopsis sp. DEM30355 by HPLC-HRMS showed the presence of a large number of secondary metabolites, in keeping with the predicted number of BGCs, including a compound with activity against Gram-positive bacteria (MW of 402Da, m/z=403 [M+H]+, m/z=425 [M+Na]+, ()-tatiomicin (3)). Fermentation of Amycolatopsis sp. DEM30355, removal of the biomass, extraction of the supernatant and bioactivity guided fractionation by multiple chromatography steps resulted in a fraction which retained antimicrobial activity and contained two closely related compounds. HRMS analysis suggested that these compounds were stereoisomers of each other, the major compound showing m/z=425.1221 [M+Na]+ corresponding to a molecular formula of C21H22O8 for both molecules (see ESI).

Structural determination of the major component was initially performed by NMR, which provided the majority of molecular connectivity with the exception of the ordering of the three contiguous quaternary centres at the C-3, C-4 and C-4a positions. Structural assignment was completed via single-crystal X-ray diffraction (XRD) analysis, revealing a highly oxygenated anthracenone polyketide, structurally consistent with the BGC of interest, which we named ()-tatiomicin (3) (Fig.1)29.

NMR and HPLC experiments demonstrated that the minor compound was the C-2 epimer, capable of equilibrating with ()-(3) under acidic conditions (see ESI).

Determination of the absolute stereochemistry of ()-3 was undertaken in parallel via vibrational and electronic circular dichroism spectroscopies and additional single-crystal X-ray diffraction (SCXRD) experiments.

Absolute configuration determination by vibrational circular dichroism (VCD) was based on a comparison of experimental and computationally predicted spectra, taking into account the presence of two epimers of ()-3. Conformational analysis (see ESI), removal of redundant geometries and final optimization at the B3LYP/6311++G(d,p) level allowed Boltzmann-weighted VCD spectra for both epimers of ()-3 to be constructed. The final predicted spectrum was obtained by applying a 3:1 ratio to account for the experimentally analysed mixture of epimers. Numerical analysis was used to establish agreement between experiment and theory, the neighbourhood similarity values (IR=92.0, VCD=71.2, ESI=57.8) suggesting an absolute stereochemical assignment of (2S,3S,4R,4aR,10R) (Fig.3 and ESI)30. The assignment was supported through similar electronic circular dichroism (ECD) experiments; however, in this case correlation between experiment and prediction was weaker (see ESI).

Experimental IR (top) and VCD spectra (bottom) of ()-tatiomicin 3 (CDCl3) with predicted spectra obtained at the B3LYP/PCM/6311++G(d,p) level of theory. VCD: Solid line=(2R,3R,4S,4aS,10S), dashed line=(2S,3S,4R,4aR,10R). Spectra have been frequency scaled Black line (=0.987) to yield maximal similarity grey line between the computed and experimental VCD spectra.

A suitable, albeit small, single-crystal of tatiomicin (3) was grown via slow evaporation from a benzene solution. Due to the crystals dimensions, diffraction data were collected at beam line I19 at the Diamond Light Source using synchrotron radiation at standard operating wavelength (=0.6889), providing a data set of sufficient quality to allow for structural confirmation. ()-Tatiomicin (3) crystallized as an H-bonded dimer in the unit cell (Z=2) along with a single molecule of solvent (benzene). To validate the absolute stereochemical assignment a further single-crystal X-ray diffraction experiment was undertaken at I19, employing non-typical, longer wavelength synchrotron radiation (=1.4879) to enhance resonant scattering contributions (also known inappropriately as anomalous dispersion). The absolute-structure (Flack) parameter (0.05(6)) was insignificantly different from zero and with a small standard uncertainty, indicating the correct absolute configuration in the refined (2S,3S,4R,4aR,10R) structure (see ESI)29. Interestingly, following extensive stereochemical debate and several reported total syntheses, the absolute stereochemistry of the congeneric (+)-rishirilide B (2) was recently revised (2S,3S,4S), matching that of ()-(3) over the three common stereocentres, suggesting a similar biosynthetic pathway for both sets of natural products (Fig.4)31,32,33,34,35.

Displacement ellipsoid plot of the molecular structure of ()-tatiomicin (3), absolute stereochemistry as shown determined by resonant scatteringthe dimer molecular structure (Flack parameter=0.05(6)). Displacement ellipsoids shown at 50% probability level.

To verify that the BGC previously identified does indeed encode the biosynthetic pathway for tatiomicin (3), a high molecular-weight P1 artificial chromosome (PAC) library was obtained, consisting of 2,688 clones with an average insert size of 138kb which contained resistant markers for kanamycin (for E. coli) and thiostreptone (for S. coelicolor). The PAC library was screened by PCR, using four primer pairs for the putative BGC. A single PAC clone was identified with the required PCR profile, which was then transferred into E. coli strain ET12567/pR9604 (dam- dcm-), the plasmid was subsequently transferred into S. coelicolor M1152 via conjugation. Exconjugants containing the plasmid integrated on the chromosome were selected for resistance to thiostrepton. Ninety-six putatively identified exconjugants were arrayed into 24 well plates and screened for the production of tatiomicin (3) by TLC, with detection based on the characteristic fluorescence upon UV irradiation at 365nm. Based on these screening parameters, S. coelicolor M1152::tat was identified as a producer of tatiomicin (3) (see ESI).

Growth of S. coelicolor M1152::tat was examined on solid medium, the agar was extracted (EtOAc) and analysed by LCMS alongside similar fermentation extracts from both the parent strain M1152, Amycolatopsis sp. DEM30355 and a tatiomicin (3) standard. An LCMS peak corresponding to tatiomicin (3) was observed in the extract from S. coelicolor M1152::tat but was absent in that of the parent strain M1152 (Fig.6).

Tatiomicin (3) was subsequently isolated from the fermentation of S. coelicolor M1152::tat in liquid medium (GYMG), as demonstrated by HRMS ([M+H]+=403.1403), with a production level in the heterologous host estimated at 0.57mg/L, confirming the identity of the tatiomicin BGC (Fig.5).

Detection of tatiomicin from the fermentation of heterologous host S. coelicolor M1152::tat. Top) Extracted ion chromatogram (EIC) based on m/z=827.25. S. coelicolor M1152 (purple), S. coelicolor M1152::tat (blue), Amycolatopsis sp. DEM30355 (black) and tatiomicin standard (red). Bottom) MS spectrum of tatiomicin (3) purified from the heterologous host S. coelicolor M1152::tat.

Based on a comparison between the tatiomicin and rishirilide BGCs22,23,24,25,26 we propose the following biosynthetic pathway operates for the assembly of tatiomicin (3) (See ESI). The modular type I polyketide synthase TatS1 is likely responsible for the biosynthesis of the polyketide starter unit, cis-crotonyl-ACP, which is then elongated via the attachment of eight malonyl-CoA by minimal PKS enzymes TatK1, TatK2, and TatK3. TatC1, TatC2, TatC3 and TatO10 show close homology to rishirilide cyclases RslC1, RslC2, and RslC3 and C9-ketoreductase RslO10, respectively. Thus, TatC1 and TatO10 likely act together to form the A ring of tatiomicin (3), whilst TatC2 and TatC3 catalyse the formation of the B and C rings. Tailoring of the polyketide core likely involves oxidation of the C ring by TatO4, and installation of the C ring epoxide by flavin mononucleotide (FMN)-dependent monooxygenase TatO1 together with a putative flavin reductase, TatO2. Opening of the epoxide is proposed to be mediated by NADPH:acceptor oxidoreductase TatO5, followed by the key BaeyerVilliger oxidation/rearrangement controlled by TatO9 and finally reduction of the B ring ketone by ketoreductase TatO8.

Three additional tailoring enzymes are present in the BGC of tatiomicin (3) for which no homologues are present in that of rishirilide, TatO11, TatM1 and TatM2. TatO11 is a cytochrome p450 oxidoreductase, likely responsible for oxidation of the A ring to the hydroquinone form, followed by double methylation by the two methyl transferases TatM1 and TatM2 to yield the completed molecule (Fig.6).

Top) Proposed pathway for the biosynthesis of ()-tatiomicin (3) based on homology with the biosynthetic gene cluster for the rishirilides. Enzymes shown in red have no direct congener in the rishirilide BGC and their biosynthetic role is hypothesised, based on BLAST analysis. Bottom) comparison of the rishirilide and (-) tatiomicin gene cluter based on BLAST analysis. (red; tatS=starter unit biosynthesis, tatK or rslK=chain biosynthesis), polyketide modification (blue; tatO or rslO=oxidoreductases, tatC or rslO=cyclases), regulation (yellow; tatR or rslR), transport (green, tatT or rslT) and others (grey; tatP or rslP=phosphorylase; tatM=methyltransferases, black; genes not assigned to the tatiomicin BGC based on homology to the rishirilide BGC and proposed biosynthetic pathway)).

The enzymes TatC4 and TatC5, which are not present in the rishirilide cluster, encode for a dehydrogenase and a monooxygenase and are located in the centre of the biosynthetic gene cluster. The tatiomicin BGC contains all orthologous genes responsible for the synthesis of rishirilide. The function of these additional genes is therefore not immediate obvious and might be a result of evolutionary divergence.

()-Tatiomicin (3) showed no detectable antimicrobial activity (MIC>64g/mL) against ten Gram-negative bacteria and two eukaryotic microorganisms (Candida spp.) (see ESI). However, antibacterial activity was observed against a sub-set of Gram-positive bacteria (MIC=48g/mL), namely Staphylococcus and Streptococcus species. Due to the interest in developing new antibiotics against drug-resistant Staphylococcus infections, we further evaluated ()-3 against a panel of MRSA clinical isolates, including twenty-four EMRSA-15 and EMRSA-16 strains (the main causative agents of nosocomial epidemic MRSA bacteraemia in the UK, with resistance to penicillin, ciprofloxacin and erythromycin)36, and twelve MRSA strains isolated from Belgian, Finnish, French and German hospitals (see SI). In all cases antibiotic activity was maintained (MIC=48g/mL), suggesting that ()-3 does not operate via a mode-of-action previously encountered by these strains, prompting us towards further investigation.

Elucidation of the mode-of-action (MOA) for a new antibacterial agent is a significant experimental challenge. The characterization of resistance mutations can be informative, however all attempts to isolate Bacillus subtilis mutants resistant to ()-tatiomicin (3) proved unsuccessful (see ESI). Also, no positive responses were seen with a panel of B. subtilis strains containing lacZ reporter genes used to indicate common antibacterial mechanisms of action, including: fatty acid synthesis (fabHA), DNA damage (105 prophage induction), RNA polymerase (RNAP) inhibition (helD), cell wall damage (ypuA), gyrase inhibition (gyrA), and cell envelope stress (liaI)) (see ESI)37,38,39.

Due to the presence of an, albeit electron-rich, ,-unsaturated carbonyl moiety, we postulated that the observed biological activity of ()-tatiomicin (3) may involve the covalent modification of thiol-containing enzymes through a conjugate or Michael addition of the thiol to the ,-unsaturated carbonyl. Thus, ()-tatiomicin (3) was reacted with L-cysteine hydrochloride, L-cysteine methyl ester hydrochloride and a short thiol-containing peptide (LcrV (271291)) as an enzyme proxy, under biologically relevant conditions. In all cases thiol adducts could be detected by LCMS, suggesting that ()-tatiomicin (3) may have biologically relevant Michael acceptor activity (see ESI).

To gain further insight into a potential mode-of-action, we undertook a bacterial cytological profiling experiment in which antibacterial induced changes in the morphology of test bacteria are compared to those induced by known mode-of-action antibacterials40,41. B. subtilis 168CA-CRW419 expresses two fusion proteins, HbsU-GFP and WALP23-mCherry, allowing simultaneous visualization of both the chromosomal DNA and the bacterial cell membrane by fluorescence microscopy. The cytoplasmic membrane was unaffected unlike in the control compound nisin, which forms large pores in the membrane42. Interestingly, treatment with ()-tatiomicin (3) induced chromosome decondensation in B. subtilis 168CA-CRW419, similar to the effects elicited by the RNAP inhibitor rifampicin (Fig.7).

Single-cell analysis of chromosome and membrane integrity. Phase contrast (top panels) and fluorescence microscopy of B. subtilis cells treated with various antibiotics (indicated above). DNA was visualized with an HsbU-GFP fusion (middle panels) and the cytoplasmic membrane with a WALP23-mCherry fusion (bottom panels).

The combination of the negative result observed with the helD reporter strain, cell lysis after prolonged incubation with the compound and the inability to create resistant mutants suggest that direct RNAP inhibition is unlikely. We therefore attempted to examine the integrity of the cytoplasmic membrane using the voltage sensitive dye DiSC3(5). This dye accumulates in well-energised cells in the cytoplasmic membrane15,43 but is released upon depolarisation of the membrane, and this release can be measured by fluorescence microscopy. DiSC3(5) is used in parallel with Sytox Green, a membrane-impermeable DNA stain used as a reporter for pore formation44. Upon addition of nisin, which forms large pores in the B. subtilis membrane42, both a loss of DiSC3(5) and uptake of Sytox Green was observed. In contrast gramicidin, which forms small cation-specific channels45, showed loss of DiSC3(5) without Sytox Green staining. Treatment with ()-tatiomicin (3) showed a similar effect to that of gramicidin, i.e. loss of DiSC3(5) without Sytox Green staining. Hence tatiomicin probably acts to dissipate the membrane potential without the formation of large pores (Fig.8).

Single-cell measurement of membrane potential and permeability. Phase contrast (top panels) and fluorescence microscopy of B. subtilis cells stained with the voltage-sensitive dye DiSC3(5) (middle panels) and the membrane permeability indicator Sytox Green (bottom panels) in the presence and absence of 32 g/mL of tatiomicin. As positive controls, the cells were treated with 5 g/mL of gramicidin (membrane depolarisation without pore formation) and 10 M nisin (membrane depolarisation through pore formation). Cellular DiSC3(5) and Sytox Green fluorescence values were quantified for cells treated with tatiomicin (32 g/mL), gramicidin (5 g/mL), and nisin (10 M) (see SI).

In an attempt to ascertain whether the observed loss of membrane potential is a downstream effect or occurs at the same time as chromosome depolarisation we performed a time-course experiment using DiSC3(5) in combination with a HsbU-GFP fusion to assess chromosome decondensation with images taken every two minutes. This showed that the loss of membrane potential occurred simultaneously with the chromosome decondensation, between 2 to 4min, suggesting that they are closely linked events (Fig.9).

Single-cell measurement of chromosome decondensation and membrane potential in a time course experiment in the presence of tatiomicin (32 g/mL). Phase contrast (top panels), fluorescence microscopy of B. subtilis HsbUGFP (chromosome marker) (middle panel) and stained with the voltage sensitive dye DiSC3(5) bottom panel. Cellular DiSC3(5) fluorescence values where quantified over time. The bar chart depicts the fluorescent intensity values of individual cells (> 30) (see SI).

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Discovery, isolation, heterologous expression and mode-of-action studies of the antibiotic polyketide tatiomicin from Amycolatopsis sp. DEM30355 |...

Bursts in microRNA (miRNA) diversity often line up with sudden increases in morphological complexity, especially in the context of the nervous system. In a 2022 bioRxiv preprint, researchers uncovered an miRNA repertoire expansion (orange) in the ancestor of coleoid cephalopodsthe group that includes squids and octopuses, generally thought to be more intelligent than any other invertebrateson par with ones seen in the ancestors of vertebrates (blue) and placental mammals (green).

The term noncoding RNA is a catch-all for sequences in the genome that are transcribed but typically not translated. These molecules, which account for the majority of the transcribed sequences in the genome, are now thought to play key roles in brain evolution and function. Noncoding RNAs can be classified based on their size, structure, location, or function, with dozens of different kinds described to date. Here are four types of noncoding RNA frequently studied in brain tissues.

Long noncoding RNAs (lncRNAs) are generally described as any noncoding RNAs greater than 200 nucleotides in length. Because of their variable size and composition, they can have complex shapes and perform a variety of cellular activities, though most lncRNAs await functional investigation.

Example: The human and chimpanzee versions of a lncRNA called HAR1 differ by 18 nucleotides, which impacts the molecules secondary structure. The human version is predicted to be more stable, but exactly how that translates into differences in brain form or function isnt yet clear.

MicroRNAs (miRNAs) are small noncoding RNAs of just ~2026 nucleotides (teal) that are cleaved from larger precursors. Their most well-described function is the regulation of gene expression via binding to messenger RNAs, where they generally inhibit translation and, therefore, reduce the amount of protein produced from a given gene.

Example: Overexpression of miRNA-124 leads to Alzheimers-like pathologies in mice, and elevated levels of the miRNA are found in the brains of people who died from the disease.

As the name suggests, circular RNAs (circRNAs) are noncoding RNAs with joined ends, creating a more stable, circular molecule. Many questions remain as to the functions of circRNAs, but some are known to bind miRNAs, likely acting as sponges to modulate the miRNAs translation-suppressing effects.

Example:The circRNA CDR1-AS fine tunes neuronal development in humans, binding microRNAs (teal) highly expressed in secretory neurons that regulate developmental gene expression.

Transfer RNAs primary job is to shuttle amino acids to growing peptide chains during translation. In the brain specifically, theres emerging evidence that modifications to tRNAs play important roles in neuronal health and disease. Furthermore,tRNA fragmentssmall chunks from tRNA breakdownseem to have their own functions, including in neurodegeneration.

Example: When researchers exposed Drosophilaneuron cultures to synthetic tRFGln-CTG(teal)a fragment of the tRNA for glutaminethe cells swelled and died, suggesting the fragment could play a role in neuronal necrosis.

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Infographic: Noncoding RNA in the Brain - The Scientist