Category Archives: Gene Medicine

Researchers from the CU School of Medicine Department of Orthopedics have been awarded a $3.4 million National Institutes of Health grant to study the genetics of bone density and to look for therapeutic targets to counter bone loss.

With the grant, Cheryl Ackert-Bicknell, PhD, associate professor of orthopedics at the CU School of Medicine, plans to map the key genes and pathways involved with bone cell activity. Using that information, researchers hope to find targets for more effective treatments to counter bone loss. Ackert-Bicknells fellow principal investigator on the grant is Charles Farber, PhD, associate professor of public health sciences at University of Virginia.

Bone is in a constant state of remodeling, Ackert-Bicknell said, and her study is designed to look at cells known as osteoblasts, which work to build bones. When a healthy body is functioning properly, osteoblasts work in balance with other cells called osteoclasts to maintain sufficient bone mineral density.

In the process of walking from your car to here, you did microcrack damage to your bone, Ackert-Bicknell explained. The osteoclasts job is to home to that site of the microcrack, eat a hole out all the way around that microcrack and the osteoblast comes and fills that in. Thats called normal bone turnover. Thats most of your life. If the osteoclasts go haywire and there is insufficient building of new bone, thats osteoporosis.

Current therapies to cause the body to build the right amount of new bone are limited. These therapies can often only be used for a limited time, and none can be used in children. There is a need for better therapies, Ackert-Bicknell said, but they cannot be developed without improved understanding of how a healthy body gets bone density just right.

Thats where Ackert-Bicknells study comes in. She plans a comprehensive analysis of how the genes influence the process. She will conduct a genome-wide association study that seeks to identify genetic variations that are associated with osteoblast function and bone mineral density.

Osteoblasts build bone. Osteoclasts chew up bone. And thats how I always teach it: Blasts build and clasts chew, Ackert-Bicknell said. Osteoblasts do two things to make bone. They make a protein matrix and then they mineralize that matrix. For that to be accomplished, the osteoblast has to get to the right place, and it has to proliferate. So, it is proliferating, it is migrating, then it is making that bone by making matrix, and it is mineralizing that matrix.

Previous studies of osteoblasts have shown that its characteristics are highly heritable, or transmissible from parents to children. But how osteoblasts form and do their work is not fully understood. What might appear to be a small change on osteoblast behavior can have significant developmental consequences. Ackert-Bicknell cited an example of how knocking out a single gene in one type of bone cell can result in an obese mouse.

You can actually knock out a gene, just in these osteoblast bone cells, and get an obese mouse, she said. In that cell only. It is the only cell in the whole body that makes that gene, and you end up with an obese mouse. This just shows how bone is tied into all of physiology.

To get a better understanding of those connections, Ackert-Bicknells new study will look at the network of all the genes expressed in this cell and their relative expression in different contexts of genetics.

To do this, we must compare bone image after bone image to identify variations that could be meaningful in relation to the genetics, said Douglas Adams, PhD, associate professor of orthopedics, who has worked with Ackert-Bicknell on previous studies that underpin the work in this new award.

Adams is also working with Ackert-Bicknell on another NIH grant studying how the leading treatment for osteoporosis might have variable efficacy because of genetic differences between patients. To conduct such studies, researchers grind through thousands of data points to discover links that have yet to be uncovered. By identifying those unknown connections, the team hopes to discover new ways to treat disorders of bone mineralization.

Its sort of along the lines of looking under the streetlamp for your keys, Ackert-Bicknell said. As long as your keys fell down where the lamp is shining light youve got a good chance of finding them. In our work, we are looking outside of the streetlight for the things we havent studied before.

Lets face it, what we know now isnt giving us enough drug targets, enough information. It isnt helping us. The most unique pathways and the ones that are going to get us drugs are not the ones we have already studied.

Ackert-Bicknells new grant provides funding for a five-year research project. Coupled with the parallel active NIH grant held by both Ackert-Bicknell and Adams, CU Orthopedics is at the forefront of the effort nationally and globally to understand and develop disease modifying approaches to address bone loss in osteoporosis patients. These projects are just part of a growing portfolio of research activity in the Department of Orthopedics, which has seen a greater than 10-fold increase in extramural grant support since the departments leadership committed to expand its research mission in 2018.

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CU Researcher Awarded NIH Grant to Study Genetics of Bone Density - CU Anschutz Today

LentiMutate identified a mutation that changes an amino acid of KRAS G12C at position 96 from tyrosine (Y) to histidine (H). This change impairs the binding of the novel lung cancer drug LUMAKRAS/AMG 510 (depicted in green) to KRAS G12C (depicted by greyscale). Credit: Kenneth D. Westover

DALLAS Aug. 10, 2021 Using a virus to purposely mutate genes that produce cancer-driving proteins could shed light on the resistance that inevitably develops to cancer drugs that target them, a new study led by UTSouthwestern scientists suggests. The findings, published online in Cancer Research, could help researchers develop drugs that circumvent resistance, validate new drug targets, or better understand the interaction between drugs and their target proteins.

Ralf Kittler, Ph.D.

We believe this approach will be a very useful tool in the fight against cancer therapeutic resistance and could have potential in a variety of other areas of drug development, said study leader Ralf Kittler, Ph.D., Associate Professor of Pharmacology in the Eugene McDermott Center for Human Growth and Development and the Harold C. Simmons Comprehensive Cancer Center. Dr. Kittler co-led the study with John D. Minna, M.D., Professor of Internal Medicine and Pharmacology, Director of the Hamon Center for Therapeutic Oncology Research and member of the Simmons Cancer Center.

Targeted therapies represent a major advance in cancer treatment for multiple tumor types, comprising drugs that specifically alter the function of oncoproteins that drive tumors to grow and spread. They are often oral agents with low toxicity that provide symptom relief and prolong survival. However, explained Dr. Kittler, these drugs have a marked drawback: they lose effectiveness over time as tumors become resistant because the genes responsible for the targeted oncoproteins inevitably mutate, producing proteins that no longer bind the drugs. For example, patients with non-small cell lung cancer are often treated with drugs that inhibit a protein known as the epidermal growth factor receptor (EGFR), providing great clinical benefit; unfortunately, most of these tumors develop resistance to the treatment within about a year. This response has led to second-, third-, and even fourth-generation versions of such EGFR-targeting drugs to try to overcome this resistance.

Although methods exist to predict mutations that will develop in cancer target genes an important step toward developing drugs that can attack the resulting mutant proteins these methods are cumbersome, expensive, time-consuming, or can only predict a limited type of mutation known as a point mutation, Dr. Kittler explained.

Looking for a better way to predict therapeutic resistance, the researchers developed a technique they call LentiMutate. This approach relies on a class of viruses called lentiviruses to cause mutations. In contrast with human cells and many other viruses, lentiviruses take RNA and convert it to DNA while infecting its target cells to eventually produce proteins; however, this process is inherently error-prone, producing mutant mistakes in the resulting DNA.

Working with a lentivirus engineered to make it even more error-prone, Dr. Kittler and his colleagues used the vector to insert EGFR RNA in human cells, causing the cells to produce mutant versions of this protein. They then dosed the cells with a commonly used inhibitor for EGFR called gefitinib to search for resistant cells. By sequencing the introduced single gene in the resistant cells, the researchers were able to identify several mutations that made EGFR resistant to gefitinib, a first-generation anti-EGFR drug, including those previously identified in human patients.

Further experiments showed that LentiMutate was able to identify mutations that conferred resistance to the fourth-generation anti-EGFR drug osimertinib, which is now the standard of care for EGFR mutant non-small cell lung cancer. The approach also identified mutations that cause resistance to imatinib, a drug that targets the BCR-ABL1 protein, which drives chronic myelogenous leukemia, and AMG 510, a drug that targets a specific mutant form of the KRAS protein, which drives non-small cell lung cancer.

Dr. Kittler noted that identifying these mutations through LentiMutate can greatly speed up the process of developing new drugs that can bind to the drug-resistant mutant proteins so that it takes weeks rather than years. LentiMutate could also be used in different ways in drug development: to confirm that new drugs are acting on the target protein and not a different one, to help researchers gain a better understanding of how drugs are interacting with their targets, or to develop new types of drugs for a variety of other diseases beyond cancer.

Precision medicine that comes from sequencing a patients tumor to identify specific proteins to target for therapy has revolutionized cancer treatment. However, we need patients to be cured and not just benefit for 10 to 15 months from such targeted therapy, said Dr. Minna. To do this, we need to deal with drug resistance mutations, including by developing new drugs, and LentiMutate gives us an important new tool in our research armamentarium to help solve this pressing problem.

Other UTSW researchers who contributed to this study include Paul Yenerall, Rahul K. Kollipara, Kimberley Avila, Michael Peyton, Yan Liu, and Kenneth D. Westover.

A patent pending for LentiMutate lists Yenerall, Dr. Minna, and Dr. Kittler as inventors. Dr. Minna receives licensing royalties from the National Cancer Institute and UTSouthwestern for cell lines.

This study was supported by funding from the Simmons Cancer Center at UTSouthwestern (P30CA142543), the Cancer Prevention and Research Institute of Texas (CPRIT) (RP120732-P3, RP160652, RP170373), the National Institutes of Health (NCI SPORE in lung cancer 5P50CA070907, R01CA200787, R01CA244341, and R01CA065823), the Margot Johnson Foundation, and the Howard Hughes Medical Institute.

Dr. Kittler is a John L. Roach Scholar in Biomedical Research and a CPRIT Scholar in Cancer Research. Dr. Minna holds the Sarah M. and Charles E. Seay Distinguished Chair in Cancer Research and the Max L. Thomas Distinguished Chair in Molecular Pulmonary Oncology.

About UTSouthwestern Medical Center

UTSouthwestern, one of the nations premier academic medical centers, integrates pioneering biomedical research with exceptional clinical care and education. The institutions faculty has received six Nobel Prizes, and includes 25 members of the National Academy of Sciences, 16 members of the National Academy of Medicine, and 13 Howard Hughes Medical Institute Investigators. The full-time faculty of more than 2,800 is responsible for groundbreaking medical advances and is committed to translating science-driven research quickly to new clinical treatments. UT Southwestern physicians provide care in about 80 specialties to more than 117,000 hospitalized patients, more than 360,000 emergency room cases, and oversee nearly 3 million outpatient visits a year.

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Hunting down the mutations that cause cancer drug resistance - UT Southwestern

When an injury occurs, damaged cells need to be replaced. Stem cells, known as the go-to cells when new specialized cells need to be produced, are rare in adult tissues, so the job often falls to differentiated, or mature, cells.

Dr. Jason Mills and his lab have been working on identifying the genes driving mature cells to return to a regenerative state, a process called paligenosis.

My lab has been promoting the idea that given that cells in all organs use similar functions like mitosis and apoptosis, theres likely to be a conserved genetic program for how mature cells become regenerative cells, said Mills, senior author of the study and professor of medicine gastroenterology,pathology and immunologyandmolecular and cellular biologyat Baylor. The research was conducted while his lab was atWashington University School of Medicine in St. Louis.

To begin paligenosis and reenter the cell cycle, mature cells must first go through the process of autodegredation, breaking down larger structures used in specialized cell function. Mills and his team, led by first author Dr. Megan Radyk, a postdoctoral associate at the Washington University School of Medicine in St. Louis at the time of research, found that the genes Atf3 and Rab7b are upregulated in gastric and pancreatic digestive-enzyme-secreting cells of mice during autodegredation, and return to normal expression before mitosis.

The researchers showed that Atf3 activates Rab7b, which directs lysosomes to begin dismantling cell parts not needed for regeneration. But when Atf3 was not present, Rab7b did not trigger autodegredation.

The team also found Atf3 and Rab7b expression were consistent in paligenosis across other organs and organisms. Similar gene expression also appeared in precancerous gastric lesions in humans. According to Mills, the discoveries in this research are foundational to understanding how repetitive injury and paligenosis may impact cancer.

The more tissue damage you have, the more youre calling mature cells back into regeneration duty, said Mills, co-director of theTexas Medical Center Digestive Disease Center. Theres emerging evidence that, when these cells go through paligenosis, they dont check for DNA damage well. The cells are storing DNA mutations when they return to their differentiated function. Over time, they become so damaged that they cant go back to normal function and instead keep replicating.

Its our belief that paligenosis is at the heart of cancer development.

This research also provides groundwork for potential therapeutic targets. Existing drugs like hydroxychloroquine can be used to inhibit autodegredation, therefore stopping paligenosis.

According to Mills, further study is required to determine whether drugs targeting autodegredation can be used in conjunction with cancer treatments to stop cells from replicating.

The complete study is published in EMBO Reports.

For a full list of authors, their contributions to this work and sources of support, see the publication.

By Molly Chiu

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Atf3 and Rab7b genes drive regeneration in mature cells - Baylor College of Medicine News

Introduction

The overuse of antibiotics and the widespread development of antibiotic resistance genes have facilitated the evolution of multidrug-resistant (MDR) Gram-negative bacteria.1 Owing to its toxicity and narrow therapeutic window, colistin has been approved for treatment only for infections in certain patients, including those with cystic fibrosis.2,3 However, the increased incidence of infections with MDR pathogens has led to increased interest in the use of colistin as a last-resort option in a larger number of patients.

Colistin is a positively charged, polypeptide drug that exerts a strong bactericidal effect against a broad-spectrum of Gram-negative bacteria by integration into the negatively charged lipid A, thereby destabilizing the outer membrane lipopolysaccharide (LPS) and leading to cell death.3 However, exposure of Enterobacterales to colistin both in vivo and in vitro has been reported to induce the emergence of colistin resistance in these strains.4,5 The main mechanism of colistin resistance occurs via the addition of cationic groups (ie, phosphoethanolamine [PEtN] or 4-amino-4-deoxy-L-arabinose [L-Ara4N]) to the LPS on bacterial membranes, preventing the high-affinity binding of colistin to LPS.3 The two-component system (TCS) of pmrAB and phoPQ, and the regulator of TCS (ie, mgrB), are primarily responsible for the development of colistin resistance in Enterobacterales.3,6 Moreover, a recently identified plasmid carrying mcr-1 resulted in the addition of PEtN to lipid A.7 Studies have assessed the development of high-level colistin-resistant mutants (HLCRMs) in MCR-1-producing Escherichia coli (MCRPEC). It is not known whether the mcr-1 gene has effects similar to those of plasmid-mediated quinolone resistance genes, which promote the evolution of strains with higher quinolone resistance.8,9 The aim of this study was to determine the impact of chromosomal modifications in pmrAB, phoPQ, and mgrB, combined with mcr-1, on colistin resistance in E. coli.

Six E. coli isolates, five mcr-1-positive clinical strains of E. coli and E. coli ATCC25922, obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) were used in this study. Isolates were re-identified as E. coli by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).10 The mcr-1 gene was amplified by PCR, and its DNA sequence was determined (Table S1). Multi-Locus Sequence Typing (MLST) was performed by comparing sequences of the seven housekeeping genes adk, fumC, gyrB, icd, mdh, purA and recA (https://enterobase.readthedocs.io/en/latest/mlst/mlst-legacy-info-ecoli.html) with the E. coli MLST database (https://enterobase.warwick.ac.uk/species/ecoli/allele_st_search) to determine the allelic types and STs of the tested isolates. None of the data in this study were linked to clinical information.

Plasmid eradication for mcr-1-positive E. coli was performed as previously described.11 Briefly, 5 mL aliquots of LuriaBertani (LB) medium were inoculated with 50 L of a suspension of wild-type E. coli. To each suspension was added 7.5 L, 15 L, or 30 L 10% SDS, and the cultures were incubated with shaking at 37C for 12 h. Subsequently, 50 L of these bacterial suspensions was inoculated into 5 mL fresh LB medium, and the cultures were incubated at 43C for 8 h. Both steps were repeated, and the incubation at 37C was performed a third time. These plasmid-cured derivative strains were plated onto MuellerHinton agar (MHA) plates with and without 4 mg/L colistin. The elimination of the mcr-1-bearing plasmid was confirmed by pulsed-field gel electrophoresis (PFGE), S1-nuclease PFGE (S1-PFGE), and Southern blotting, as described.12,13

Antibiotic susceptibility, except for colistin, was evaluated by Vitek2 (bioMrieux, Marcy-lEtoile, France). The results were in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines.14 The minimum inhibitory concentrations (MICs) of colistin against the tested strains were determined using the broth microdilution according to CLSI. In addition, the MICs of colistin against the multi-stepwise solutions were determined using the agar dilution method.

The parental and plasmid-curing strains were grown in antibiotic-free MuellerHinton broth at 37C for 68 h, and ~1010 CFU/mL of each strain was spread onto MHA in the presence or absence of colistin. The colistin concentrations used for mutant induction ranged from 1MIC to the concentration at which growth of the parental strain or a sub-parental mutant strain isolated from the prior induction step was fully inhibited. After 4872 h incubation at 37C, colonies growing on the plates were randomly selected, and their MICs of colistin were determined using both the broth microdilution and agar dilution methods. Isolates with the highest MIC were subjected to next-step induction. These induction/selection cycles were terminated when mutants with significantly high MIC were selected, or when their growth on plates with 1MIC colistin concentration was completely inhibited.

The TCS of pmrAB and phoPQ, the negative regulator of the phoPQ system (mgrB) and mcr-1 in parental strains, and their respective mutants, were PCR amplified using primers (listed in Table S1) and 2X A9 LongHiFi PCR MasterMix (Aidlab Biotechnologies Co., Ltd.). Following DNA sequencing, the presumed amino acid sequences of the mutants were compared with those of parental strains using the web platforms of the NCBI (National Center for Biotechnological Information) and ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Sorting Intolerant From Tolerant (SIFT) scores were calculated (http://sift.jcvi.org) to evaluate whether amino acid alterations in PmrAB and PhoPQ affected protein function. Moreover, the TCS domains of PmrA/PmrB and PhoP/PhoQ were subjected to SMART analysis (http://smart.embl.de/).

To determine the effect of mcr-1-bearing plasmids on the evolution of HLCRMs, conjugation experiments were performed as previously described.15 Briefly, a culture of mcr-1-producing isolates was mixed 1:9 with a culture of the recipient strain E. coli C600 in LB broth, followed by overnight incubation on LB agar plates. The resulting transconjugants were selected on MHA plates containing 150 g/mL sodium azide and 2 g/mL colistin. The colonies were identified as E. coli via MALDI-TOF MS, and the DNA of these colonies were sequenced to determine the presence of the mcr-1 gene. Plasmid sizes and numbers were determined using S1-nuclease PFGE. The colonies containing only mcr-1-bearing plasmids (E63-C600 and E66-C600) and E. coli C600 were used to select for colistin-resistant mutants (MuC600, MuE63-C600, and MuE66-C600). Total RNA was extracted from cells grown to mid-log phase in drug-free MHB using the TaKaRa RNAiso Plus (TaKaRa, Japan), according to the manufacturers instructions. The RNA was reverse transcribed to cDNA using PrimeScriptTM RT Reagent kits (TaKaRa). Transcripts of the pmrABC, phoP, mgrB, and mcr-1 genes were quantified by RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI7300 Sequence Detection System (Applied Biosystems), using the primers listed in Table S1. Transcription abundance was calculated by the 2CT method16 using gapA as the internal control, and the respective wild-type pmrABC, phoP, mgrB, and mcr-1 genes as references.

Four MCRPECs were subjected to the plasmid eradication test, which successfully eliminated the mcr-1 gene from strains EC18398 and EC26207 (Figure S1). The MICs and STs of the parental and plasmid-cured strains are shown in Table 1. Loss of the mcr-1 gene had little effect on the susceptibility of plasmid-curing strains to other antimicrobial agents, but reduced colistin MIC 416 fold, resulting in MICs of 0.5 and 2 mg/L for strains EC18398E and EC26207E, respectively. In addition, the strains EC1002 and EC2474, co-harboring the mcr-1, blaNDM-1, and blaCTX-M genes, were resistant to colistin, carbapenems, and cephalosporins.

Table 1 Antimicrobial Susceptibility of the E. coli Strains Used in This Study

Following a series of in vitro colistin selection steps, all the tested strains, including MCRPEC and non-MCRPEC strains, successfully evolved to HLCRMs, with MICs of 32 and 64 mg/L, respectively, as determined by the broth microdilution method, and 64 and 64 mg/L, respectively, as determined by the agar dilution method (Table 2 and Figure 1). Colistin inhibited first-step mutants at concentrations of 8 to 32 mg/L, resulting in a 16- to 32-fold increase in susceptibility for these non-MCRPEC mutants compared with their parental strains. By contrast, the in vitro first-step induction had little effect on the MCRPECs, which had MICs equal to or 2-fold higher than their parental strains. For the second cycle, the colistin MIC of all mutants was 32 mg/L, as determined by the broth microdilution method. Second-step mutants were subjected to further repeated inductions, while Mu2EC26207, Mu2EC24990, and Mu3EC18398 failed to grow on the plates containing 1MIC (64 mg/L). Interestingly, all three non-MCRPECs successfully grew on plates containing 1MIC (64 mg/L) after in vitro multi-stepwise induction and selection. The MICs of these non-MCRPEC mutants were 64- to 128-fold higher than those of their parental strains (Figure 2 and Table 2). In addition to determining MIC for colistin, the MICs of various antibiotics with diverse modes of action were also evaluated. Compared with their parental strains, the mutants had equivalent MICs for carbapenems, cephalosporins, levofloxacin, and tigecycline.

Table 2 Phenotypic and Genotypic Profiles of the in vitro Selected Mutants of mcr-1-Positive and mcr-1-Negative E. coli

Figure 1 Changes in the colistin susceptibility of selected mutants. Five mcr-1-positive and three mcr-1-negative E. coli strains were exposed to colistin in a multi-stepwise manner. MIC was measured by the broth dilution method. Mutants with the highest MIC were used for next-step induction and selection processes. Mu1, Mu2, Mu3, and Mu4 indicate the first, second, third, and fourth cycles of induction, respectively.

Figure 2 Mutation frequencies of mcr-1-positive and negative strains when cultured with colistin at its MICs for the parent strains and sub-parental mutants. The colistin MICs of the tested strains were determined by the agar dilution method. Solid line, mcr-1-positive strains; dotted line, mcr-1-negative strains.

In the tested E. coli strains, the mutation rates decreased significantly with increasing colistin concentrations on the selection plates (Figure 2). These results revealed that the frequency of mutation of non-MCRPEC strains to colistin resistance ranged from 106 to 102, whereas the frequency of mutation of MCRPEC strains to colistin resistance was 108 to 10.4 The non-MCRPEC strains could grow on plates containing colistin concentrations of 16 or 32 mg/L, and showed higher mutation rates than their parental strains. For example, EC26207E had a mutation rate of 102 to 106 at 1MIC, which was much higher than that of EC26207 (108 to 106). Additionally, the frequency of non-MCRPEC mutants on plates containing 32 or 64 mg/L was higher than that of MCRPEC mutants.

Comparative genomic analysis of parental and mutants strains showed that non-synonymous mutations in the major TCS associated with colistin resistance were more frequent in non-MCRPEC than in MCRPEC strains. None of the TCS mutations were found in any mutants of EC18398 and EC24990, with only single amino acid changes found in PmrA at position 15 (Gly15Arg) in EC26207, and in PmrB at position 86 (Pro86Gln) in EC1002. By contrast, the mutants of non-MCRPEC strains acquired more non-synonymous mutations in the target regions, including in PmrAB, PhoPQ, and MgrB in EC25922; PmrA in EC18398E; and PmrAB and PhoQ in EC26207E (Table 2). No amino acid substitutions were observed in MCR-1, and neither frameshift mutations nor deletions were identified in any of these strains. The alterations in the TCS regions of EC25922 were predicted to have little impact on protein function, as determined by SIFT score. Interestingly, the amino acid substitution in the mutant of EC25922 was also detected in other tested parental strains, including both MCRPEC and non-MCRPEC strains, suggesting that non-synonymous mutations may occur frequently in PmrA at positions 31, 128, and 144; in PmrB at positions 123 and 351; in PhoQ at positions 6 and 482; and in MgrB at position 36. Interestingly, PmrA at position 144 (Ser144Gly) and PhoQ at position 482 (Ala482Thr) could convert to each other when exposed to colistin plates (Table 2 and S2). The non-synonymous mutations in the plasmid-curing isolates were easily detected when compared with their parental strains. Amino acid alterations were observed in PmrA Gly144Ser, PmrB Pro94Gln, Asn358Tyr, and PhoQ Thr482Ala in the EC26207E mutant, and in PmrA Gly53Arg in the EC18398E mutant. The EC26207 and 18398 mutants had 1 or 0 amino acid variations, respectively. However, the second- and third-step mutants showed no further mutational changes in PmrAB, PhoPQ, and MgrB, except for those in EC26207E and EC25922.

SMART analysis revealed the major domains of the PmrA/PmrB and PhoP/PhoQ TCS, and the positions of all the mutations in colistin-resistant mutants (Figure 3). Our results showed that non-synonymous mutations were mainly found in the HAMP and ATPase domains of PmrB and PhoQ, and in the receiver domain of PmrA and PhoQ. SIFT analysis predicted that the PmrA Gly15Arg, Gly53Arg, PmrB Pro94Gln, and PhoP Asp86Gly mutations would affect protein function.

Figure 3 Domains of the PmrA/PmrB and PhoP/PhoQ two-component system and the positions of all mutations in colistin-resistant mutants. *These substitutions are predicted to affect protein function by SIFT. #These substitutions are predicted to affect protein function by SIFT because the sequences used were not sufficiently diverse. Red, EC18398E; Fuchsin, EC26207; Blue, EC26207E; Black, EC25922; Green, EC1002; Brown, EC2474. Domains of PmrA/PmrB and PhoP/PhoQ are indicated as REC, CheY-homologous receiver domain; Trans_reg_c, transcriptional regulatory C-terminal domain; TM1, first transmembrane domain; TM2, second transmembrane domain; HAMP, histidine kinases, adenylyl cyclases, methyl-binding proteins, and phosphatases domain; HisKA, histidine kinase domain; HATPase_c, histidine kinase-like ATPase C-terminal domain.

To better understand the impact of mcr-1-bearing plasmids on the evolution of HLCRMs, E63-C600, E66-C600, and E. coli C600 were used for the selection of HLCRMs. Transcription of the pmrCAB, phoP, mgrB, and mcr-1 genes in HLCRMs was evaluated by qRTPCR. The levels of expression of pmrCAB and phoP were higher in MuC600 than in E. coli C600, with the level of expression of pmrA being 200-fold higher in MuC600 than in E. coli C600 (Figure 4). Moreover, the magnitude of pmrCAB up-regulation was higher than that of phoP and mgrB, indicating that pmrCAB may play more important roles in the evolution of HLCRMs than phoPQ and mgrB. However, the levels of expression of the pmrCAB, phoP, mgrB, and mcr-1 genes in MuE63-C600 and MuE66-C600 were not significantly higher than those in their parental strains.

Figure 4 Transcriptional activities of pmrABC, phoP, mgrB, and mcr-1 in wild-type isolates and their derivative colistin-resistant mutants (MuC600, MuE63-C600, and MuE66-C600) grown in drug-free MHB. The fold change in transcription was calculated as 2CT. Means and standard deviations were determined for three independent replicates.

The clinical use of colistin is being re-evaluated because of the increasing prevalence of infections caused by MDR organisms.17 Plasmid-mediated colistin resistance via the mcr-1 gene was found to provide a horizontal transfer mechanism for rapid dissemination.7 The prevalence of colistin resistance has become of great concern because of the location of the mcr-1 gene on highly mobile genetic elements and its coexistence with other resistance determinants. However, the phenotype of HLCRMs in mcr-1-harboring E. coli is not fully understood. Moreover, the impact of chromosomal modifications in TCS combined with mcr-1 on colistin resistance has not been determined.1820

The present study found that HLCRMs could be successfully isolated from MCRPEC and non-MCRPEC strains by multi-stepwise induction under conditions of colistin exposure. Unexpectedly, the absence of the mcr-1 gene from E. coli resulted in higher mutation rates and facilitated the selection of HLCRMs, in contrast to the role of plasmid-mediated quinolone resistance genes in Enterobacteriae. Quinolone resistance may be due to the presence of a plasmid-carried quinolone resistance determinant Qnr, which has been shown to bind to and protect both DNA gyrase and topoisomerase IV from inhibition by ciprofloxacin. In addition, because of their additive nature, the concentration required for mutant prevention is increased.8,21,22 Conversely, mcr-1, which encodes a pEtN transferase, confers colistin resistance via the addition of pEtN to LPS, similar to the chromosomal colistin resistance mechanism that constitutively activates PhoPQ and PmrAB.19,23 Thus MCR-1-associated LPS modifications may impair the role of TCS in the evolution of HLCRMs. These findings demonstrated that non-synonymous mutations by TCS were more easily observed in non-MCRPECs than in MCRPECs. Furthermore, pmrABC and phoP expression levels were higher in non-MCRPECs. Taken together, these findings indicated that the presence of mcr-1 limited the up-regulation of TCS genes related to colistin resistance. Usually, the MIC of colistin against MCRPECs is 2 to 8 mg/L, whereas the MIC of colistin mediated by chromosomal resistance mechanisms, such as mutations in pmrAB or phoPQ, is 16 to 256 mg/L.3,23 Because chromosomal resistance mechanisms, rather than mcr-1, may have an important impact on the evolution of HLCRMs, HLCRMs in the present study were more easily generated by non-MCRPECs. The presence of the mcr-1 gene may, however, facilitate the selection of HLCRMs. These findings suggest that the dilution of overnight cultures was too low (105 CFU/mL) to prevent E. coli TOP10 from generating HLCRMs.24

Mutations related to colistin resistance in PmrAB and PhoPQ TCS play crucial roles in the development of MCRPEC and non-MCRPEC into HLCRMs, as mutations in these systems can cause their constitutive overexpression, resulting in the activation of arnBCADTEF and pmrCAB and the modification of lipid A.23 Various genetic alterations have been associated with an increased MIC of colistin, including Ser39Ile and Arg81Ser in PmrA; Glu375lys in PhoQ; several mutations in PmrB, including Leu10Gly, Glu, 41::Tn5 (insertion of Tn5 at nucleotide 41), Cys84Tyr, a 12 bp deletion from nucleotide 258 to nucleotide 269 (GlnAlaValArgArg), Ile91Thr92 ins Ile (an insertion of isoleucine at position 92), Asp149Tyr, Thr156Lys, Ala159Val, and Val161Gly.3,4,25 Although none of these non-synonymous mutations were detected in the present study, SIFT determined that the Gly15Arg and Gly53Arg mutations in PmrA, the Pro94Gln mutation in PmrB, and the Asp86Gly mutation in PhoP affect protein function. Except for PmrA Gly15Arg, which was found in MCRPEC strains, these mutations were found in non-MCRPEC strains. The Gly15Arg and Gly53Arg mutations in PmrA, and the Pro94Gln mutation in PmrB, were found to be involved in colistin resistance in Salmonella enterica.6 Position 53 in the PmrA has also been described as being responsible for acquired colistin resistance in Klebsiella pneumoniae and Enterobacter aerogenes.3 Gly53 of PmrA is located in its phosphate receiver domain, close to the active site at Asp51.26 An amino acid substitution at Gly53, whether to Arg or Ala, prevented the Asp active site from being dephosphorylated by the phosphatase activity of PmrB. Pro94 of PmrB is located in its HAMP domain, which is crucial for signal transduction from the periplasmic input to the kinase domain.27 A mutation in the HAMP domain might therefore lead to constitutive activation of PmrA. In addition, several non-synonymous mutations were identified in PmrAB and PhoPQ, especially in non-MCRPEC strains, but SIFT showed that these mutations had little impact on protein function.

These results are in agreement with studies showing that not all the mutations in pmrAB and phoPQ result in colistin resistance.4,28 The MICs of mutants were progressively elevated by in vitro multi-stepwise induction and selection, whereas the second- and third-steps did not yield further mutations in pmrAB, phoPQ, and mgrB. This analysis may have been unable to identify mutations in other regulatory pathways that led to colistin resistance.

Previous genetic analysis revealed that Etk, a tyrosine-kinase, can phosphorylate Ugd, the starting material for L-Ara4N synthesis and can activate the PmrAB system, resulting in colistin resistance and the deletion of mgrR (influenced by the PhoPQ system).2931 Therefore, different mechanisms mediating or contributing to colistin resistance may be responsible for the development of greater resistance to colistin, especially for MCRPEC strains, inasmuch as non-synonymous mutations in pmrAB, phoPQ, and mgrB were not detected in EC24990 or EC18398. Thus, increasing the clinical use of colistin may result in the spread of colistin-resistant organisms. The present findings suggested that acquisition of the mcr-1 gene partly lowered the target mutation to impede the evolution of HLCRMs. The difficulty of a chromosomal mutation related to further colistin resistance in MCRPEC strains may provide further support for the use of colistin-based combination strategies to treat infections caused by MCR-1-producing isolates. Exposure of MCR-1- and NDM-5-producing E. coli to polymyxin B monotherapy did not result in the acquisition of a chromosomal polymyxin resistance mutation, with polymyxin B MIC remaining stable at 4 mg/L in the hollow-fiber infection model.18 The triple combination of polymyxin B, aztreonam, and amikacin resulted in undetectable bacterial counts and suppression of colistin resistance.

The present study had several limitations. First, the number of tested strains in this study was limited. Moreover, our findings showed that the presence of the mcr-1 gene may limit the evolution of MCRPEC strains into HLCRMs. Further investigations are required to determine the effects on colistin resistance of a combination of chromosomal modifications in TCS and the mcr-1 gene. Additionally, the colistin MICs of mutants in this study were further improved by in vitro multi-stepwise induction and selection, with non-synonymous mutations and other resistance mechanisms not detected. Further research is required to determine the internal molecular mechanisms of colistin resistance.

The acquisition by E. coli of the mcr-1 gene usually results in a low-level colistin resistance (28 mg/L), while having a negative impact on the development of HLCRMs. This may support the use of colistin-based combination regimens to combat infections with MCR-1-producing isolates.

The datasets used and analyzed during the current study are available from the corresponding author, Yonghong Xiao, upon reasonable request.

All named authors meet the criteria of the International Committee of Medical Journal Editors (ICMJE) for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.

All authors contributed to the data analysis, and the drafting and revising of the article; agreed on the journal to which the article will be submitted; gave final approval for the version to be published; and agreed to be accountable for all aspects of this work.

This work was supported by the Key research and development program of Zhejiang province (no. 2021C03068) and the Natural Science Foundation of Ningbo (no. 2019A610232).

The authors report no conflicts of interest in this work.

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2. Tangden T, Giske CG. Global dissemination of extensively drug-resistant carbapenemase-producing Enterobacteriaceae: clinical perspectives on detection, treatment and infection control. J Intern Med. 2015;277(5):501512. doi:10.1111/joim.12342

3. Poirel L, Jayol A, Polymyxins: NP, Activity A, Testing S. Resistance mechanisms encoded by plasmids or chromosomes. Clin Microbiol Rev. 2017;30:557596. doi:10.1128/CMR.00064-16

4. Phan MD, Nhu NTK, Achard MES, et al. Modifications in the pmrB gene are the primary mechanism for the development of chromosomally encoded resistance to polymyxins in uropathogenic Escherichia coli. J Antimicrobial Chemother. 2017;72:27292736. doi:10.1093/jac/dkx204

5. Cannatelli A, Di Pilato V, Giani T, et al. In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment. Antimicrob Agents Chemother. 2014;58(8):43994403. doi:10.1128/AAC.02555-14

6. Olaitan AO, Morand S, Rolain J-M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol. 2014;5:643. doi:10.3389/fmicb.2014.00643

7. Liu YY, Wang Y, Walsh TR, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16(2):161168. doi:10.1016/S1473-3099(15)00424-7

8. Briales A, Rodrguez-Martnez JM, Velasco C, et al. In vitro effect of qnrA1, qnrB1, and qnrS1 genes on fluoroquinolone activity against isogenic Escherichia coli isolates with mutations in gyrA and parC. Antimicrob Agents Chemother. 2011;55(3):12661269. doi:10.1128/AAC.00927-10

9. Robicsek A, Strahilevitz J, Jacoby GA, et al. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med. 2006;12(1):8388. doi:10.1038/nm1347

10. Wattal C, Oberoi JK, Goel N, Raveendran R, Khanna S. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory. Eur J Clin Microbiol. 2017;36(5):807812. doi:10.1007/s10096-016-2864-9

11. Sun Y, Liu Q, Chen S, et al. Characterization and plasmid elimination of NDM-1-producing Acinetobacter calcoaceticus from China. PLoS One. 2014;9(9):e106555. doi:10.1371/journal.pone.0106555

12. Shen C, Feng S, Chen H, et al. Transmission of mcr-1-producing multidrug-resistant enterobacteriaceae in public transportation in Guangzhou, China. Clin Infect Dis. 2018;67(suppl_2):S217S224. doi:10.1093/cid/ciy661

13. Shen P, Wei Z, Jiang Y, et al. Novel genetic environment of the carbapenem-hydrolyzing -Lactamase KPC-2 among enterobacteriaceae in China. Antimicrob Agents Chemother. 2009;53:43334338. doi:10.1128/AAC.00260-09

14. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. Available from: http://www.clsi.org/. Accessed January 21, 2020.

15. Zheng B, Huang C, Xu H, et al. Occurrence and genomic characterization of ESBL-producing, MCR-1-harboring Escherichia coli in farming soil. Front Microbiol. 2017;8:2510. doi:10.3389/fmicb.2017.02510

16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402408. doi:10.1006/meth.2001.1262

17. Falagas ME, Rafailidis PI, Matthaiou DK. Resistance to polymyxins: mechanisms, frequency and treatment options. Drug Resist Updat. 2010;13(45):132138. doi:10.1016/j.drup.2010.05.002

18. Bulman ZP, Chen L, Walsh TJ, et al. Polymyxin combinations combat Escherichia coli harboring mcr-1 and blaNDM-5: preparation for a postantibiotic era. MBio. 2017;8. doi:10.1128/mBio.00540-17

19. Smith NM, Bulman ZP, Sieron AO, et al. Pharmacodynamics of dose-escalated front-loading polymyxin B regimens against polymyxin-resistant mcr-1-harbouring Escherichia coli. J Antimicrob Chemother. 2017;72(8):22972303. doi:10.1093/jac/dkx121

20. Zhou Y-F, Tao M-T, Feng Y, et al. Increased activity of colistin in combination with amikacin against Escherichia coli co-producing NDM-5 and MCR-1. J Antimicrob Chemother. 2017;72(6):17231730. doi:10.1093/jac/dkx038

21. Rodriguez-Martinez JM, Velasco C, Garca I, et al. Mutant prevention concentrations of fluoroquinolones for Enterobacteriaceae expressing the plasmid-carried quinolone resistance determinant qnrA1. Antimicrob Agents Chemother. 2007;51(6):22362239. doi:10.1128/AAC.01444-06

22. Jacoby GA. Mechanisms of resistance to quinolones. Clin Infect Dis. 2005;41 Suppl 2:S120126. doi:10.1086/428052

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25. Cannatelli A, Giani T, Aiezza N, et al. An allelic variant of the PmrB sensor kinase responsible for colistin resistance in an Escherichia coli strain of clinical origin. Sci Rep. 2017;7(1):5071. doi:10.1038/s41598-017-05167-6

26. Sun S, Negrea A, Rhen M, Andersson DI. Genetic analysis of colistin resistance in Salmonella enterica Serovar Typhimurium. Antimicrob Agents Chemother. 2009;53(6):22982305. doi:10.1128/AAC.01016-08

27. Aravind L, Ponting CP. The cytoplasmic helical linker domain of receptor histidine kinase and methyl-accepting proteins is common to many prokaryotic signalling proteins. FEMS Microbiol Lett. 1999;176(1):111116. doi:10.1111/j.1574-6968.1999.tb13650.x

28. Agerso Y, Torpdahl M, Zachariasen C, et al. Tentative colistin epidemiological cut-off value for Salmonella spp. Foodborne Pathog Dis. 2012;9(4):367369. doi:10.1089/fpd.2011.1015

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Acquisition of the mcr-1 gene lowers the target mutation | IDR - Dove Medical Press

2021 UBC Faculty of Medicine recipients of the BC Knowledge Development Fund (clock-wise): Dr. Samuel Aparicio, Dr. Hilla Weidberg, Dr. Nozomu Yachie, Dr. Carl de Boer, Dr. Thibault Mayor, Dr. Vivien Measday, and Dr. Don Sin.

Faculty of medicine members have been awarded more than $6.5 million in funding from the B.C. Knowledge Development Fund (BCKDF) to drive innovation in B.C.

More than $22 million was awarded to 24 projects at UBC. The funding will help provide students and researchers access to the latest technology, tools and equipment to drive research. Past recipients of the BCKDF include faculty of medicine professor Dr. Pieter Cullis, who developed the lipid nanoparticle technology that allows the Pfizer-BioNTech mRNA vaccine to enter human cells.

UBC is home to some of the worlds top researchers, and this investment gives them access to cutting-edge scientific infrastructure that will support breakthroughs in fields like health care, clean technology, quantum science and agriculture, said Santa Ono, UBC president and vice-chancellor in a release. Whether its developing life-saving new drugs, ensuring literacy for all or creating novel technologies that give B.C. companies a competitive edge, this investment will promote a more healthy, innovative and sustainable society for all British Columbians.

The BCKDF enables B.C.s public post-secondary institutions and affiliated research hospitals to compete successfully for federal and private sector funding. This funding matches Government of Canada investments made through the Canada Foundation for Innovation.

The BCKDF plays a crucial role in the modernization of our universities research infrastructure capacity and capabilities, said Anne Kang, Minister of Advanced Education and Skills Training, in a release. By investing in technologically-advanced equipment and buildings, B.C. institutions will be well positioned to develop successful collaborations with industry and other partners.

We are proud to partner with the B.C. Knowledge Development Fund to invest in British Columbias teaching and research facilities, said the Honourable Franois-Philippe Champagne, Minister of Innovation, Science and Industry in a release. This partnership is helping B.C. universities rise to the challenges facing Canadians across the country from combatting climate change to conserving our precious water resources, from fighting cancer to maintaining a high quality of life for our growing senior population all while cultivating the top-notch talent we need to excel on the global stage.

The research projects will contribute to B.C.s economic plan to rebuild and grow the economy by improving B.C.s productivity and competitiveness. Other benefits include potential commercialization, spin-offs and patents, as well as discoveries that directly impact the lives of British Columbians.

The BCKDF funding will accelerate cancer research by providing researchers with specialized technology that analyses the genomes of single cells. This will advance the development of precision oncology, which uses the genomes of the patient and tumour to inform the choice of therapy that is most likely to benefit the patient. The research will provide insight into how cancer changes over time and factors that cause treatment resistance, leading to improved diagnostics and therapeutics for cancer patients in British Columbia.

Principal Investigator: Dr. Samuel Aparicio, department of pathology and laboratory medicine

BCKDF award: $2,396,810

The BCKDF funding will be used to shed light on the complex genetic underpinnings behind common inherited diseases affecting British Columbians, such as autoimmunity and heart disease, which will pave the way for the development of cellular therapies and targeted treatments for patients.

Principal Investigator: Dr. Carl de Boer, School of Biomedical Engineering

BCKDF award: $125,000

The BCKDF funding will support the development of new genetic circuit devices that will advance understanding of complex biological systems and enable the development of innovative cell-based therapies for cancer and cardiovascular diseases.

Principal Investigator: Dr. Nozomu Yachie, School of Biomedical Engineering

BCKDF award: $400,000

The BCKDF funding will help uncover better ways to treat patients with chronic obstructive pulmonary disease (COPD) using new molecular and imaging technologies. The research will support the development of innovative precision therapies that have the potential to improve the lives and enhance the health outcomes of millions of Canadians with COPD.

Principal Investigator: Dr. Don Sin, department of medicine

BCKDF award: $185,935

The BCKDF funding will be used to study the role that mitochondrial damage plays in neurodegenerative diseases such as Parkinsons and Alzheimers. The research will help uncover mechanisms to prevent this damage and develop new therapeutics to fight these otherwise incurable diseases.

Principal Investigator: Dr. Hilla Weidberg, department of cellular and physiological sciences

BCKDF award: $125,000

The BCKDF funding supports the development of new technologies that will expand the use of yeast for bioprocessing applications that benefit the environment, economy and health of British Columbians. These applications include the food and beverage industry (e.g., wine, beer, dough), removal of pollutants from the environment and the production of non-animal proteins, enzymes and new medicines.

Principal Investigator: Dr. Thibault Mayor and Dr. Vivien Measday, department of biochemistry and molecular biology

BCKDF award: $3,276,459

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Faculty of medicine researchers receive more than $6.5M from BC Knowledge Development Fund - UBC Faculty of Medicine

FREMONT, Calif., Aug. 12, 2021 /PRNewswire/ -- The premium market intelligence report published by BIS Research on the title Global Direct-to-Consumer Genetic Testing Markethighlights that the market is projected to reach $6.60 billion by 2031. The study also highlights that the market is set to witness a CAGR of 17.30% during the period 2021-2031. The growth of the market is aided by rising government initiatives for the implementation of large sequencing initiatives coupled with the increasing requirement of genetic testing, including the current COVID-19 pandemic.

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Key insights are drawn from in-depth interviews with the key opinion leaders of more than 15 leading companies, market participants, and vendors. The key players profiled in the report include 23andme, Inc., Ancestry.com LLC, 24Genetics, Atlas Biomed, Color Genomics, DNAfit, Gene by Gene, 10.9 Chengdu Twenty-Three Rubik's Cube Biotechnology Co., Ltd., Easy DNA, Mapmygenome, Laboratory Corporation of American Holdings, Myriad Genetics, Inc., Konika Minolta, Inc., and XCODE Lifescience, Inc.

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Global Direct-to-Consumer Genetic Testing Market to Reach $6.60 Billion by 2031, Says BIS Research Study - PRNewswire

PITTSBURGH, Aug. 12, 2021 (GLOBE NEWSWIRE) -- NeuBase Therapeutics, Inc. (Nasdaq: NBSE) (NeuBase or the Company), a biotechnology platform company Drugging the Genome to address disease at the base level using a new class of precision genetic medicines, today reported its financial results for the three- and nine-month periods ended June 30, 2021.

In June, we presented preclinical in vivo data of novel compounds demonstrating selective silencing of disease-causing mutations at the DNA or RNA level in three diseases, each of which is caused by a different underlying genetic mechanism. These new data further illustrate the broad applicability of our genetic medicine platform, said Dietrich A. Stephan, Ph.D., Founder, CEO, and Chairman of NeuBase. Following intravenous or subcutaneous dosing, these compounds were well tolerated at pharmacologically active doses. In addition, the compounds achieved targeted delivery into brain and muscle, which further support our claim of offering the unique ability to deliver genetic medicines throughout the body.

For our lead program in DM1, recent data support a differentiated therapeutic approach to maintain DMPK function while selectively silencing the disease-driving mutation. With these positive data in hand, we believe we have a clear path towards entering the clinic and are planning for an IND filing in the fourth quarter of calendar year 2022, continued Dr. Stephan. We are continuing to advance our therapeutic program for Huntingtons disease and we believe our proprietary delivery technology will allow our compounds to advance beyond intrathecal delivery, overcoming challenges seen with other programs.

Dr. Stephan concluded, Finally, we have shown that we can silence activating KRAS point mutations in vivo to inhibit protein production, which has the potential to target G12D and G12V, the two most common and historically undruggable KRAS driver mutations that represent the majority of KRAS-driven tumors. This sets the stage for generating new precision genetic medicines capable of selectively targeting mutations at the single-base level to treat both rare and common diseases.

Third Quarter of Fiscal Year 2021 and Recent Operating Highlights

Financial Results for the Third Fiscal Quarter Ended June 30, 2021

Financial Results for the Nine-Month Period Ended June 30, 2021

About NeuBase TherapeuticsNeuBase is accelerating the genetic revolution by developing a new class of precision genetic medicines which can be designed to increase, decrease, or change gene function, as appropriate, to resolve genetic defects that drive disease. NeuBases targeted PATrOL therapies are centered around its proprietary drug scaffold to address genetic diseases at the DNA or RNA level by combining the highly targeted approach of traditional genetic therapies with the broad organ distribution capabilities of small molecules. With an initial focus on silencing disease-causing mutations in debilitating neuromuscular, neurological and oncologic disorders, NeuBase is committed to redefining medicine for the millions of patients with both common and rare conditions. To learn more, visit http://www.neubasetherapeutics.com.

Use of Forward-Looking StatementsThis press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act. These forward-looking statements are distinguished by use of words such as will, would, anticipate, expect, believe, designed, plan, or intend, the negative of these terms, and similar references to future periods. These forward-looking statements include, among others, those related to the prospects of DM1 and the Companys expectation to make an IND filing for DM1 in the fourth quarter of CY 2022, the Companys therapeutic program for Huntingtons disease, the Companys ability to target G12D and G12V and the Companys expectation that its cash will fund currently planned operating and capital expenditures into the first quarter of CY 2023. These views involve risks and uncertainties that are difficult to predict and, accordingly, our actual results may differ materially from the results discussed in our forward-looking statements. Our forward-looking statements contained herein speak only as of the date of this press release. Factors or events that we cannot predict, including those risk factors contained in our filings with the U.S. Securities and Exchange Commission (the SEC), may cause our actual results to differ from those expressed in forward-looking statements. The Company may not actually achieve the plans, carry out the intentions or meet the expectations or projections disclosed in the forward-looking statements, and you should not place undue reliance on these forward-looking statements. Because such statements deal with future events and are based on the Companys current expectations, they are subject to various risks and uncertainties, and actual results, performance or achievements of the Company could differ materially from those described in or implied by the statements in this press release, including: the Companys plans to develop and commercialize its product candidates; the timing of initiation of the Companys planned clinical trials; the risks that prior data will not be replicated in future studies; the timing of any planned investigational new drug application or new drug application; the Companys plans to research, develop and commercialize its current and future product candidates; the clinical utility, potential benefits and market acceptance of the Companys product candidates; the Companys commercialization, marketing and manufacturing capabilities and strategy; global health conditions, including the impact of COVID-19; the Companys ability to protect its intellectual property position; and the requirement for additional capital to continue to advance these product candidates, which may not be available on favorable terms or at all, as well as those risk factors contained in our filings with the SEC. Except as otherwise required by law, the Company disclaims any intention or obligation to update or revise any forward-looking statements, which speak only as of the date hereof, whether as a result of new information, future events or circumstances or otherwise.

NeuBase Investor Contact:Dan FerryManaging DirectorLifeSci Advisors, LLCdaniel@lifesciadvisors.com OP: (617) 430-7576

NeuBase Media Contact:Jessica Yingling, Ph.D.Little Dog Communications Inc.(858) 344-8091jessica@litldog.com

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NeuBase Therapeutics Reports Financial Results for the Third Quarter of Fiscal Year 2021 and Recent Operating - GlobeNewswire

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Thursday, August 5, 2021

Mutation in a single gene appears to account for a form of male infertility in which men fail to produce sperm, according to an international study funded in part by the National Institutes of Health. Males with the condition, known as non-obstructive azoospermia, fail to produce any sperm, even though they do not have any obstruction in the ducts through which sperm are released. The gene, PNLDC1, codes for an enzyme that processes a class of non-coding ribonucleic acids (RNA) so they can function. These non-coding RNAs are not involved in making proteins but are believed to be involved in various functions that occur during spermatogenesis the process by which cells in the testes produce sperm cells. The findings may provide insight into how sperm is produced and may one day lead to information helpful for the diagnosis and treatment of non-obstructive azoospermia. Similarly, greater understanding of the genes function may contribute to the development of new methods of male contraception.

The study was conducted by an international team of researchers and appears in The New England Journal of Medicine. Funding from NIHs Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) was provided to two authors institutions, the University of Utah School of Medicine, Salt Lake City (Kenneth I. Aston, Ph.D.) and Oregon Health and Science University, Portland (Donald F. Conrad, Ph.D.).

In the search for the genetic foundations of the condition, the researchers sequenced the exomes protein coding regions of the genome of 924 men with non-obstructive azoospermia. They found that four of the men had mutations in the PNLDC1 gene. Analysis of testicular tissue from the men showed that spermatagonia (sperm producing cells) failed to complete meiotic cell division and develop sperm cells. The authors theorized that other genes coding for enzymes involved in processing non-coding RNAs also might be involved in infertility due to azoospermia.

Stuart B. Moss, Ph.D., Health Scientist Administrator, NICHD Fertility and Infertility Branch, is available for comment.

Nagirnaja, L. Variant PNLDC1, Defective piRNA Processing, and Azoospermia. New England Journal of Medicine. 2021.

About the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): NICHD leads research and training to understand human development, improve reproductive health, enhance the lives of children and adolescents, and optimize abilities for all. For more information, visit https://www.nichd.nih.gov.

About the National Institutes of Health (NIH):NIH, the nation's medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit http://www.nih.gov.

NIHTurning Discovery Into Health

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funded study discovers gene involved in male infertility - National Institutes of Health

Limb-girdle muscular dystrophy type 2A is the most common form of LGMD, accounting for a third of LGMD diagnoses

Sareptas unrivaled portfolio of investigational gene therapies for LGMD offers the potential to address six LGMD subtypes, which together represent more than 70% of all known LGMDs

CAMBRIDGE, Mass., Aug. 04, 2021 (GLOBE NEWSWIRE) -- Sarepta Therapeutics, Inc. (NASDAQ:SRPT), the leader in precision genetic medicine for rare diseases, today announced that upon completion of a number of preclinical and safety studies, it had executed an exclusive license agreement for an investigational gene therapy candidate, calpain 3 (CAPN-3), to treat Limb-girdle muscular dystrophy type 2A (LGMD2A), developed by the Abigail Wexner Research Institute at Nationwide Childrens Hospital (Nationwide Childrens).

LGMDs represent a group of distinct genetic neuromuscular diseases with a generally common set of symptoms, including progressive, debilitating weakness and wasting that begins in muscles around the hips and shoulders before progressing to muscles in the arms and legs. Many LGMD sub-types are significantly life-limiting and often life-ending diseases. Also known as calpainopathy, LGMD2A is caused by mutations in the CAPN-3 gene and is the most common type of LGMD, accounting for almost a third of cases.

Treatment plans for LGMD2A are currently limited to physical therapy, assistive devices and surgery for complications. Were excited about the opportunity to transform patient care for this significantly life-limiting disease by advancing the CAPN-3 program following extensive pre-clinical work by the team at Nationwide Childrens. Preclinical research conducted to date has provided early proof of concept for CAPN-3 in LGMD2A and supports further advancement, said Louise Rodino-Klapac, Sareptas executive vice president and chief scientific officer. We intend to build off the knowledge we have gained from our lead investigational gene transfer programs for Duchenne muscular dystrophy and LGMD2E, as the CAPN-3 program also uses the AAVrh74 vector to address another well-characterized genetic disease. Sareptas commitment and research investment in LGMD is unparalleled and we continue to work towards advancing all of our LGMD programs as quickly as possible.

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Like SRP-9001, Sareptas lead investigational gene transfer therapy for Duchenne muscular dystrophy, and the Companys five other LGMD programs, the LGMD2A program uses the AAVrh74 vector, designed to systematically and robustly deliver treatment to skeletal muscle, including the diaphragm, making it an ideal candidate to treat muscle disease.

The preclinical work on the CAPN-3 program in LGMD2A has been led by Zarife Sahenk, M.D., Ph.D., attending neurologist at Nationwide Childrens, Director of Clinical and Experimental Neuromuscular Pathology at The Research Institute at Nationwide Childrens and Professor of Pediatrics, Pathology and Neurology at The Ohio State University College of Medicine.

About Limb-girdle Muscular DystrophyLimb-girdle muscular dystrophies are genetic diseases that cause progressive, debilitating weakness and wasting that begins in muscles around the hips and shoulders before progressing to muscles in the arms and legs. Sareptas six LGMD gene therapy programs in development include LGMD2E, LGMD2D, LGMD2C, LGMD2B, LGMD2L and LGMD2A, which together represent more than 70 percent of known LGMD cases.

About Sarepta TherapeuticsSarepta is on an urgent mission: engineer precision genetic medicine for rare diseases that devastate lives and cut futures short. We hold leadership positions in Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophies (LGMDs), and we currently have more than 40 programs in various stages of development. Our vast pipeline is driven by our multi-platform Precision Genetic Medicine Engine in gene therapy, RNA and gene editing. For more information, please visit http://www.sarepta.com or follow us on Twitter, LinkedIn, Instagram and Facebook.

Internet Posting of InformationWe routinely post information that may be important to investors in the 'For Investors' section of our website at http://www.sarepta.com. We encourage investors and potential investors to consult our website regularly for important information about us.

Forward-Looking StatementsThis press release contains "forward-looking statements." Any statements contained in this press release that are not statements of historical fact may be deemed to be forward-looking statements. Words such as "believes," "anticipates," "plans," "expects," "will," "intends," "potential," "possible" and similar expressions are intended to identify forward-looking statements. These forward-looking statements include statements regarding the potential benefits of the licensing agreement; the design of the AAVrh74 vector to systematically and robustly deliver treatment to skeletal muscle, including the diaphragm, making it an ideal candidate to treat muscle disease; the potential of our portfolio of investigational gene therapies for LGMD to address six LGMD subtypes, which together represent more than 70% of all known LGMDs; and our plan to continue to advance all of our LGMD programs as quickly as possible.

These forward-looking statements involve risks and uncertainties, many of which are beyond our control. Known risk factors include, among others: the expected benefits and opportunities related to the licensing agreement may not be realized or may take longer to realize than expected due to challenges and uncertainties inherent in product research and development. In particular, activities under the license may not result in any viable treatments suitable for commercialization due to a variety of reasons, including any inability of the parties to perform their commitments and obligations under the agreement; success in preclinical trials does not ensure that later clinical trials will be successful; Sarepta may not be able to execute on its business plans and goals, including meeting its expected or planned regulatory milestones and timelines, clinical development plans, and bringing its product candidates to market, due to a variety of reasons, many of which may be outside of Sareptas control, including possible limitations of company financial and other resources, manufacturing limitations that may not be anticipated or resolved for in a timely manner, regulatory, court or agency decisions, such as decisions by the United States Patent and Trademark Office with respect to patents that cover Sareptas product candidates and the COVID-19 pandemic; even if Sareptas programs result in new commercialized products, Sarepta may not achieve the expected revenues from the sale of such products; if the actual number of patients living with LGMD2A is smaller than estimated, Sareptas revenue and ability to achieve profitability may be adversely affected; and those risks identified under the heading Risk Factors in Sareptas most recent Annual Report on Form 10-K for the year ended December 31, 2020, and most recent Quarterly Report on Form 10-Q filed with the Securities and Exchange Commission (SEC) as well as other SEC filings made by the Company which you are encouraged to review.

Any of the foregoing risks could materially and adversely affect the Companys business, results of operations and the trading price of Sareptas common stock. For a detailed description of risks and uncertainties Sarepta faces, you are encouraged to review the SEC filings made by Sarepta. We caution investors not to place considerable reliance on the forward-looking statements contained in this press release. Sarepta does not undertake any obligation to publicly update its forward-looking statements based on events or circumstances after the date hereof.

Source: Sarepta Therapeutics, Inc.

Investor Contact: Ian Estepan, 617-274-4052iestepan@sarepta.com

Media Contact: Tracy Sorrentino, 617-301-8566tsorrentino@sarepta.com

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Sarepta Therapeutics Executes Licensing Agreement for Gene Therapy Program from Nationwide Childrens Hospital to Treat Limb-Girdle Muscular Dystrophy...

A large new study scanned the genes of hundreds of thousands of women near the age of menopause and turned up hundreds of genetic signals that the researchers said might help predict and prevent early menopause, as well as treat infertility and improve womens reproductive health in the future.

The study, published in Nature, identified 290 genetic variants, many of them part of a pathway that repairs DNA, associated with the age at which women enter menopause. Researchers also found that changing the levels of two of these DNA repair genes delayed menopause in mice. The study broadens the understanding of how genes, specifically those in the DNA-damage response pathway, could influence the reproductive life span in women.

The average age at which women start menopause is about 51 years, and is brought on by a decrease of ovarian reserve, the capacity to produce healthy eggs. But there is significant variation in the age of menopause onset, determined by genetics and environmental factors. Although the environmental factors that influence menopause, like smoking and chemotherapy, are well-studied, the genetic factors had remained a black box.

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Studying the underlying biology and genetics of menopause has proven difficult because a womans supply of eggs are mostly formed before birth and studying it in adult humans often means taking a sample of ovarian tissue. If you were studying muscle or skin, you can take a biopsy of those tissues, said Anna Murray, a geneticist at the University of Exeter in the U.K. and author of the new study. Nobodys going to biopsy a womans ovaries its very precious tissue.

To get around these difficulties, researchers looked to genetic studies called genome-wide association studies, or GWAS. Two such previous studies had identified around 60 genetic regions associated with the timing of menopause.

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Now the multi-institutional team looked at the genes of a much larger group of women, about 200,000, between 40 and 60 years old, and found nearly 300 genetic signals associated with menopause timing. Similar to the results of their previous studies, many of the genetic regions they identified are involved in processes that respond to DNA damage to maintain cells health or induce cell death if necessary. Still, the researchers were surprised by how prevalent this pathway was in their findings. I dont know if other traits have found this level of enrichment for one particular process, said Murray.

Using the identified variants, the authors produced a risk score to see if they could predict which individuals were likely to have premature ovarian insufficiency, which occurs when women reach menopause before the age of 40. Although it was a weak predictor, the risk score identified women who started menopause by 40 better than smoking status.

Two DNA-repair genes, Chek1 and Chek2, stood out for their strong association with menopause timing. Women who lacked a working Chek2 protein had menopause three and a half years later than those who had normal Chek2, and female mice bred without the Chek2 gene had more eggs than normal mice when they were older, effectively extending their reproductive life span.

On the other hand, introducing a copy of the Chek1 gene into the mice also extended their reproductive life span, but by enabling production of more eggs after birth, which took longer to deplete. These different mechanisms really highlight the complexity in the processes that go into the ovarian reserve, said Rong Li, cell biologist at Johns Hopkins University and professor of the National University of Singapore who studies cellular processes of development.

In the future, researchers hope these findings could lead to therapeutics to extend fertility in women, though it may not be a straightforward process, Murray said. While early menopause was associated with increased risk of type 2 diabetes and worse bone health, it was also associated with decreased risk of breast and ovarian cancer. To avoid detrimental effects of delaying menopause, researchers suggest that therapeutics could be used in the short-term like targeting certain genes to enhance egg production during IVF cycles, for example.

Targeting DNA-repair genes with treatments could also have unintended consequences. Li suggests that other genes may be better targets. [DNA-damage response genes] are a bit scary to manipulate because [by inhibiting them] you could get cancer, said Li, who was not involved with the study but has collaborated with Perry. Other pathways might be better and safer targets for intervention.

More simply, the findings could also be used to provide women with more information about the approximate age when they would get menopause. Predictions about menopause age could inform women of their risk of developing conditions like breast cancer and help their decisions about when to have children, which could help them avoid unnecessary procedures, like infertility treatments.

But because most of the study was done on women of European ancestry, the findings need to be replicated in different populations, said Corrine Welt, an endocrinologist at the University of Utah who studies the genetics of early menopause and who was not involved in the study. When the study did look at women from East Asian ancestry, it found that many of the genetic signals held up but the size of the effect of these genetic regions on menopause timing was different than those in women with European ancestry.

Murray hopes future studies could improve menopause age prediction by also including non-genetic factors that are known to influence ovarian reserve like smoking. Researchers are hopeful that womens reproductive health is finally getting the attention it deserves, which could open the door for more studies and ultimately better health outcomes.

I think with studies like this, we are making a lot of headway, said Welt. Lets study all the female reproductive problems with genetics, because, unfortunately, its been an underserved aspect of medicine.

See more here:
Study finds genetic signals that might be used to predict early menopause - STAT