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Bacterial multidrug resistance problem solved by a broad-spectrum synthetic antibiotic
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
There is an increasing demand for new antibiotics that effectively treat patients with refractory bacteremia, do not evoke bacterial resistance, and can be readily modified to address current and anticipated patient needs. Recently scientists described a promising compound of COE (conjugated oligo electrolytes) family, COE2-2hexyl, that exhibited broad-spectrum antibacterial activity. COE2-2hexyl effectively-treated mice infected with bacteria derived from sepsis patients with refractory bacteremia, including a CRE K. pneumoniae strain resistant to nearly all clinical antibiotics tested. Notably, this lead compound did not evoke drug resistance in several pathogens tested. COE2-2hexyl has specific effects on multiple membrane-associated functions (e.g., septation, motility, ATP synthesis, respiration, membrane permeability to small molecules) that may act together to abrogate bacterial cell viability and the evolution of drug-resistance. Impeding these bacterial properties may occur through alteration of vital protein–protein or protein-lipid membrane interfaces – a mechanism of action distinct from many membrane disrupting antimicrobials or detergents that destabilize membranes to induce bacterial cell lysis. The diversity and ease of COE design and chemical synthesis have the potential to establish a new standard for drug design and personalized antibiotic treatment.
Recent studies have shown that small molecules can preferentially target bacterial membranes due to significant differences in lipid composition, presence of a cell wall, and the absence of cholesterol. The inner membranes of Gram-negative bacteria are generally more negatively charged at their surface because they contain more anionic lipids such as cardiolipin and phosphatidylglycerol within their outer leaflet compared to mammalian membranes. In contrast, membranes of mammalian cells are largely composed of more-neutral phospholipids, sphingomyelins, as well as cholesterol, which affords membrane rigidity and ability to withstand mechanical stresses; and may stabilize the membrane against structural damage to membrane-disrupting agents such as COEs. Consistent with these studies, COE2-2hexyl was well tolerated in mice, suggesting that COEs are not intrinsically toxic in vivo, which is often a primary concern with membrane-targeting antibiotics. The COE refinement workflow potentially accelerates lead compound optimization by more rapid screening of novel compounds for the iterative directed-design process. It also reduces the time and cost of subsequent biophysical characterization, medicinal chemistry and bioassays, ultimately facilitating the discovery of novel compounds with improved pharmacological properties.
Additionally, COEs provide an approach to gain new insights into microbial physiology, including membrane structure/function and mechanism of drug action/resistance, while also generating a suite of tools that enable the modulation of bacterial and mammalian membranes for scientific or manufacturing uses. Notably, further COE safety and efficacy studies are required to be conducted on a larger scale to ensure adequate understanding of the clinical benefits and risks to assure clinical efficacy and toxicity before COEs can be added to the therapeutic armamentarium. Despite these limitations, the ease of molecular design, synthesis and modular nature of COEs offer many advantages over conventional antimicrobials, making synthesis simple, scalable and affordable. It enables the construction of a spectrum of compounds with the potential for development as a new versatile therapy for the emergence and rapid global spread of pathogens that are resistant to all, or nearly all, existing antimicrobial medicines.
Use of Systems Biology for Design of inhibitor of Galectins as Cancer Therapeutic – Strategy and Software
Curator:Stephen J. Williams, Ph.D.
Below is a slide representation of the overall mission 4 to produce a PROTAC to inhibit Galectins 1, 3, and 9.
Using A Priori Knowledge of Galectin Receptor Interaction to Create a BioModel of Galectin 3 Binding
Now after collecting literature from PubMed on “galectin-3” AND “binding” to determine literature containing kinetic data we generate a WordCloud on the articles.
This following file contains the articles needed for BioModels generation.
From the WordCloud we can see that these corpus of articles describe galectin binding to the CRD (carbohydrate recognition domain). Interestingly there are many articles which describe van Der Waals interactions as well as electrostatic interactions. Certain carbohydrate modifictions like Lac NAc and Gal 1,4 may be important. Many articles describe the bonding as well as surface interactions. Many studies have been performed with galectin inhibitors like TDGs (thio-digalactosides) like TAZ TDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside). This led to an interesting article
.
Dual thio-digalactoside-binding modes of human galectins as the structural basis for the design of potent and selective inhibitors
Human galectins are promising targets for cancer immunotherapeutic and fibrotic disease-related drugs. We report herein the binding interactions of three thio-digalactosides (TDGs) including TDG itself, TD139 (3,3′-deoxy-3,3′-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside, recently approved for the treatment of idiopathic pulmonary fibrosis), and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside) with human galectins-1, -3 and -7 as assessed by X-ray crystallography, isothermal titration calorimetry and NMR spectroscopy. Five binding subsites (A-E) make up the carbohydrate-recognition domains of these galectins. We identified novel interactions between an arginine within subsite E of the galectins and an arene group in the ligands. In addition to the interactions contributed by the galactosyl sugar residues bound at subsites C and D, the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterised dual binding modes demonstrate how binding potency, reported as decreased Kd values of the TDG inhibitors from μM to nM, is improved and also offer insights to development of selective inhibitors for individual galectins.
Figures
Figure 1. Chemical structures of L3, TDG…
Figure 2. Structural comparison of the carbohydrate…
The following paper in Cells describes the discovery of protein interactors of endoglin, which is recruited to membranes at the TGF-β receptor complex upon TGF-β signaling. Interesting a carbohydrate binding protein, galectin-3, and an E3-ligase, TRIM21, were found to be unique interactors within this complex.
Gallardo-Vara E, Ruiz-Llorente L, Casado-Vela J, Ruiz-Rodríguez MJ, López-Andrés N, Pattnaik AK, Quintanilla M, Bernabeu C. Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners. Cells. 2019 Sep 13;8(9):1082. doi: 10.3390/cells8091082. PMID: 31540324; PMCID: PMC6769930.
Abstract
Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-β receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-β receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-β family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.
Endoglin is an auxiliary TGF-β co-receptor predominantly expressed in endothelial cells, which is involved in vascular development, repair, homeostasis, and disease [1,2,3,4]. Heterozygous mutations in the human ENDOGLIN gene (ENG) cause hereditary hemorrhagic telangiectasia (HHT) type 1, a vascular disease associated with nasal and gastrointestinal bleeds, telangiectases on skin and mucosa and arteriovenous malformations in the lung, liver, and brain [4,5,6]. The key role of endoglin in the vasculature is also illustrated by the fact that endoglin-KO mice die in utero due to defects in the vascular system [7]. Endoglin expression is markedly upregulated in proliferating endothelial cells involved in active angiogenesis, including the solid tumor neovasculature [8,9]. For this reason, endoglin has become a promising target for the antiangiogenic treatment of cancer [10,11,12]. Endoglin is also expressed in cancer cells where it can behave as both a tumor suppressor in prostate, breast, esophageal, and skin carcinomas [13,14,15,16] and a promoter of malignancy in melanoma and Ewing’s sarcoma [17]. Ectodomain shedding of membrane-bound endoglin may lead to a circulating form of the protein, also known as soluble endoglin (sEng) [18,19,20]. Increased levels of sEng have been found in several vascular-related pathologies, including preeclampsia, a disease of high prevalence in pregnant women which, if left untreated, can lead to serious and even fatal complications for both mother and baby [2,18,19,21]. Interestingly, several lines of evidence support a pathogenic role of sEng in the vascular system, including endothelial dysfunction, antiangiogenic activity, increased vascular permeability, inflammation-associated leukocyte adhesion and transmigration, and hypertension [18,22,23,24,25,26,27]. Because of its key role in vascular pathology, a large number of studies have addressed the structure and function of endoglin at the molecular level, in order to better understand its mechanism of action.
Galectin-3 Interacts with Endoglin in Cells
Galectin-3 is a secreted member of the lectin family with the capacity to bind membrane glycoproteins like endoglin and is involved in the pathogenesis of many human diseases [52]. We confirmed the protein screen data for galectin-3, as evidenced by two-way co-immunoprecipitation of endoglin and galectin-3 upon co-transfection in CHO-K1 cells. As shown in Figure 1A, galectin-3 and endoglin were efficiently transfected, as demonstrated by Western blot analysis in total cell extracts. No background levels of endoglin were observed in control cells transfected with the empty vector (Ø). By contrast, galectin-3 could be detected in all samples but, as expected, showed an increased signal in cells transfected with the galectin-3 expression vector. Co-immunoprecipitation studies of these cell lysates showed that galectin-3 was present in endoglin immunoprecipitates (Figure 1B). Conversely, endoglin was also detected in galectin-3 immunoprecipitates (Figure 1C).
Figure 1. Protein–protein association between galectin-3 and endoglin. (A–C). Co-immunoprecipitation of galectin-3 and endoglin. CHO-K1 cells were transiently transfected with pcEXV-Ø (Ø), pcEXV–HA–EngFL (Eng) and pcDNA3.1–Gal-3 (Gal3) expression vectors. (A) Total cell lysates (TCL) were analyzed by SDS-PAGE under reducing conditions, followed by Western blot (WB) analysis using specific antibodies to endoglin, galectin-3 and β-actin (loading control). Cell lysates were subjected to immunoprecipitation (IP) with anti-endoglin (B) or anti-galectin-3 (C) antibodies, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin or anti-galectin-3 antibodies, as indicated. Negative controls with an IgG2b (B) and IgG1 (C) were included. (D) Protein-protein interactions between galectin-3 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 7.3 µM recombinant human galectin-3/6xHis at the C-terminus (LGALS3), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.
Figure 2.Galectin-3 and endoglin co-localize in human endothelial cells. Human umbilical vein-derived endothelial cell (HUVEC) monolayers were fixed with paraformaldehyde, permeabilized with Triton X-100, incubated with the mouse mAb P4A4 anti-endoglin, washed, and incubated with a rabbit polyclonal anti-galectin-3 antibody (PA5-34819). Galectin-3 and endoglin were detected by immunofluorescence upon incubation with Alexa 647 goat anti-rabbit IgG (red staining) and Alexa 488 goat anti-mouse IgG (green staining) secondary antibodies, respectively. (A) Single staining of galectin-3 (red) and endoglin (green) at the indicated magnifications. (B) Merge images plus DAPI (nuclear staining in blue) show co-localization of galectin-3 and endoglin (yellow color). Representative images of five different experiments are shown.
Endoglin associates with the cullin-type E3 ligase TRIM21
Figure 3.Protein–protein association between TRIM21 and endoglin. (A–E) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin (A). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin (B). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (E) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin (C). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included (D). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. (F) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.
Table 1. Human protein-array analysis of endoglin interactors1.
1 Microarrays containing over 9000 unique human proteins were screened using recombinant sEng as a probe. Protein interactors showing the highest scores (Z-score ≥2.0) are listed. GeneBank (https://www.ncbi.nlm.nih.gov/genbank/) and UniProtKB (https://www.uniprot.org/help/uniprotkb) accession numbers are indicated with a yellow or green background, respectively. The cellular compartment of each protein was obtained from the UniProtKB webpage. Proteins selected for further studies (TRIM21 and galectin-3) are indicated in bold type with blue background.
Note: the following are from NCBI Genbank and Genecards on TRIM21
Official Symbol TRIM21provided by HGNC Official Full Name tripartite motif containing 21provided by HGNC Primary source HGNC:HGNC:11312 See related Ensembl:ENSG00000132109MIM:109092;AllianceGenome:HGNC:11312 Gene type protein coding RefSeq status REVIEWED Organism Homo sapiens Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo Also known as SSA; RO52; SSA1; RNF81; Ro/SSA Summary This gene encodes a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The encoded protein is part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus. RoSSA interacts with autoantigens in patients with Sjogren syndrome and systemic lupus erythematosus. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. [provided by RefSeq, Jul 2008] Expression Ubiquitous expression in spleen (RPKM 15.5), appendix (RPKM 13.2) and 24 other tissues See more Orthologs mouseall NEW Try the new Gene table Try the new Transcript table
This gene encodes a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The encoded protein is part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus. RoSSA interacts with autoantigens in patients with Sjogren syndrome and systemic lupus erythematosus. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. [provided by RefSeq, Jul 2008]
E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2. Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination. Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes. A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B (‘Thr-187’ phosphorylated-form), thereby promoting its degradation by the proteasome. Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling. Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway. Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages. Plays a role in the regulation of the cell cycle progression. Enhances the decapping activity of DCP2. Exists as a ribonucleoprotein particle present in all mammalian cells studied and composed of a single polypeptide and one of four small RNA molecules. At least two isoforms are present in nucleated and red blood cells, and tissue specific differences in RO/SSA proteins have been identified. The common feature of these proteins is their ability to bind HY RNAs.2. Involved in the regulation of innate immunity and the inflammatory response in response to IFNG/IFN-gamma. Organizes autophagic machinery by serving as a platform for the assembly of ULK1, Beclin 1/BECN1 and ATG8 family members and recognizes specific autophagy targets, thus coordinating target recognition with assembly of the autophagic apparatus and initiation of autophagy. Acts as an autophagy receptor for the degradation of IRF3, hence attenuating type I interferon (IFN)-dependent immune responses (PubMed:26347139, 16297862, 16316627, 16472766, 16880511, 18022694, 18361920, 18641315, 18845142, 19675099). Represses the innate antiviral response by facilitating the formation of the NMI-IFI35 complex through ‘Lys-63’-linked ubiquitination of NMI (PubMed:26342464). ( RO52_HUMAN,P19474 )
Molecular function for TRIM21 Gene according to UniProtKB/Swiss-Prot
Function:
E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2. Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination. Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes. A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B (‘Thr-187’ phosphorylated-form), thereby promoting its degradation by the proteasome. Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling. Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway. Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages. Plays a role in the regulation of the cell cycle progression.
Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners
Gallardo-Vara E, Ruiz-Llorente L, Casado-Vela J, Ruiz-Rodríguez MJ, López-Andrés N, Pattnaik AK, Quintanilla M, Bernabeu C. Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners. Cells. 2019 Sep 13;8(9):1082. doi: 10.3390/cells8091082. PMID: 31540324; PMCID: PMC6769930.
Abstract
Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-β receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-β receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-β family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.
Endoglin is an auxiliary TGF-β co-receptor predominantly expressed in endothelial cells, which is involved in vascular development, repair, homeostasis, and disease [1,2,3,4]. Heterozygous mutations in the human ENDOGLIN gene (ENG) cause hereditary hemorrhagic telangiectasia (HHT) type 1, a vascular disease associated with nasal and gastrointestinal bleeds, telangiectases on skin and mucosa and arteriovenous malformations in the lung, liver, and brain [4,5,6]. The key role of endoglin in the vasculature is also illustrated by the fact that endoglin-KO mice die in utero due to defects in the vascular system [7]. Endoglin expression is markedly upregulated in proliferating endothelial cells involved in active angiogenesis, including the solid tumor neovasculature [8,9]. For this reason, endoglin has become a promising target for the antiangiogenic treatment of cancer [10,11,12]. Endoglin is also expressed in cancer cells where it can behave as both a tumor suppressor in prostate, breast, esophageal, and skin carcinomas [13,14,15,16] and a promoter of malignancy in melanoma and Ewing’s sarcoma [17]. Ectodomain shedding of membrane-bound endoglin may lead to a circulating form of the protein, also known as soluble endoglin (sEng) [18,19,20]. Increased levels of sEng have been found in several vascular-related pathologies, including preeclampsia, a disease of high prevalence in pregnant women which, if left untreated, can lead to serious and even fatal complications for both mother and baby [2,18,19,21]. Interestingly, several lines of evidence support a pathogenic role of sEng in the vascular system, including endothelial dysfunction, antiangiogenic activity, increased vascular permeability, inflammation-associated leukocyte adhesion and transmigration, and hypertension [18,22,23,24,25,26,27]. Because of its key role in vascular pathology, a large number of studies have addressed the structure and function of endoglin at the molecular level, in order to better understand its mechanism of action.
Galectin-3 Interacts with Endoglin in Cells
Galectin-3 is a secreted member of the lectin family with the capacity to bind membrane glycoproteins like endoglin and is involved in the pathogenesis of many human diseases [52]. We confirmed the protein screen data for galectin-3, as evidenced by two-way co-immunoprecipitation of endoglin and galectin-3 upon co-transfection in CHO-K1 cells. As shown in Figure 1A, galectin-3 and endoglin were efficiently transfected, as demonstrated by Western blot analysis in total cell extracts. No background levels of endoglin were observed in control cells transfected with the empty vector (Ø). By contrast, galectin-3 could be detected in all samples but, as expected, showed an increased signal in cells transfected with the galectin-3 expression vector. Co-immunoprecipitation studies of these cell lysates showed that galectin-3 was present in endoglin immunoprecipitates (Figure 1B). Conversely, endoglin was also detected in galectin-3 immunoprecipitates (Figure 1C).
Figure 1. Protein–protein association between galectin-3 and endoglin. (A–C). Co-immunoprecipitation of galectin-3 and endoglin. CHO-K1 cells were transiently transfected with pcEXV-Ø (Ø), pcEXV–HA–EngFL (Eng) and pcDNA3.1–Gal-3 (Gal3) expression vectors. (A) Total cell lysates (TCL) were analyzed by SDS-PAGE under reducing conditions, followed by Western blot (WB) analysis using specific antibodies to endoglin, galectin-3 and β-actin (loading control). Cell lysates were subjected to immunoprecipitation (IP) with anti-endoglin (B) or anti-galectin-3 (C) antibodies, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin or anti-galectin-3 antibodies, as indicated. Negative controls with an IgG2b (B) and IgG1 (C) were included. (D) Protein-protein interactions between galectin-3 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 7.3 µM recombinant human galectin-3/6xHis at the C-terminus (LGALS3), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.
Figure 2.Galectin-3 and endoglin co-localize in human endothelial cells. Human umbilical vein-derived endothelial cell (HUVEC) monolayers were fixed with paraformaldehyde, permeabilized with Triton X-100, incubated with the mouse mAb P4A4 anti-endoglin, washed, and incubated with a rabbit polyclonal anti-galectin-3 antibody (PA5-34819). Galectin-3 and endoglin were detected by immunofluorescence upon incubation with Alexa 647 goat anti-rabbit IgG (red staining) and Alexa 488 goat anti-mouse IgG (green staining) secondary antibodies, respectively. (A) Single staining of galectin-3 (red) and endoglin (green) at the indicated magnifications. (B) Merge images plus DAPI (nuclear staining in blue) show co-localization of galectin-3 and endoglin (yellow color). Representative images of five different experiments are shown.
Endoglin associates with the cullin-type E3 ligase TRIM21
Figure 3.Protein–protein association between TRIM21 and endoglin. (A–E) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin (A). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin (B). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (E) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin (C). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included (D). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. (F) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.
Table 1. Human protein-array analysis of endoglin interactors1.
1 Microarrays containing over 9000 unique human proteins were screened using recombinant sEng as a probe. Protein interactors showing the highest scores (Z-score ≥2.0) are listed. GeneBank (https://www.ncbi.nlm.nih.gov/genbank/) and UniProtKB (https://www.uniprot.org/help/uniprotkb) accession numbers are indicated with a yellow or green background, respectively. The cellular compartment of each protein was obtained from the UniProtKB webpage. Proteins selected for further studies (TRIM21 and galectin-3) are indicated in bold type with blue background.
Note: the following are from NCBI Genbank and Genecards on TRIM21
This gene encodes a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The encoded protein is part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus. RoSSA interacts with autoantigens in patients with Sjogren syndrome and systemic lupus erythematosus. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. [provided by RefSeq, Jul 2008]
Expression
Ubiquitous expression in spleen (RPKM 15.5), appendix (RPKM 13.2) and 24 other tissues See more
This gene encodes a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The encoded protein is part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus. RoSSA interacts with autoantigens in patients with Sjogren syndrome and systemic lupus erythematosus. Alternatively spliced transcript variants for this gene have been described but the full-length nature of only one has been determined. [provided by RefSeq, Jul 2008]
E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2. Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination. Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes. A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B (‘Thr-187’ phosphorylated-form), thereby promoting its degradation by the proteasome. Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling. Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway. Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages. Plays a role in the regulation of the cell cycle progression. Enhances the decapping activity of DCP2. Exists as a ribonucleoprotein particle present in all mammalian cells studied and composed of a single polypeptide and one of four small RNA molecules. At least two isoforms are present in nucleated and red blood cells, and tissue specific differences in RO/SSA proteins have been identified. The common feature of these proteins is their ability to bind HY RNAs.2. Involved in the regulation of innate immunity and the inflammatory response in response to IFNG/IFN-gamma. Organizes autophagic machinery by serving as a platform for the assembly of ULK1, Beclin 1/BECN1 and ATG8 family members and recognizes specific autophagy targets, thus coordinating target recognition with assembly of the autophagic apparatus and initiation of autophagy. Acts as an autophagy receptor for the degradation of IRF3, hence attenuating type I interferon (IFN)-dependent immune responses (PubMed:26347139, 16297862, 16316627, 16472766, 16880511, 18022694, 18361920, 18641315, 18845142, 19675099). Represses the innate antiviral response by facilitating the formation of the NMI-IFI35 complex through ‘Lys-63’-linked ubiquitination of NMI (PubMed:26342464). ( RO52_HUMAN,P19474 )
Molecular function for TRIM21 Gene according to UniProtKB/Swiss-Prot
Function:
E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2. Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination. Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes. A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B (‘Thr-187’ phosphorylated-form), thereby promoting its degradation by the proteasome. Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling. Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway. Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages. Plays a role in the regulation of the cell cycle progression.
Other Articles in this Open Access Scientific Journal on Galectins and Proteosome Include
Accelerating PROTAC drug discovery: Establishing a relationship between ubiquitination and target protein degradation
Curator: Stephen J. Williams, Ph.D.
PROTACs have been explored in multiple disease fields with focus on only few ligases like cereblon (CRBN), Von Hippel-Lindau (VHL), IAP and MDM2. Cancer targets like androgen receptor, estrogen receptor, BTK, BCL2, CDK8 and c-MET [[6], [7], [8], [9], [10], [11]] have been successfully targeted using PROTACs. A variety of BET family (BRD2, BRD3, and BRD4)- PROTACs were designed using multiple ligases; MDM2-based BRD4 PROTAC [12], CRBN based dBET1 [13] and BETd-24-6 [14] for triple-negative breast cancer, enhanced membrane permeable dBET6 [15], and dBET57 PROTAC [16]. PROTACs for Hepatitis c virus (HCV) protease, IRAK4 and Tau [[17], [18], [19]] have been explored for viral, immune and neurodegenerative diseases, respectively. Currently, the PROTAC field expansion to vast undruggable proteome is hindered due to narrow focus on select E3 ligases. Lack of reliable tools to rapidly evaluate PROTACs based on new ligases is hindering the progress. Screening platforms designed must be physiologically relevant and represent true PROTAC cellular function, i.e., PROTAC-mediated target ubiquitination and degradation.
In the current study, we employ TUBEs as affinity capture reagents to monitor PROTAC-induced poly-ubiquitination and degradation as a measure of potency. We established and validated proof-of-concept cell-based assays in a 96-well format using PROTACS for three therapeutic targets BET family proteins, kinases, and KRAS. To our knowledge, the proposed PROTAC assays are first of its kind that can simultaneously 1) detect ubiquitination of endogenous, native protein targets, 2) evaluate the potency of PROTACs, and 3) establish a link between the UPS and protein degradation. Using these TUBE assays, we established rank order potencies between four BET family PROTACs dBET1, dBET6, BETd246 and dBET57 based on peak ubiquitination signals (“UbMax”) of the target protein. TUBE assay was successful in demonstrating promiscuous kinase PROTACs efficiency to degrade Aurora Kinase A at sub-nanomolar concentrations within 1 h. A comparative study to identify changes in the ubiquitination and degradation profile of KRAS G12C PROTACs recruiting two E3 ligases (CRBN and VHL). All of the ubiquitination and degradation profiles obtained from TUBE based assays correlate well with traditional low throughput immunoblotting. Significant correlation between DC50 obtained from protein degradation in western blotting and UbMax values demonstrates our proposed assays can aid in high-throughput screening and drastically eliminate artifacts to overcome bottlenecks in PROTAC drug discovery.
To successfully set up HTS screening with novel PROTACs without pre-existing knowledge, we recommend the following steps. 1. Identify a model PROTAC that can potentially demonstrate activity based on knowledge in PROTAC design or in vitro binding studies. 2. Perform a time course study with 2–3 doses of the model PROTAC based on affinities of the ligands selected. 3. Monitor ubiquitination and degradation profiles using plate-based assay and identify time point that demonstrates UbMax. 4. Perform a dose response at selected time point with a library of PROTACs to establish rank order potency.
INTRODUCTION
Ubiquitination is a major regulatory mechanism to maintain cellular protein homeostasis by marking proteins for proteasomal-mediated degradation [1]. Given ubiquitin’s role in a variety of pathologies, the idea of targeting the Ubiquitin Proteasome System (UPS) is at the forefront of drug discovery [2]. “Event-driven” protein degradation using the cell’s own UPS is a promising technology for addressing the “undruggable” proteome [3]. Targeted protein degradation (TPD) has emerged as a new paradigm and promising therapeutic option to selectively attack previously intractable drug targets using PROteolytic TArgeting Chimeras (PROTACs) [4]. PROTACs are heterobifunctional molecules with a distinct ligand that targets a specific E3 ligase which is tethered to another ligand specific for the target protein using an optimized chemical linker. A functional PROTAC induces a ternary E3-PROTAC-target complex, resulting in poly-ubiquitination and subsequent controlled protein degradation [5]. Ability to function at sub-stoichiometric levels for efficient degradation, a significant advantage over traditional small molecules.
PROTACs have been explored in multiple disease fields with focus on only few ligases like cereblon (CRBN), Von Hippel-Lindau (VHL), IAP and MDM2. Cancer targets like androgen receptor, estrogen receptor, BTK, BCL2, CDK8 and c-MET [[6], [7], [8], [9], [10], [11]] have been successfully targeted using PROTACs. A variety of BET family (BRD2, BRD3, and BRD4)- PROTACs were designed using multiple ligases; MDM2-based BRD4 PROTAC [12], CRBN based dBET1 [13] and BETd-24-6 [14] for triple-negative breast cancer, enhanced membrane permeable dBET6 [15], and dBET57 PROTAC [16]. PROTACs for Hepatitis c virus (HCV) protease, IRAK4 and Tau [[17], [18], [19]] have been explored for viral, immune and neurodegenerative diseases, respectively. Currently, the PROTAC field expansion to vast undruggable proteome is hindered due to narrow focus on select E3 ligases. Lack of reliable tools to rapidly evaluate PROTACs based on new ligases is hindering the progress. Screening platforms designed must be physiologically relevant and represent true PROTAC cellular function, i.e., PROTAC-mediated target ubiquitination and degradation.
Cellular PROTAC screening is traditionally performed using cell lines harboring reporter genes and/or Western blotting. While Western blotting is easy to perform, they are low throughput, semi-quantitative and lack sensitivity. While reporter gene assays address some of the issues, they are challenged by reporter tags having internal lysines leading to artifacts. Currently, no approaches are available that can identify true PROTAC effects such as target ubiquitination and proteasome-mediated degradation simultaneously. High affinity ubiquitin capture reagents like TUBEs [20] (tandem ubiquitin binding entities), are engineered ubiquitin binding domains (UBDs) that allow for detection of ultralow levels of polyubiquitinated proteins under native conditions with affinities as low as 1 nM. The versatility and selectivity of TUBEs makes them superior to antibodies, and they also offer chain-selectivity (-K48, -K63, or linear) [21]. High throughput assays that can report the efficacy of multiple PROTACs simultaneously by monitoring PROTAC mediated ubiquitination can help establish rank order potency and guide chemists in developing meaningful structure activity relationships (SAR) rapidly.
In the current study, we employ TUBEs as affinity capture reagents to monitor PROTAC-induced poly-ubiquitination and degradation as a measure of potency. We established and validated proof-of-concept cell-based assays in a 96-well format using PROTACS for three therapeutic targets BET family proteins, kinases, and KRAS. To our knowledge, the proposed PROTAC assays are first of its kind that can simultaneously 1) detect ubiquitination of endogenous, native protein targets, 2) evaluate the potency of PROTACs, and 3) establish a link between the UPS and protein degradation. Using these TUBE assays, we established rank order potencies between four BET family PROTACs dBET1, dBET6, BETd246 and dBET57 based on peak ubiquitination signals (“UbMax”) of the target protein. TUBE assay was successful in demonstrating promiscuous kinase PROTACs efficiency to degrade Aurora Kinase A at sub-nanomolar concentrations within 1 h. A comparative study to identify changes in the ubiquitination and degradation profile of KRAS G12C PROTACs recruiting two E3 ligases (CRBN and VHL). All of the ubiquitination and degradation profiles obtained from TUBE based assays correlate well with traditional low throughput immunoblotting. Significant correlation between DC50 obtained from protein degradation in western blotting and UbMax values demonstrates our proposed assays can aid in high-throughput screening and drastically eliminate artifacts to overcome bottlenecks in PROTAC drug discovery.
Fig. 1. Schematic representation of TUBE assay to monitor PROTAC mediated cellular ubiquitination of target proteins.Fig. 2. TUBE based assay screening of PROTACs: Jurkat cell lysates were treated with BRD3-specific PROTACs A) dBET1, B) dBET6, C) BETd24-6, and D) dBET57. Polyubiquitination profiles and Ubmax of BRD3 for each PROTAC were represented as relative CL intensity. Relative CL intensities were calculated by dividing raw CL signals from a given PROTAC dose over DMSO treated samples. Error bars represent standard deviations, n = 3.Fig. 3. PROTAC mediated degradation of bromodomain proteins analyzed by anti-BRD3 western blotting. Dose response of PROTACs dBET1, dBET6, Betd-24-6 and dBET57 at 45 min in Jurkat cells demonstrates degradation of BRD3, Acting as loading control.
Fig. 4. PROTAC mediated ubiquitination and degradation of AURKA in K562 cells. (A) Time course study to evaluate intracellular ubiquitination and degradation. (B) Western blot analysis of time course study: degradation kinetics (C) A dose response study to evaluate DC50 of the promiscuous kinase PROTAC in K562 cells. (D) Western blot analysis of dose response study to monitor degradation, GAPDH as loading control. Error bars represent standard deviation, n = 3.
2022 Albert Lasker Basic Medical Research Award for Integrins—Mediators of Cell-Matrix and Cell-Cell Adhesion
Reporter: Aviva Lev-Ari, PhD, RN
The three recipients of 2022 Albert Lasker Basic Medical Research Award For discoveries concerning the integrins—key mediators of cell-matrix and cell-cell adhesion in physiology and disease are:
Richard O. Hynes, Massachusetts Institute of Technology
Erkki Ruoslahti, Sanford Burnham Prebys
Timothy A. Springer, Boston Children’s Hospital/Harvard Medical School
Ruoslahti, E., Vaheri. A., Kuusela, P., and Linder, E. (1973). Fibroblast surface antigen: a new serum protein. Biochim. Biophys. Acta. 322, 352-358.
Hynes, R.O. (1973). Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc. Natl. Acad. Sci. USA. 70, 3170-3174.
Pierschbacher, M.D., and Ruoslahti, E. (1984). Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 309, 30-33.
Pytela, R., Pierschbacher, M.D., and Ruoslahti, E. (1985). A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin. Proc. Natl. Acad. Sci. USA. 82, 5766-5770.
Pytela, R., Pierschbacher, M.D., Ginsberg, M.H., Plow, E.F., and Ruoslahti, E. (1986). Platelet membrane glycoprotein IIb/IIIa: member of a family of Arg-Gly-Asp-specific adhesion receptors. Science. 231, 1559-1562.
Tamkun, J.W., DeSimone, D.W., Fonda, D., Patel, R.S., Buck, C., Horwitz, A.F., and Hynes, R.O. (1986). Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. 46, 271-282.
Hynes, R.O. (1987). Integrins: a family of cell surface receptors. Cell. 48, 549-554.
Ruoslahti, E. (1996). RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 12, 697-715.
Kurzinger, K., Reynolds, T., Germain, R.N., Davignon, D., Martz, E., and Springer, T.A. (1981). A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure. J. Immunol. 127, 596-600.
Sanchez-Madrid, F., Nagy, J., Robbins, E., Simon, P., and Springer, T.A. (1983). A human leukocyte differentiation antigen family with distinct alpha subunits and a common beta subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J. Exp. Med. 158, 1785-1789.
Thompson, W.S., Miller, L.J., Schmalstieg, F.C., Anderson, D.C., and Springer, T.A. (1984). Inherited deficiency of the Mac-1, LFA-1, p150,95 glycoprotein family and its molecular basis. J. Exp. Med. 160, 1901-1905.
Kishimoto, T.K., Lee, A., Roberts, T.M., and Springer, T.A. (1987). Cloning of the beta subunit of the leukocyte adhesion proteins: homology to an extra-cellular matrix receptor defines a novel supergene family. Cell. 48, 681-690.
Makgoba. M.W., Sanders, M.E., Luce, G.E.G., Dustin, M.L., Springer, T.A., Clark, E.A., Mannoni, P., and Shaw, S. (1988). ICAM-1 a ligand for LFA-1-dependent adhesion of B, T and myeloid cells. Nature. 331, 86-88.
Staunton, D.E., Dustin, M.L., and Springer, T.A. (1989). Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. Nature. 339, 61-65.
Springer, T.A. (1990). Adhesion receptors of the immune system. Nature. 346, 425-434.
Lawrence, M.B., and Springer, T.A. (1991). Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell. 65, 859-873.
Luo, B.-H., Carman, C.V., and Springer, T.A. (2007) Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25, 619-647.
Li, J., Yan, J., and Springer, T.A. (2021). Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state. eLife. 10, 1-22. e73359. doi: 10.7554/eLife.73359.
Infertility has been primarily treated as a female predicament but around one-half of infertility cases can be tracked to male factors. Clinically, male infertility is typically determined using measures of semen quality recommended by World Health Organization (WHO). A major limitation, however, is that standard semen analyses are relatively poor predictors of reproductive capacity and success. Despite major advances in understanding the molecular and cellular functions in sperm over the last several decades, semen analyses remain the primary method to assess male fecundity and fertility.
Chronological age is a significant determinant of human fecundity and fertility. The disease burden of infertility is likely to continue to rise as parental age at the time of conception has been steadily increasing. While the emphasis has been on the effects of advanced maternal age on adverse reproductive and offspring health, new evidence suggests that, irrespective of maternal age, higher male age contributes to longer time-to-conception, poor pregnancy outcomes and adverse health of the offspring in later life. The effect of chronological age on the genomic landscape of DNA methylation is profound and likely occurs through the accumulation of maintenance errors of DNA methylation over the lifespan, which have been originally described as epigenetic drift.
In recent years, the strong relation between age and DNA methylation profiles has enabled the development of statistical models to estimate biological age in most somatic tissue via different epigenetic ‘clock’ metrics, such as DNA methylation age and epigenetic age acceleration, which describe the degree to which predicted biological age deviates from chronological age. In turn, these epigenetic clock metrics have emerged as novel biomarkers of a host of phenotypes such as allergy and asthma in children, early menopause, increased incidence of cancer types and cardiovascular-related diseases, frailty and cognitive decline in adults. They also display good predictive ability for cancer, cardiovascular and all-cause mortality.
Epigenetic clock metrics are powerful tools to better understand the aging process in somatic tissue as well as their associations with adverse disease outcomes and mortality. Only a few studies have constructed epigenetic clocks specific to male germ cells and only one study reported that smokers trended toward an increased epigenetic age compared to non-smokers. These results indicate that sperm epigenetic clocks hold promise as a novel biomarker for reproductive health and/or environmental exposures. However, the relation between sperm epigenetic clocks and reproductive outcomes has not been examined.
There is a critical need for new measures of male fecundity for assessing overall reproductive success among couples in the general population. Data shows that sperm epigenetic clocks may fulfill this need as a novel biomarker that predicts pregnancy success among couples not seeking fertility treatment. Such a summary measure of sperm biological age is of clinical importance as it allows couples in the general population to realize their probability of achieving pregnancy during natural intercourse, thereby informing and expediting potential infertility treatment decisions. With the ability to customize high throughput DNA methylation arrays and capture sequencing approaches, the integration of the epigenetic clocks as part of standard clinical care can enhance our understanding of idiopathic infertility and the paternal contribution to reproductive success and offspring health.
Read key takeaways from the 2022 World Medical Innovation Forum in this report from the Bank of America Institute. #WMIF2022
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Bank of America News
@BofA_News
· May 6
What are the 12 emerging #GeneAndCellTherapy technologies with the greatest potential to transform #healthcare? Read our report for key takeaways from #WMIF2022. @MassGenBrigham
Read key takeaways from the 2022 World Medical Innovation Forum in this report from the Bank of America Institute. #WMIF2022
Quote Tweet
Bank of America News
@BofA_News
· May 6
What are the 12 emerging #GeneAndCellTherapy technologies with the greatest potential to transform #healthcare? Read our report for key takeaways from #WMIF2022. @MassGenBrigham
The Disruptive Dozen 12 #GCT Breakthroughs that are revolutionizing Healthcare
Liz Everett Krisberg, Head of the Bank of America Institute
The Disruptive Dozen 12 GCT breakthroughs that are revolutionizing healthcare 05 May 2022 Key Takeaways • Gene and cell therapy (GCT) is widely recognized as a transformational opportunity in medicine, with the potential to stop or slow the effects of disease by targeting it at the genetic level. • The “Disruptive Dozen” identifies 12 emerging GCT technologies with the greatest potential to transform healthcare over the next several years • These breakthroughs range from restoration of sight and increasing the supply of donor organs, to treating brain cancer, hearing loss and autoimmune diseases that currently lack few or any treatment alternatives. Gene and cell therapy (GCT) technologies are transforming medicine and the approach to severe diseases like cancer, hereditary conditions including Huntington Disease and Sickle Cell, as well as rare disorders that currently have no treatment alternatives. GCT has the potential to stop or slow the effects of disease by targeting it at the genetic level, either replacing, inactivating or modifying the genetic material or by transferring live or intact cells into a patient to treat or cure disease. Even in cases where the GCT approach does not fully cure a condition, GCT has the potential to be life changing. This is because GCT treatments are often “one and done,” only requiring a single administration, which may enable a patient to manage their disease without onerous ongoing treatment cycles. While some of the first GCT applications were focused on rare and orphan diseases, recent advancements show tremendous potential opportunity for use cases with more broad applications. Beyond the messenger ribonucleic acid or mRNA vaccines that protect against infectious disease including COVID-19, GCT technologies exhibit promise to address prevalent chronic diseases such as diabetes and hearing loss, as well as central nervous system (CNS) disorders and Alzheimer’s. This week, Bank of America joined Mass General Brigham to present the World Medical Innovation Forum in Boston, where over 1,000 clinical experts, industry leaders and investors explored how to advance GCT technologies that may lead to breakthrough medical advancements and solutions. We highlight the twelve emerging GCT technologies – the “Disruptive Dozen” – with the greatest potential to impact and transform healthcare in the next several years. These breakthroughs range from restoration of sight and increasing the supply of donor organs, to treating brain cancer, hearing loss and autoimmune diseases. Restoring sight by mending broken genes Roughly 200 genes are directly linked to vision disorders. In the last several years, groundbreaking new gene therapies have emerged that can compensate for faulty genes in the eye by adding new, healthy copies — a molecular fix that promises to restore sight to those who have lost it. The approach, known as CRISPR-Cas-9 gene editing, could open the door to treating genetic forms of vision loss that are not suited to conventional gene therapy, and a host of other medical conditions. A clinical trial is now underway to evaluate a CRISPR-Cas 9 gene-editing therapy for a severe form of childhood blindness for which there currently are no treatments. Although this treatment is still experimental, it is already historic — it is the first medicine based on CRISPR-Cas-9 to be delivered in vivo, or inside a patient’s body. Similar gene-editing therapies are also under development that correct genes within blood cells. A gene editing solution to increase the supply of donor organs In the U.S. alone, more than 100,000 people need a life-saving organ transplant. But the supply of donor organs is quite limited, and every day, patients die waiting for a donor organ. One way to address this crisis is xenotransplantation — harvesting organs from animals and placing them into human patients. Advances in gene editing technology make it possible to remove, insert, or replace genes with relative ease and precision. This molecular engineering can sidestep the human immune system, which is highly adept at recognizing foreign tissues and triggering rejection. Over the last 20 years, scientists have been working to devise successful gene editing strategies that will render pig organs compatible with humans. The field has taken another major step forward in the past year: transplanting gene-edited pig organs, including the heart and kidney, into humans. While extensive clinical testing is needed before xenotransplantation becomes a reality, that future now seems within reach. I NSTI TUTE Accessible version 2 05 May 2022 I NSTI TUTE Cell therapies to conquer common forms of blindness The eye has been a proving ground for pioneering gene therapies and is also fueling new cell-based therapies than can restore sight, offering a functional cure by replacing critical cells that have been lost or injured. One approach involves stem cells from the retina that can give rise to light-sensitive cells, called photoreceptors, which are required for healthy vision. Scientists are harnessing retinal stem cells to develop treatments for incurable eye diseases, including retinitis pigmentosa. Because the immune system doesn’t patrol the eye as aggressively as other parts of the body, retinal stem cells from unrelated, healthy donors can be transplanted into patients with vision disorders. Other progress includes cell therapies that harness patients’ own cells, for example, from blood or skin, that can be converted into almost any cell type in the body, including retinal cells. Another novel treatment being tested utilizes stem cells from a patient’s healthy eye to repair the affected cornea of the other eye. Harnessing the power of RNA to treat brain cancer RNA is widely known for its helper functions, carrying messages from one part of a cell to another to make proteins. But scientists now recognize that RNA plays a more central role in biology and are tapping its hidden potential to create potent new therapies for a range of diseases, including a devastating form of brain cancer called glioblastoma. This cancer is extremely challenging to treat and highly adaptable. New approaches that either target RNA or mimic its activity could hold promise, including an intriguing class of RNA molecules called microRNAs. One team identified a trio of microRNAs that plays important roles in healthy neurons but is lost when brain cancer develops. These microRNAs can be stitched together into a single unit and delivered into the brain using a virus. Initial studies in mice reveal that this therapeutic can render tumors more vulnerable to existing treatments, including chemotherapy. Another team is also exploring a microRNA called miR-10b. Blocking its activity causes tumor cells to die. Now, scientists are working to develop a targeted therapeutic against miR-10b that can be tested in clinical trials. Realizing the promise of gene therapy for brain disorders Gene therapy holds enormous promise for serious and currently untreatable diseases, including those of the brain and central nervous system. But some big obstacles remain. For example, a commonly-used vehicle for gene therapy — a virus called AAV — cannot penetrate a major biological roadblock, the blood-brain barrier. Now, researchers are engineering new versions of AAV that can cross the blood-brain barrier. Using various molecular strategies, a handful of teams have modified the protein shell that surrounds the virus so it can gain entry and become broadly distributed within the brain. These modified viral vectors are now under development and could begin clinical testing within a few years. Scientists are also tinkering with the inner machinery of AAV to sidestep potential toxicities. With a safe, effective method for accessing the brain, researchers will be able to devise gene therapies for a range of neurological conditions, including neurodegenerative diseases, cancers, and devastating rare diseases that lack any treatment. A flexible, programmable approach to fighting viruses The COVID-19 pandemic has laid bare the tremendous need for rapidly deployable therapies to counteract emerging viruses. Scientists are now developing a novel form of anti-viral therapy that can be programmed to target a range of different viruses — from well-known human pathogens, such as hepatitis C, to those less familiar, such as the novel coronavirus SARS-CoV-2. This new approach harnesses a popular family of gene editing tools, known as CRISPR-Cas. While CRISPR-based systems have gained attention for their capacity to modify human genes, their original purpose in nature was to defend bacteria from viral infections. As a throwback to these early roots, scientists are now adapting CRISPR tools to tackle a variety of viruses that infect humans. Researchers are studying the potential of these programmable anti-viral agents in the context of several different viruses, including ones that pose significant threats to global health, such as SARS-CoV-2, hepatitis C, and HIV. On the move: Cell therapies to restore gut motility The human digestive tract — or “gut” — has its own nervous system. This second brain, known as the enteric nervous system, is comprised of neurons and support cells that carry out critical tasks, like moving food through the gut. When enteric neurons are missing or injured, gut motility can be impaired. Now, scientists are developing an innovative cell replacement therapy to treat diseases of gut motility. Donor cells can be isolated from a patient’s own gut or from a more readily available source, such as subcutaneous fat. These cells are then cultivated in the laboratory and coaxed to form the progenitors that give rise to enteric neurons. Researchers are also devising “off-the-shelf” approaches, which could create a supply of donor cells that are shielded from the immune system and can therefore be transplanted universally across different patients. Early research shows that transplanted enteric neurons can also take up residence in the brain. That means these forays in cell therapy for the gut could also help pave a path toward cell therapies for the brain and spinal cord. CAR-T cell therapies take aim at autoimmune diseases CAR-T cells have emerged as powerful treatments for some forms of cancer, especially blood cancers. By harnessing the same underlying concept — rewiring patients’ own T cells to endow them with therapeutic properties — scientists are working to develop novel CAR-T therapies for a variety of autoimmune diseases. Several research teams are engineering CAR-T cells so they can seek out and destroy harmful immune cells, such as those that produce auto-antibodies — immune proteins that help coordinate the attack on the body’s own tissues. For example, one team is using CAR-T cells to destroy certain immune cells, called B cells, as a potential treatment for lupus, a serious autoimmune disease that mainly affects women. Scientists are also 05 May 2022 3 I NSTI TUTE developing CAR-T therapies that take aim at other rogue members of the immune system. These efforts could yield novel treatments for multiple sclerosis and type 1 diabetes. Regrowing cells in the inner ear to treat hearing loss In the U.S. alone, some 37 million people suffer from a hearing deficit. Currently, there are no drugs that can halt, prevent, or even reverse hearing loss. Scientists are working on a novel regenerative approach that could restore the cells in the inner ear required for normal hearing, offering hope to millions of patients who grapple with hearing loss. Healthy hearing requires specialized cells in the inner ear called hair cells, which have fine, hair-like projections. If the cells are damaged or lost, which often happens with age or after repeated exposure to loud sounds, the body cannot repair them. But researchers have discovered a potential workaround that can stimulate existing cells in the ear to proliferate and give rise to new hair cells. Scientists are now working to convert this molecular strategy, which is being studied in animal models, into a therapeutic that is safe and effective for hearing loss patients. New technologies for delivering gene therapies A formidable challenge in the field of gene therapy is delivery — getting gene-based therapeutics into the body and into the right target cells. Researchers are exploring the potential of new delivery methods that could expand the reach of gene therapy, including microneedles. When applied to the skin, a microneedle patch can penetrate the outermost layer with minimal pain and discomfort. This novel delivery method can readily access the legion of immune cells that reside in the skin — important targets for vaccines as well as for the treatment of various diseases, including cancer and autoimmune conditions. Another emerging technology involves an implantable device made of biodegradable materials. When placed inside the body, this device can provide localized, sustained release of therapeutics with few side effects. The approach is now being tested for the first time in cancer patients using standard chemotherapy drugs administered directly at tumor sites. In the future, this method could be customized for the delivery of gene therapy payloads, an advance that could revolutionize cancer treatment, particularly for difficult-to-treat forms like pancreatic cancer. Engineering cancer-killing cells that target solid tumors CAR-T cells are a revolutionary form of cell therapy that has yielded some remarkable cures of difficult-to-treat blood cancers. But the outcomes in other cancers have been lackluster. Now, scientists are enhancing this technology to enable new ways of treating solid tumors. One approach involves making CAR-T cells more like computers, relying on simple logic to decide which cells are cancer — and should be destroyed — and which cells are healthy and should be spared. By building several logic gates and combining them together, researchers are hoping to pave the way toward targeting new tumor types. Scientists are also devising other groundbreaking forms of cancer-killing cell therapy, including one that uses cancer cells themselves. This approach exploits a remarkable feature: once disseminated within the body, cancer cells can migrate back to the original tumor. Researchers are now harnessing this rehoming capability and, with the help of gene editing, turning tumor cells into potent cancer killers. An early version of this technology uses patients’ own cells. Now, the scientists are developing an off-the-shelf version that can be universally applied to patients. Reawakening the X-chromosome: a therapeutic strategy for devastating neurodevelopmental diseases The X chromosome is one of two sex-determining chromosomes in humans, and it carries hundreds of disease-causing genes. These diseases often affect males and females differently. In females, one X chromosome is naturally, and randomly, chosen and rendered inactive. Although X-inactivation was once thought to be permanent, scientists are uncovering ways to reverse it. Scientists are now exploiting this unusual biology to reawaken the dormant X chromosome — a strategy that could yield muchneeded treatments for a group of rare, yet devastating neurodevelopmental disorders, which predominantly affect females. This new approach could hold promise for females with Rett syndrome, a severe X-linked disorder. A similar strategy could also hold promise for other serious X-linked disorders, including fragile X syndrome and CDKL5 syndrome.
Other related articles published in this Open Access Online Scientific Journal include the following:
UPDATED on 5/7/2022
Tweets at #WMIF2022 by @pharma_BI & @AVIVA1950 and All Retweets of these Tweets – 2022 World Medical Innovation Forum, GENE & CELL THERAPY • MAY 2–4, 2022 • BOSTON
2022 World Medical Innovation Forum, GENE & CELL THERAPY • MAY 2–4, 2022 • BOSTON • IN-PERSON
Reporter: Aviva Lev-Ari, PhD, RN
World Medical Innovation Forum as we bring together global leaders to assess the latest opportunities and challenges, from the investment landscape to key technology developments to manufacturing and regulatory barriers. Gain first-hand insights on medicine’s ultimate game changer.
World Medical Innovation Forum will be held June 12 – 14 in Boston, MA. We hope you’ll join us for #WMIF2023!
From: “Rieck, Lucy (BOS-WSW)” <LRieck@webershandwick.com> Date: Tuesday, April 12, 2022 at 10:25 AM To: Aviva Lev-Ari <avivalev-ari@alum.berkeley.edu> Subject: You’re Invited: Mass General Brigham’s World Medical Innovation Forum
Hi Aviva,
I’m reaching out to extend free registration for you or a colleague to the 8th annual World Medical Innovation Forum (WMIF), taking place May 2-4 at the Westin Copley Place in Boston. This year’s event, co-sponsored with Bank of America, will explore gene and cell therapies (GCT), including the latest opportunities and challenges – from the investment landscape to key technology developments to manufacturing and regulatory barriers.
The event will feature 200 speakers – including CEOs of leading companies in the GCT and biotech fields, investors, entrepreneurs, Harvard clinicians and scientists, government officials and other key influencers – who discover, invest in, and cultivate GCT breakthroughs. Notable speakers include:
Peter Marks: Director, Center for Biologics Evaluation and Research at the FDA
Brian Moynihan: CEO, Bank of America
Anne Klibansky: President & CEO, Mass General Brigham
Senior executives from biopharma and academic institutions of all sizes (including Novartis, BMS, Takeda, Verve, UPenn)
You can view the full list of speakers here and the program agenda here.
WMIF is hosted by the Mass General Brigham health system, which comprises 14 hospitals, including two world-renowned medical centers: Mass General and Brigham & Women’s. Since 2015, the Forum has brought together global leaders to assess medical breakthroughs, the investment landscape and technology developments that have the potential to transform the industry.
In addition to a packed agenda, the 2022 “Disruptive Dozen” – 12 breakthrough technologies most likely to have significant impact on gene and cell therapy in the next 18 months – will also be announced.
Please let me know if you would be interested in attending.
Understanding long-term Gene and Cell Therapy investment complexities requires a keen awareness of where the science and the markets are headed. That’s why “The Doctor is In” in these updates on the latest GCT technologies. Presented by Mass General Brigham clinicians and innovators from the front lines of care, the sessions are co-hosted by expert analysts from Bank of America and include interactive discussion and Q&A.
In this session, Dr. Eichler will discuss the impact of gene defects across the lifespan and how timing and delivery of new genetic therapies is transforming the field of neurogenetics.
In this session, hear from experts in their field as they discuss the need and importance of regenerative medicine for the advancement in the treatment of diseases such as diabetes, kidney disease and blood disorders.
In this session, Dr. Artzi will share how an integrative approach of combining materials science, chemistry, imaging, and biology enables targeted delivery of gene therapy.
In this session, Drs. Robert Green and Adam Shaywitz will discuss how the early detection and prevention of rare diseases is imminent and represents an enormous public health opportunity.
In this session, Dr. Poznansky will share how research and development of vaccines and immunotherapy are safely accelerated from research lab to the patient leveraging novel mechanisms.
This panel features industry leaders who will discuss what the future may hold for gene and cell therapy. Which applications are likely to have the greatest impact? What are the key hurdles to be overcome? What specific platforms and technologies may enable optimal solutions? In what disease areas? Learn more about these and other questions as the panelists discuss the future potential of GCT.
Manufacturing quality and cost are critical for enabling rapid growth in GCT. Panelists will explore a variety of critical questions in this space. For example, are there historic parallels that can be drawn between GCT manufacturing and other groundbreaking technologies? How do key manufacturing concerns in GCT differ from those for more conventional pharmaceutical? What are the long-term opportunities for non-viral vectors? Will manufacturing capacity be a limiting factor in GCT growth over the next 5 to 10 years?
At the end of 2021, roughly 410 novel drugs had been approved in the past decade. On average, there were 40 approvals per year with over 150 of them being between 2018 and 2020. What has changed in the approval process and what is the vision of the future state? What will happen over the next 1–3 years? What does the new iteration of the Prescription Drug User Fees Act (PDUFA) need to do in this area and which fields show the greatest potential for innovation in CGT?
This panel will delve into clinical trials for GCT. How do these trials differ from those for conventional therapeutics? What are the key lessons learned from completed GCT trials? How is the regulatory landscape shifting and what will that mean for the future of GCT?
Dr. Bourla will share what Pfizer has learned from its leadership on mRNA and the development of the Covid vaccine that can be extrapolated to other R&D.
As we enter the third year of the coronavirus pandemic, the world is shifting to a new strategy: living with and managing COVID as a part of our everyday lives. What will the coming year look like? How will mitigation measures differ in this new phase? What about treatment strategies? Should we be bracing for another surge?
This panel will feature a discussion of global biotech clusters with a deep dive into the New England/Boston area. How does the capital availability, scale, and density of New England drive local growth in GCT? Also, the influx of large biopharmaceutical companies into the region has fueled global outcomes. What is the future impact of these investments and when will they peak? How will the biopharmaceutical landscape in New England appear in 2030?
The role of patients and their experiences are critical as the promise of GCT unfolds. This panel will discuss the patient experience and explore the challenges different patient populations face, both in rare diseases and more common conditions. Panelists will also discuss financial considerations, clinical trial access, and the role of advocacy groups in GCT.
As many countries begin to turn the corner on COVID-19, they face a resurgence of chronic illnesses, such as cancer and cardiovascular disease, that were not adequately addressed during the pandemic, and for which new treatments are urgently needed. Population aging – and the resulting increase in chronic diseases associated with aging – has compounded the challenge. There’s never been a greater need for biopharmaceutical innovation – or, fortunately, a greater ability to innovate. Amgen is investing in new discovery research capabilities that portend a revolution in drug design and development.
Understanding long-term Gene and Cell Therapy investment complexities requires a keen awareness of where the science and the markets are headed. That’s why “The Doctor is In” in these updates on the latest GCT technologies. Presented by Mass General Brigham clinicians and innovators from the front lines of care, the sessions are co-hosted by expert analysts from Bank of America and include interactive discussion and Q&A.
In this session, Dr. Vavvas will discuss examples of clinical trials in rare diseases and share insights into how clinical trials should be approached for rare and ultra-rare diseases and how study design is not a one-size fits all.
In this session, hear experts weigh in on the possibilities of cell therapy development and transplantation for the treatment of Parkinson’s Disease. What does the futures hold and how do we get there?
In this session, Dr. Nikiforow will provide insights into the world of gene therapy manufacturing and the complexities of scaling, costs and insurance reimbursement.
In this session, Dr. Marks will discuss the ins and outs of regulatory challenges for biological products and therapies in gene and cell therapy and the responsibility to assure safety and effectiveness.
Dark genome, accounting for ~98.5% of the human genome and containing the non-coding part, offers unprecedented opportunity to look for novel elements that could play a role in human health. This non-coding region consists of repeat elements, enhancers, regulatory sequences and non-coding RNAs. This session will explore this exciting new frontier in biology and how to translate this so called “junk” and previously ignored genome into potential novel therapeutics.
Panelists will discuss the life sciences capital markets environment with particular emphasis on private and public fundraising for GCT companies. What trends do panelists observe that will impact the availability and cost of capital for GCT? Are there novel fundraising structures that will serve GCT in the future?
As one of the foremost researchers of CAR-T cancer treatments, Dr. June will share what he believes is the next wave of cell-and-gene based oncology research and how his work set the stage for breakthrough developments in cancer.
Richard W. Vague Professor in Immunotherapy, Director, Center for Cellular Immunotherapies, Director, Parker Institute for Cancer Immunotherapy, University of Pennsylvania Perelman School of Medicine
This panel will examine the role of academia in driving the promise of GCT. How does academic innovation contribute to the success of GCT? What are the risks and opportunities? Which models have proven most successful and what is the impact on clinical translation? How can these partnerships be accelerated?
Richard W. Vague Professor in Immunotherapy, Director, Center for Cellular Immunotherapies, Director, Parker Institute for Cancer Immunotherapy, University of Pennsylvania Perelman School of Medicine
This panel will bring together gene and cell therapy leaders from across the world to discuss the latest opportunities and challenges in the field, from the investment landscape to key technology developments to manufacturing and regulatory barriers. These global experts will offer first-hand insights on the systemic complexity of this advancing field and its therapeutic promise.
Chronic inflammation in the brain is now recognized as a contributor to many neurodegenerative diseases, ranging from Parkinson’s disease to multiple sclerosis to Alzheimer’s disease. Are solutions to these historically intractable neurological diseases imminent or several years away? Are market-making platforms identifiable for neurological diseases? Are there novel genetic targets that can be explored? What are the prospects for cell therapies?
Cell therapies, ranging from CAR-T cells to stem-cell-based approaches, are emerging as a transformative therapeutic modality. Panelists will examine this emerging landscape and discuss a range of key topics. What drives differentiation in this space given the high number of competing technologies? How will the uptake of autologous cell therapies and allogeneic versions evolve? When will the regenerative medicine market mature?
This panel will explore how GCT technology could lead to disruptions in other areas of medicine, including surgery and medical devices, over the next several years. Could cell replacement therapy in diabetes advance enough to reduce the need for diabetes pumps or insulin? Will stem-cell-based methods for regenerating cartilage advance rapidly enough to disrupt the number of patients seeking hip and knee replacements? How is GCT driving innovations in surgical techniques?
What is the new generation of approaches to gene therapy manufacturing and delivery? What are the lessons learned from Covid and how can it be applied to custom disease response and the ability to custom design biologic organisms?
This panel will feature an in-depth discussion of the safety of gene and cell therapies. What are the unique safety concerns in this field, both acute and potential long-term risks? Which of these concerns are supported by clinical data versus the presumption of theoretical risk? What are the key issues for AAV-based gene therapies? Will redosing become feasible? What are the predominant safety concerns for in vivo versus ex vivo GCT modalities, including base editing?
The label “RNA” encompasses a wide array of biologically active agents spanning therapeutic modalities, vaccines, non-coding controls, and other forms. In this panel we will discuss a number of these forms, discuss examples of recent developments and illustrate why RNA developments represent a promising source of novel therapies and therapeutic approaches.
The Disruptive Dozen identifies and ranks the GCT technologies that Mass General Brigham faculty feel will break through over the next one to five years to significantly improve health care.
Recent genetic studies have identified variants associated with bipolar disorder (BD), but it remains unclear how brain gene expression is altered in BD and how genetic risk for BD may contribute to these alterations. Here, we obtained transcriptomes from subgenual anterior cingulate cortex and amygdala samples from post-mortem brains of individuals with BD and neurotypical controls, including 511 total samples from 295 unique donors. We examined differential gene expression between cases and controls and the transcriptional effects of BD-associated genetic variants. We found two coexpressed modules that were associated with transcriptional changes in BD: one enriched for immune and inflammatory genes and the other with genes related to the postsynaptic membrane. Over 50% of BD genome-wide significant loci contained significant expression quantitative trait loci (QTL) (eQTL), and these data converged on several individual genes, including SCN2A and GRIN2A. Thus, these data implicate specific genes and pathways that may contribute to the pathology of BP.
Gene Expression Markers for Bipolar Disorder Pinpointed
The work was led by researchers at Johns Hopkins’ Lieber Institute for Brain Development. The findings, published this week in Nature Neuroscience, represent the first time that researchers have been able to apply large-scale genetic research to brain samples from hundreds of patients with bipolar disorder (BD). They used 511 total samples from 295 unique donors.
“This is the first deep dive into the molecular biology of the brain in people who died with bipolar disorder—studying actual genes, not urine, blood or skin samples,” said Thomas Hyde of the Lieber Institute and a lead author of the paper. “If we can figure out the mechanisms behind BD, if we can figure out what’s wrong in the brain, then we can begin to develop new targeted treatments of what has long been a mysterious condition.”
Bipolar disorder is characterized by extreme mood swings, with episodes of mania alternating with episodes of depression. It usually emerges in people in their 20s and 30s and remains with them for life. This condition affects approximately 2.8% of the adult American population, or about 7 million people. Patients face higher rates of suicide, poorer quality of life, and lower productivity than the general population. Some estimates put the annual cost of the condition in the U.S. alone at $219.1 billion.
While drugs can be useful in treating BD, many patients find they have bothersome side effects, and for some patients, current medications don’t work at all.
In this study, researchers measured levels of messenger RNA in the brain samples. They observed almost eight times more differentially expressed gene features in the sACC versus the amygdala, suggesting that the sACC may play an especially prominent role—both in mood regulation in general and BD specifically.
In patients who died with BD, the researchers found abnormalities in two families of genes: one containing genes related to the synapse and the second related to immune and inflammatory function.
“There finally is a study using modern technology and our current understanding of genetics to uncover how the brain is doing,” Hyde said. “We know that BD tends to run in families, and there is strong evidence that there are inherited genetic abnormalities that put an individual at risk for bipolar disorder. Unlike diseases such as sickle-cell anemia, bipolar disorder does not result from a single genetic abnormality. Rather, most patients have inherited a group of variants spread across a number of genes.”
“Bipolar disorder, also known as manic-depressive disorder, is a highly damaging and paradoxical condition,” said Daniel R. Weinberger, chief executive and director of the Lieber Institute and a co-author of the study. “It can make people very productive so they can lead countries and companies, but it can also hurl them into the meat grinder of dysfunction and depression. Patients with BD may live on two hours of sleep a night, saving the world with their abundance of energy, and then become so self-destructive that they spend their family’s fortune in a week and lose all friends as they spiral downward. Bipolar disorder also has some shared genetic links to other psychiatric disorders, such as schizophrenia, and is implicated in overuse of drugs and alcohol.”
New studies link cell cycle proteins to immunosurveillance of premalignant cells
Curator: Stephen J. Williams, Ph.D.
The following is from a Perspectives article in the journal Science by Virinder Reen and Jesus Gil called “Clearing Stressed Cells: Cell cycle arrest produces a p21-dependent secretome that initaites immunosurveillance of premalignant cells”. This is a synopsis of the Sturmlechener et al. research article in the same issue (2).
Complex organisms repair stress-induced damage to limit the replication of faulty cells that could drive cancer. When repair is not possible, tissue homeostasis is maintained by the activation of stress response programs such as apoptosis, which eliminates the cells, or senescence, which arrests them (1). Cellular senescence causes the arrest of damaged cells through the induction of cyclin-dependent kinase inhibitors (CDKIs) such as p16 and p21 (2). Senescent cells also produce a bioactive secretome (the senescence-associated secretory phenotype, SASP) that places cells under immunosurveillance, which is key to avoiding the detrimental inflammatory effects caused by lingering senescent cells on surrounding tissues. On page 577 of this issue, Sturmlechner et al. (3) report that induction of p21 not only contributes to the arrest of senescent cells, but is also an early signal that primes stressed cells for immunosurveillance.Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).
Senescence is a complex program that is tightly regulated at the epigenetic and transcriptional levels. For example, exit from the cell cycle is controlled by the induction of p16 and p21, which inhibit phosphorylation of the retinoblastoma protein (RB), a transcriptional regulator and tumor suppressor. Hypophosphorylated RB represses transcription of E2F target genes, which are necessary for cell cycle progression. Conversely, production of the SASP is regulated by a complex program that involves super-enhancer (SE) remodeling and activation of transcriptional regulators such as nuclear factor κB (NF-κB) or CCAAT enhancer binding protein–β (C/EBPβ) (4).
Sturmlechner et al. found that activation of p21 following stress rapidly halted cell cycle progression and triggered an internal biological timer (of ∼4 days in hepatocytes), allowing time to repair and resolve damage (see the figure). In parallel, C-X-C motif chemokine 14 (CXCL14), a component of the PASP, attracted macrophages to surround and closely surveil these damaged cells. Stressed cells that recovered and normalized p21 expression suspended PASP production and circumvented immunosurveillance. However, if the p21-induced stress was unmanageable, the repair timer expired, and the immune cells transitioned from surveillance to clearance mode. Adjacent macrophages mounted a cytotoxic T lymphocyte response that destroyed damaged cells. Notably, the overexpression of p21 alone was sufficient to orchestrate immune killing of stressed cells, without the need of a senescence phenotype. Overexpression of other CDKIs, such as p16 and p27, did not trigger immunosurveillance, likely because they do not induce CXCL14 expression.In the context of cancer, senescent cell clearance was first observed following reactivation of the tumor suppressor p53 in liver cancer cells. Restoring p53 signaling induced senescence and triggered the elimination of senescent cells by the innate immune system, prompting tumor regression (5). Subsequent work has revealed that the SASP alerts the immune system to target preneoplastic senescent cells. Hepatocytes expressing the oncogenic mutant NRASG12V (Gly12→Val) become senescent and secrete chemokines and cytokines that trigger CD4+ T cell–mediated clearance (6). Despite the relevance for tumor suppression, relatively little is known about how immunosurveillance of oncogene-induced senescent cells is initiated and controlled.
Source of image: Reen, V. and Gil, J. Clearing Stressed Cells. Science Perspectives 2021;Vol 374(6567) p 534-535.
References
2. Sturmlechner I, Zhang C, Sine CC, van Deursen EJ, Jeganathan KB, Hamada N, Grasic J, Friedman D, Stutchman JT, Can I, Hamada M, Lim DY, Lee JH, Ordog T, Laberge RM, Shapiro V, Baker DJ, Li H, van Deursen JM. p21 produces a bioactive secretome that places stressed cells under immunosurveillance. Science. 2021 Oct 29;374(6567):eabb3420. doi: 10.1126/science.abb3420. Epub 2021 Oct 29. PMID: 34709885.
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