银牌赞助

Targeting DNA repair pathways in cancer using oligonucleotides

Genome stability pathways offer a promising area of therapeutic intervention in cancer. Specific inhibitors of key DNA repair proteins have shown promising results as combination therapies and more recently also as single agents. We use nucleic acid-based technology to target DNA repair proteins in cancer and explore this technology as a potential cancer therapeutic. We show that specific oligonucleotides inhibit DNA repair, induce DNA damage, genomic fragmentation and apoptosis in multiple cancer cell lines, and suppress tumour growth in mouse xenograft models of prostate and lung cancer without any apparent toxicity to the animals.

Nuclear architecture by long noncoding RNAs through phase separation

Specific long noncoding RNAs have been identified as the structural scaffold of distinct membraneless nuclear bodies, therefore termed as architectural RNAs (arcRNAs). The arcRNAs build massive nuclear bodies by sequestrating multiple RNA-binding proteins possessing intrinsically disordered regions (IDRs). This process likely proceeds via liquid-liquid phase separation (LLPS) where arcRNA locally concentrates IDR containing RNA binding proteins. I will discuss the molecular mechanism of arcRNAs function revealed by identifying functional elements embedded in the arcRNA sequences as well as interacting RNA-binding proteins that induce LLPS.

Functional and mechanistic roles of long noncoding RNAs (lncRNAs) in cancer development and progression

Despite rapid improvements in unveiling the importance of long noncoding RNAs (lncRNA) in all aspects of cancer biology, there is still a lot of void in how lncRNAs mechanistically influence various biological processes associated with cancer development. Towards this, by using RNA-seq data from cell cycle-based high-throughput functional screens and different neuroblastoma (NB) prognostic sub-groups, we have identified several cancer-relevant, therapeutically applicable lncRNAs. Functional investigations of these lncRNAs revealed their nexus with oncogenic and tumor-suppressor pathways. I will discuss how these cancer relevant lncRNAs can be used as potential therapeutic targets in different cancer types.

Spacer acquisition mechanism in type II-A CRISPR system.

Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. The prespacer is bound by Cas1-Cas2 as a dual-forked DNA, and the terminal 3′-OH of each 3′ overhang serves as an attacking nucleophile during integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. I will present our structural and biochemical efforts in revealing the spacer acquisition mechanism in the Enterococcus faecalis type II-A CRISPR system.

Delivering Therapeutic Oligonucleotides across Biobarriers: Opportunities and Challenges in Drug Development

• Macromolecular biopharmaceuticals such as oligonucleotides have a huge pharmacological potential but their widespread therapeutic application has been very limited due to pharmacokinetic and drug disposition limitations at both the tissue and cellular level.
• The presentation will address these delivery limitations and summarize the work of a European consortium (www.compact-research.org) of pharma companies and academic partners to improve nanocarrier-based delivery technologies that can overcome these limitations.

Asymmetric siRNA Targeting Fibrotic and Ocular Disorders

OLX10010, an anti-fibrotic cell-penetrating asymmetric siRNA (cp-asiRNA) targeting connective tissue growth factor (CTGF), effectively reduces target gene expression as well as expression of fibrotic markers in animal model study. Preclinical as well as clinical study update of OLX10010 in anti-skin scar will be presented. In addition to skin scar, OLX10010 has a potential to be developed as therapeutics targeting various fibrotic disorders. We will present preclinical study data of OLX10010 in other fibrotic diseases in lung and eye, such as idiopathic pulmonary fibrosis (IPF) and subretinal fibrosis.

Alternative polyadenylation (APA) is emerging as a pervasive mechanism in the regulation of more than 70% of human genes. The role of APA in human cancer is largely overlooked due to the lack of genome-wide APA profiling on large clinical cohorts. To overcome this limitation, we recently developed DaPars algorithm (Nature Communications 2014) for the de novo identification of APA from standard RNA-seq. Through DaPars analyses of thousands of TCGA tumor RNA-seq data, we revealed ~1,300 highly recurrent 3’UTR shortening genes, nominated CFIm25 (Nature 2014) as a master regulator of 3`-UTR shortening that links APA to glioblastoma tumor suppression, and suggested a surprising enrichment of 3’UTR shortening among transcripts that are predicted to act as competing-endogenous RNAs (ceRNAs) for tumor suppressor genes (Nature Genetics 2018).

TRiMTM Platform Based RNAi Therapeutics in Treating HBV and Cardiometabolic Diseases

One of the more important recent advances in biology, RNA interference is a natural cellular process where short oligonucleotides can be used to silence gene expression and regulate the production of proteins. Arrowhead’s RNAi-based therapeutics leverage this natural pathway to shut down specific genes that cause disease.
The presentation will focus on Arrowhead’s clinical candidate ARO-HBV for the treatment of Hepatitis B Virus infection. HBV infection presents a huge unmet medical need, as it affects over 250 million people worldwide and currently there is no cure for this devastating disease. In the presentation, some of our earliest clinical data on ARO-HBV will be presented, and the journey in discovering this powerful drug candidate will be discussed. The presentation will also discuss Arrowhead’s preclinical candidates ARO-ANG3 and ARO-APOC3 for the treatment of cardiometabolic disease such as hypertriglyceridemia. Gene knockdown, reduction of protein levels, and reduction of triglycerides in disease models for Arrowhead’s preclinical candidates ARO-ANG3 and ARO-APOC3 will be presented.

Advances in RNAi Therapeutics Using Chemistry

RNA interference (RNAi) based therapeutics has evolved for the past two decades, and this year finally there is one approved siRNA-based drug representing the success of RNAi mechanisms of action: ONPATTRO or patisiran. On celebrating this occasion, we will review the key milestones of the past 16 years of drug discovery at Alnylam. This includes
• Chemical modifications, their functions, and pharmacological properties
• Delivery platforms (lipid nanoparticles and conjugates)
• Mechanisms of Delivery of RNAi therapeutics targeted to liver
• Emerging new approaches of overcoming RNAi-mediated, hybridization based off-target effects
• Use of Reversirs to modulate the RNAi effects

Phase-separated Domains of mRNA Regulation and Epigenetic Memory

In the C. elegans germline, three distinct Argonaute (AGO) systems act in concert with nearly 1 million distinct guide RNAs to scan the entire transcriptome inclusive of the coding regions. These AGO systems include a scanning AGO (Piwi) and its genomically encoded piRNA cofactors, and two ‘memory’ AGO systems whose guides are synthesized and maintained by RNA-dependent RNA polymerase, (RdRP). The CSR-1 memory Argonaute targets but does not silence expressed germline mRNAs, while the WAGO memory Argonautes target mRNAs that are silenced in the germline. These RNA-surveillance pathways reside in, and help drive the association of perinuclear nuage, (P-granules), which form liquid-like condensates around nuclei in transcriptionally active germ cells.

In mature gametes, CSR-1 and WAGO small RNA complexes are transmitted in cytoplasmic P-granules (in oocytes), and in a perinuclear halo of Ago/RNPs in sperm. Interestingly piRNA targeting induces a muted recruitment of RdRP on CSR-1 targets, suggesting that CSR-1 protects its targets from silencing. In contrast, piRNAs induce the robust localized recruitment of RdRP on WAGO targets (or foreign mRNAs) to amplify WAGO guide RNAs that maintain (or induce) the silencing of the target mRNA. In addition to completely silencing its targets, experimental studies reveal that germline-expressed mRNAs can respond to artificially-increased piRNA targeting with partial reductions in mRNA and protein expression.

piRNAs are abundantly expressed in the germlines of diverse animals, and because they tolerate mismatched pairing, have a vast potential target space. Thus, the repertoire of piRNAs in metazoan germlines provides a wealth of potential post-transcriptional regulatory capacity. Our findings suggest that piRNAs can access and regulate mRNAs throughout the mature transcript, including the ORF, and that even the coding regions of mRNAs are free to sample regulatory inputs from piRNAs. Importantly, because piRNAs are expressed as independent genes, they are also free to evolve independently, unconstrained by the coding requirements of their target regions. Thus piRNAs may unlock a vast regulatory space, the coding regions of genes, for the control of gene expression. Thus all germline mRNAs appear to undergo Argonaute surveillance and regulation within peri-nuclear nuage as they export to the cytoplasm. The assembly of the nuage around the nuclear periphery may facilitate access of this scanning machinery to RNAs as they emerge, prior to Ribosome loading, thus permitting regulation through the entire transcript. The ubiquity of AGO scanning within nuage begs the question of whether the condensate forms to drive the function, or whether the function drives the form.

In other words, perhaps the massive scale of protein-RNA and RNA-RNA targeting within nuage drives the exclusion of water (which always happens on a molecular scale) but simply becomes evident in the light microscope—function drives the form?

How mRNA controls the dynamics of translation

Translation by the ribosome is a central and final step in gene expression in all organisms. The goal of our research is to understand the underlying conformational and compositional dynamics of translation. We have applied single-molecule fluorescence and structural approaches to both prokaryotic and eukaryotic translation systems. We have explored the dynamics of translation initiation, elongation and termination. Here we will describe recent investigations of eukaryotic translation initiation in both yeast and humans that reveal the underlying dynamics of translational control.

Architecture of the HIV-1 reverse transcriptase initiation complex

Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in infection and a common target of antiretroviral therapy. The reaction is catalyzed by viral reverse transcriptase (RT) that is packaged in an infectious virion along with 2 copies of dimeric viral genomic RNA and host tRNALys3, which acts as a primer for initiation of reverse transcription. Upon viral entry, initiation is slow and non-processive compared to elongation. We applied cryo-electron microscopy (cryo-EM) to determine the three-dimensional structure of the HIV RT initiation complex. RT is in an inactive polymerase conformation with open fingers and thumb and the nucleic acid primer-template complex is shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV RNA lies in the cleft of RT, extended by additional pairing interactions. The 5’ end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.

Innovation at Vertex: Nucleic Acid Therapies as Transformative Medicines

Vertex is a global biotechnology company that invests in scientific innovation to create transformative medicines for people with serious and life-threatening diseases, through internal research and external partnerships. We discovered and developed the first medicines to treat the underlying cause of cystic fibrosis (CF), a rare, life-threatening genetic disease. In addition to cystic fibrosis, the following are diseases of further research interest: adrenoleukodystrophy, alpha-1 antitrypsin deficiency, polycystic kidney disease, and sickle cell disease.

Nucleic Acid Therapies expand our ability to target genes that are considered undruggable or difficult to drug with small molecules and biologics. Vertex established strategic research collaborations with Moderna and CRISPR to discover and develop potential new treatments aimed at the underlying genetic causes of human disease. Highlights from these efforts will be presented, in particular, our gene editing clinical program. As part of the CRISPR collaboration, we are co-developing CTX001, an investigational ex vivo CRISPR gene-edited therapy for patients suffering from β-thalassemia or sickle cell disease in which a patient’s hematopoietic stem cells are engineered to produce high levels of fetal hemoglobin (HbF; hemoglobin F) in red blood cells. HbF is a form of the oxygen carrying hemoglobin that is naturally present at birth and is then replaced by the adult form of hemoglobin. The elevation of HbF by CTX001 has the potential to alleviate transfusion-requirements for β-thalassemia patients and painful and debilitating sickle crises for sickle cell patients. CTX001 in on track to initiate a Phase 1/2 clinical study in SCD by the end of 2018 and are currently enrolling patients with transfusion dependent β-thalassemia in a Phase 1/2 trial in β-thalassemia in Europe.

piRNA biogenesis and functions

Female sterile (1) Yb (Yb) is a primary component of Yb bodies, perinuclear foci known as the site for piRNA biogenesis in Drosophila ovarian somatic cells. Yb consists of Helicase C-terminal (Hel-C), RNA helicase and extended Tudor (eTud) domains. We previously showed that the RNA helicase domain is necessary for Yb RNA interaction, Yb body formation and piRNA biogenesis. Here, we investigated functions of two other domains and found that Hel-C and eTud are necessary for Yb to self-associate and to interact with RNAs and other Yb body components, respectively. Both domains were essential for Yb body formation and transposon silencing. Without eTud, piRNA production was totally impaired. Loss of Hel-C severely reduced the levels of transposon-targeting piRNAs while non-transposon-targeting piRNAs were fairly produced. Yb bodies seem to be liquid-like multivalent condensates whose assembly depends on Yb self-association and Yb-flam RNA binding.

Towards targeting MCL-1 for cancer therapy

Impaired apoptosis is considered one of the prerequisites for the development of most, if not all, cancers, but the mechanisms that guarantee the sustained survival of most cancer cells remain unknown. We examined the roles of individual pro-survival BCL-2 family members (when expressed under endogenous control) in lymphoma development. BCL-2 was found to be dispensable for the development of lymphoma. In contrast, loss of BCL-XL and even more remarkably, loss of a single allele of Mcl-1 greatly impaired lymphoma development in Eµ-Myc mice. Experiments with inducible knockout mice demonstrated that MCL-1 is essential for the sustained survival and expansion of MYC-driven malignant pore-B/B lymphoma. In collaboration with the pharma company Servier we have been testing S63845, a novel potent and selective inhibitor of MCL-1. S63845 was well tolerated in mice and could potently kill many human multiple myeloma, acute myeloid leukaemia and several other haematological and even solid cancer cell lines as single agent. These results reveal that the targeting of MCL-1 could be a promising strategy for the treatment of diverse cancers.

Contributors to this work and affiliations:

 G Kelly*, S Glaser*, S Grabow*, JM Adams*, M Herold*, M Brennan*, O Geneste#, AL Maragno#, A Kotschy# and A Strasser*

 *The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia; #Servier, Paris, France

MicroRNAs as cell fate controllers and cellular reprogramming factors

Brain-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), are potent cell fate regulators that can induce direct conversion (reprogramming) of non-neural human somatic cells to neurons. As such, ectopic expression of miR-9/9*-124 in human adult fibroblasts has been shown to directly target multiple components of chromatin modifiers leading to an extensive reconfiguration of the chromatin landscape while erasing the pre-existing fibroblast fate and adopting the neuronal identity. Our recent efforts in identifying direct targets of miR-9/9*-124 during neuronal conversion of human fibroblasts indicate multifactorial components in maintaining the non-neuronal fibroblast fate and repressors of neuronal fate. Some of the targeted pathways that converge to promote the fate switch include the downregulation of the USP14-EZH2-REST protein stability cascade, switching of subunit components of BAF chromatin remodeling complexes, alternative-splicing factors, and DNA methyltransferases. The resulting miRNA-induced chromatin then becomes permissive to the inputs of additional subtype-defining transcription factors that could guide the conversion to specific types of neurons including cortical neurons, striatal medium spiny neurons, and spinal cord motor neurons. Generation of specific types of human neurons through miRNA-mediated reprogramming then serves as an effective platform to model neurological disorders. Importantly for modeling late-onset diseases, directly converted human neurons have been shown to maintain the age signatures of fibroblast donors. As such, medium spiny neurons, a primary target of Huntington’s disease (HD), generated from direct conversion of HD fibroblasts reliably recapitulated hallmarks of adult-onset HD including the aggregation of Huntingtin protein, increased DNA damage, mitochondrial dysfunction and spontaneous neuronal death. Collectively, our studies demonstrate the potent activity of microRNAs as cell fate regulators and its utility to generate a patient-derived cellular platform to model diseases.