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.

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?

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.

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.

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

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.