Session One: Molecular Oncology

---

Talk Title: CRISPR in action: engineering bowel cancer models and bacteria for tumour detection

Abstract Title: Using cutting edge CRISPR base editor technology to explore development of lymphoma

Authors:  Christina Koenig #1#2#3, Andrew Kueh #1#2#3#4, Sarah Diepstraten #3#4, Maggie Potts #1#2#3#4, Lin Tai #1, Lauren Whelan #3, Marco Herold #1#2#3#4 

Affiliations:  #1Olivia Newton-John Cancer Research Institute, Heidelberg, Melbourne, Australia. #2School of Cancer Medicine, La Trobe University, Bundoora, Melbourne, Australia. #3The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Australia. #4Department of Medical Biology, University of Melbourne, Parkville, Melbourne, Australia. 

Full Abstract: Neoplastic diseases result from the unrestricted growth of cells that have been transformed into a malignant state. Genetic defects mainly caused by single nucleotide variants (SNPs) in tumour suppressor genes or in proto-oncogenes allow cancer cells to acquire essential biological properties and deregulate several cellular processes, ensuring their survival and efficient growth. To model these SNPs and identify cancer-driving mutations, we employ the recently described CRISPR base editing (BE) technology.  

We established experimental procedures using the CRISPR BE technology together with single guide (sg)RNA libraries specifically designed to introduce the top mutations in human cancer-causing genes into the mouse genome in vitro and in vivo. In vitro, we transduce tumour prone EµMYC cells (a model of B cell lymphoma) with lentiviral BE plasmids able to introduce base changes and a respective sgRNA library for targeting the individual mutations in the genes of interest. We then treat this pool of engineered cells with diverse chemotherapeutic drugs, such as DNA-damaging agents or BCL-2 family inhibitors. In a major in vivo approach, we isolate hematopoietic stem and progenitor cells (HSPCs) from E14.5 embryos of newly developed EµMYC/TRE-CBE double-transgenic animals (harbouring an inducible BE system) and transduce them with BE sgRNA libraries to reconstitute lethally irradiated recipient mice. To activate the base editing that will create the specific mutations in pre-leukemic cells, recipient animals receive doxycycline food. Tumours that arise at an accelerated pace will be isolated and the tumour promoting sgRNA and genetic mutation identified. 

Using this approach will reveal critical mutations in tumour suppressor pathways and oncogenes involved in the transformation of haematological malignancies. This will enhance our understanding of malignant transformation and provide novel targets for anti-cancer therapies.

Abstract Title: Uncovering the Functions of Alternative Splicing in EMT using RNA-Targeting CRISPR Technology

Authors: Jasleen Rajpal1, Caroline A. Phillips1, John Toubia1, Katherine A. Pillman1, Gregory J. Goodall1,2 and Philip A. Gregory1,2

Affiliations: 1Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, 5000, Australia 2Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA 5000, Australia.

Full Abstract: Epithelial-Mesenchymal Transition (EMT) is a tightly regulated cell differentiation process that plays crucial roles in embryonic development and cancer progression. During EMT, the RNA-binding protein Quaking (QKI) orchestrates widespread changes in alternative splicing (AS) which influences cell plasticity, migration, and invasion. Despite the importance of AS in EMT, the functional roles of most alternatively spliced proteins remain unclear due to challenges in manipulating AS. To address this, we are developing RNA-targeting CRISPR technology to facilitate precise manipulation of alternative splicing at the transcript level and enable large-scale functional studies. Our approach utilizes catalytically inactive "dead" CasRx (dCasRx) to bind specific RNA sequences, block splice machinery access, and induce exon skipping. Additionally, we have developed a dCasRx-QKI fusion protein to facilitate exon inclusion by targeting QKI to selected binding motifs. As a proof of concept, we show that dCasRx effectively blocked the inclusion of exon 3 in the transcription factor NFYA which results in increased cell proliferation. This CRISPR-based strategy will be applied to pooled single-cell screening to evaluate the functional consequences of hundreds of individual splicing alterations on cell proliferation, migration, EMT, and drug resistance.

Talk Title: Using CRISPR technology to model complex chromosomal aberrations: a step towards better understanding leukemia pathobiology

Abstract Title: Identification of the synthetic lethality for chemotherapy drugs to develop novel combination therapy for rhabdomyosarcoma through CRISPR technology

Authors: Aqsa Mazhar 1,2, Daenikka Ravindrarajah 1,2, Twishi Gulati 4, Glenn M. Marshall1,3, Belamy B. Cheung 1,2

Affiliations: 1.Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, NSW, Australia: 2.School of Clinical Medicine, UNSW, Australia : 3.Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, Australia: 4.Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre Victoria Australia

Full Abstract: Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, comprising 4.5% of all childhood cancers. Alveolar tumours (ARMS) contribute to approximately one-third of RMS, and it is clinically more aggressive due to a propensity for metastasis and recurrence. Most ARMS expresses one of two oncogenic gene fusions: PAX3 or PAX7 with FOXO1, which act as a dominant-acting oncogene in driving tumorigenesis. To date, there is no inhibitors which directly bind to PAX3-FOXO1. Currently, CRISPR-Cas9 screens are an increasingly valuable method for determining synthetic lethality and drug-resistant genes across the entire genome in the present of chemotherapy drugs or target therapy drugs.

Objective: To identify drug resistant genes in alveolar RMS by CRISPR-Cas9 screen and develop the effective novel combination therapies with currently clinical used therapeutic drugs for alveolar RMS.

Methods and Results: We have successfully generated stable Cas9 expressing Rh41 cell line by transducing cells with lentiCas9- mCherry plasmid After determining the lentivirus titre of pXPR-011 construct using FACS analysis, we confirmed the success of transduction of Rh41 with Cas9-mCherry lentivirus by Western Blot assay. We have performed CRISPR KO screen and transduced cas9-Rh41 cells with sgRNA library (whole genome library) by using 0.3 MOI with irinotecan treatments by using IC30 and collected cell pellets at different time points. After extraction of genomic DNA and amplification of PCR, we have sent samples for sequencing to identify the candidate genes that play a role in drug sensitivity in alveolar RMS cell line. We are validating candidate genes obtained from CRISPR KO screen by performing in vitro phenotypic and molecular analysis to determine the efficacy and mechanism of synergy in the drug combinations for further in vivo study.

Conclusion: This study will provide new avenues for the treatment of alveolar RMS by identifying the drug-resistant mechanism and developing novel combination therapy

Abstract Title: CRISPR editing of adenosine receptor expression in CAR T cells to enhance therapy of solid tumours

Authors: Kevin Sek, Paul Beavis, Phil Darcy

Affiliations: Peter MacCallum Cancer Center

Full Abstract: Tumour-infiltrating CAR-T cells face many immunosuppressive conditions within the solid tumour microenvironment, including the accumulation of hypoxia-driven metabolites such as extracellular adenosine. This metabolite acts primarily through the A2A receptor (A2AR) and suppresses adoptive cellular immunotherapies including CAR-T cells. We hypothesized that using CRISPR gene-editing technologies to modulate the adenosine receptor immunosuppressive axis could enhance CAR-T cell function in solid tumours.

The A1 receptor (A1R) is an alternative adenosine receptor that signals inversely to A2AR. Earlier work demonstrated that CRISPR homology-directed repair (HDR) to knock-in A1R into an endogenous T cell promoter achieved tightly tumour-restricted A1R expression by hijacking endogenous transcriptional regulatory mechanisms. This approach enhanced effector function, cytokine production, and prolonged persistence of CAR-T cells in human solid tumour models. Through this investigation we discovered novel transcription factors activated by A1R signalling in CAR T cells that are targetable with CRISPR KO to enhance therapy even further.

Subsequent testing was conducted in murine CAR-T cells in models that recapitulate the immunosuppressive tumour microenvironment. The use of the same promoter did not effectively overcome the suppressive influence of endogenous A2AR signalling. However, by using alternative promoters with stronger but less specific transgene expression, we improved CAR-T effector function in vitro, though at the expense of depleting the memory pool crucial for in vivo efficacy.

We consequently explored a dual CRISPR strategy: A2AR ablation combined with A1R HDR to synergistically enhance CAR-T cell function by strengthening the A1R bias. This approach resulted in amplification of the A1R transcriptional phenotype and further augmented CAR T cell effector function. In summary our study leverages CRISPR gene-editing to remodel the adenosine receptor profile of CAR-T cells, providing a promising strategy for enhancing their efficacy in solid tumours.

Abstract Title: Targeting the hepatitis B RNAs using CRISPR-Cas13b to suppress hepatitis B virus replication and protein expression in vitro and in vivo

Authors: McCoullough LC1,2,3, Fareh M4,5, Hu W4,5, Sozzi V1, Makhlouf C1, Droungas Y1,2, Lee CL1,5, Takawy M1,3, Fabb SA6, Payne TJ6, Pouton CW6, Netter HJ1, Lewin SR3,7,8, Purcell DFJ2, Holmes JA9, Trapani JA4,5, Littlejohn M1,3*, Revill PA1,3*

Affiliations: 1Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. 2Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. 3Department of Infectious Diseases, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. 4Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 5Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia. 6Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. 7Victorian Infectious Diseases Service, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. 8Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, Victoria, Australia. 9Department of Gastroenterology, St. Vincent’s Hospital, Melbourne, Victoria, Australia. *: These authors contributed equally to this work.

Full Abstract: Background: Bacterial CRISPR-Cas13b endonuclease has been repurposed to target RNA in mammalian cells by designing highly specific 30 nucleotide CRISPR RNAs (crRNAs) complementary to the target RNAs of interest, which reduces the possibility of off-target effects. Recent preclinical studies have used CRISPR-Cas13b as a novel antiviral to target viral RNAs such as SARS-CoV-2 and influenza RNAs to reduce viral replication. Hepatitis B virus (HBV) is a DNA virus that replicates through an RNA intermediate known as the pregenomic RNA (pgRNA). The pgRNA and viral mRNAs represent novel antiviral targets, which may be targeted by CRISPR-Cas13b. Here, in a world first study, we used CRISPR-Cas13b to target the HBV RNAs to reduce HBV replication and protein expression in vitro and in vivo.

Methods: Cas13b crRNAs were designed to target the HBV RNAs. Hepatoma cells were transfected with wildtype (WT) HBV of multiple genotypes, Cas13b and crRNA plasmids. A HBV stable cell line and HBV infection model were transfected with Cas13b and crRNA plasmids. The impact on HBV replication and protein expression was determined. WT HBV, Cas13b and crRNA plasmids were hydrodynamically co-injected into CBA mice and sera hepatitis B surface antigen (HBsAg) was measured. Cas13b mRNA and crRNA were delivered by lipid nanoparticles (LNPs) in a HBsAg-expressing stable cell line and secreted HBsAg was measured. 

Results: Cas13b strongly suppressed HBV replication and protein expression in all cell lines tested. The effect was pan-genotypic. Sera HBsAg was reduced by ~50% in vivo. LNP-encapsulated Cas13b mRNA reduced secreted HBsAg by 87% in a HBsAg-expressing stable cell line. 

Conclusion: CRISPR-Cas13b successfully targeted the HBV RNAs to significantly reduce HBV replication and protein expression in vitro and in vivo which, together with other studies that have used CRISPR-Cas13b to target viral RNAs, further demonstrates its potential as a novel antiviral.

Abstract Title: Efficient CRISPR-Cas9 therapies for the correction of Duchenne Muscular Dystrophy  amendable to exon 45 targeting

Authors: Ryan HB Lee1,2, Fatwa Adikusuma1,2, Paul Q Thomas1,2,3

Affiliations: 1. School of Biomedicine, University of Adelaide 2. South Australian Health and Medical Research Institute 3. South Australian Genome Editing Facility

Full Abstract: Duchenne Muscular Dystrophy (DMD) is a monogenic muscle-wasting disorder caused by mutations that disrupt Dystrophin production. Since no curative treatments are available, DMD is universally fatal around 30 years old. CRISPR therapies for DMD install loci-specific edits which could restore the production of a truncated yet functional Dystrophin, through exon skipping or reframing. Importantly, the long-lasting nature of these edits are potentially curative and overcome the lifelong administration required for current treatments.  Streptococcus pyogenes Cas9 (SpCas9) remains the most utilised system for therapeutics due to its established efficiency. Smaller Cas9s like Staphylococcus aureus Cas9 (SaCas9) allow for single Adeno-associated virus (AAV) delivery, which holds promise in vivo as therapeutic benefit can be achieved with a lower AAV dosage.  

This study aims to identify SpCas9 and SaCas9 gRNAs which efficiently target exon 45 of the human DMD gene and to validate the dual-gRNA approach we devised to maximise Dystrophin restoration. These therapies would be applicable to 9% of DMD patients harbouring exon 46-47, 46-51 and 44 deletions (Δ44).  Here, we present a clonal Δ44 DMD model, generated through paired SpCas9 activity on wildtype immortalised, human myoblast and validated through PCR and Western blotting. 

gRNAs were transfected in triplicate into this Δ44 model, and the resulting editing profile was analysed through Next Generation Sequencing (NGS). Dystrophin restoration post-treatment was also assessed through cDNA PCR and Western blotting.  

Through NGS, several SpCas9 & SaCas9 gRNAs with mean on-target editing efficiencies exceeding 90% were identified, of which up to 55.8% of therapeutic editing was observed. In accordance with our dual-gRNA approach, the co-delivery of these gRNAs led to a significant enhancement of therapeutic exon skipping in RNA and Dystrophin restoration in protein.  

Collectively, these experiments demonstrate the therapeutic promise of our CRISPR candidates, thus justifying future in vivo studies. 

Abstract Title: Functional Mapping of PRC2 Mutations with Unbiased Base Editing.

Authors: Ali Motazedian, Maria Faleeva, Henrietta Holze, Kapil Mcinerney Mark Dawson

Affiliations: Peter Maccallum Cancer Centre VCCC

Full Abstract: Polycomb Repressive Complex 2 (PRC2) regulates gene expression through its histone methyltransferase activity. It consists of three core proteins: EZH2, SUZ12, and EED. EZH2 is the catalytic subunit that methylates histone H3 at lysine 27 (H3K27), crucial for gene silencing. SUZ12 provides structural support and interacts with accessory proteins, whilst EED recognises existing H3K27 methylation, allosterically boosting EZH2 activity to maintain repressive chromatin states. PRC2's structure and function are highly conserved across eukaryotes, highlighting its vital role in cellular processes. Mutations in PRC2 can cause both loss (LoF) and gain (GoF) of function, inducing either hypo- or hyper-methylating activity in various cancers and congenital disorders. The effects of DNA mutations on PRC2's activity and stability necessitate further research to provide insights into the evolutionary mechanisms that enable PRC2's role in epigenetic regulation.

This project aimed to systematically alter the coding sequence of PRC2 complex members using base editors (BEs) and characterise LoF and GoF mutations. We used five different BEs to perform A to G and C to T mutations, inducing random mutations across a significant portion of the gene for a comprehensive analysis of PRC2 function at single cell resolution. Experiments were conducted in K562 cells which tolerate GoF and LoF mutations without a discernible effect on growth and survival. 

Our screen identified synonymous, stop, and missense mutations, several of which are hypothesised to be dominant negative. We identified LoF mutations in both structured and unstructured domains of PRC2 proteins. Guides and mutations frequently clustered in specific 'hotspots' within disordered domains of SUZ12 and EED, highlighting key regions not identifiable by conventional structure determination methods. Finally, transcriptome sc-RNA sequencing revealed whether PRC2 disruption has binary or variable effect on gene expression.

In summary, this BE-driven mutagenesis screen revealed diverse PRC2 LoF and GoF mutations undetectable by conventional methods.

Abstract Title: Developing a pipeline for generating gene knockout and knock-in fluorophore fusion proteins using CRISPR-Cas9 to study T cell development in primary mouse thymocytes

Authors: Lucas M. Newton1,2, Mirren Charnley1,2, Sarah M. Russell1,2

Affiliations:  1. Organogenesis and Cancer Program, Cancer Research, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 2. Optical Sciences Centre, Swinburne University of Technology, Melbourne, Victoria, Australia.

Full Abstract: β-selection is a critical checkpoint during T cell development that determines the fate and functional competence of mature T cells. This process occurs in the thymus, where developing T cells undergo stringent selection based on their ability to successfully rearrange and express a functional T cell receptor (TCR) β-chain. β-selection not only ensures the generation of a diverse T cell repertoire capable of recognising a wide array of antigens but also plays a fundamental role in shaping immune tolerance and responsiveness. Our previous work has identified new regulators of β-selection using a transgenic Cre-LoxP system in mice to generate protein knockdown primary mouse thymocytes. However, this method is infamously leaky and requires the costly generation of new transgenic mouse lines for each gene of interest. Therefore, we are now working towards applying CRISPR-Cas9 technology to genetically manipulate primary mouse developing T cells and the mouse stromal cell line OP9-DL1/4, which represents the thymic niche. Our goal is to produce a pipeline to genetically manipulate human and mouse developing T cells and the stroma. Here, we present an update on our progress in generating CD5 knockout primary mouse thymocytes by applying CRISPR-Cas9 to foetal liver-derived hematopoietic stem cells prior to expansion and differentiation. In addition, we share our initial results into the generation of StayGold and mCardinal fluorescently tagged endogenous Numb and Bcl6, which both play significant roles in regulating asymmetric cell divisions during T cell β-selection. Furthermore, this work provides insights into our efforts to generate E-cadherin knockout OP9-DL1/4 stromal cells, which has proven problematic using conventional CRISPR-Cas9 approaches in mouse cells. We believe this work will prove helpful to those interested in genetically manipulating haematopoietic cell systems and we look forward to much needed feedback on our current achievements.

Abstract Title: Uncovering the design principles of CRISPR/Cas13d as an effective antiviral strategy

Authors: Emily Hann1,2*, Debolina Majumdar1, Daniel Layton1, Mark Ziemann3, David Cahill2, Beata Ujvari2, Mohamed Fareh4, Karel A Schat5, Arjun Challagulla1

Affiliations:  1Australian Centre for Disease Preparedness, CSIRO Health and Biosecurity, Geelong, VIC, Australia, 2School of Life and Environmental Sciences, Deakin University, Geelong, VIC, Australia 3Burnet Institute, Melbourne, VIC, Australia 4Peter MacCallum Cancer Centre, Melbourne, VIC, Australia 5Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

Full Abstract: The CRISPR/Cas13d is a programmable RNA endonuclease system that has been harnessed for facile and efficient targeting of cellular and exogenous RNAs, including RNA viruses such as influenza virus. Given the robust catalytic activity and specificity of Cas13d, coupled with the ease to design crRNAs, CRISPR/Cas13d has the potential to offer an advantage over conventional antiviral strategies by rapidly designing antiviral effectors. However, there is a considerable knowledge gap in our current understanding of the principles governing the effectiveness of crRNAs in the context of mutation-driven influenza virus evolution and emerging strains. In this study, we delineated the principles for the development of effective crRNAs by targeting a DsRed fluorescence reporter gene in chicken fibroblast DF1 cells. To systematically determine the optimal design for crRNAs, we designed multiple versions of crRNAs to investigate the minimum length of the crRNA, protospacer flanking sequence, degree of mismatch tolerance, and collateral effects. Our data revealed variable knockdown levels between crRNAs, in which a few crRNAs achieved over 95% DsRed knockdown. Other crRNAs exhibited moderate to no effects, although they targeted adjacent RNA locations. crRNAs showed a preference for length requirements and sequences with fewer than 21-nt failed to knockdown the reporter gene. We demonstrated that crRNAs exhibit a high degree of tolerance to single-nucleotide mismatches, regardless of the position at which the single-nt mismatch was introduced. However, 4-nt mismatches within crRNA significantly reduced targeting efficacy, and eight nucleotide mismatches completely diminished targeting efficacy. Finally, Cas13d induced collateral degradation of bystander RNA, suggesting that additional studies are necessary to understand its pleotropic effects. This study extends our understanding of the Cas13d targeting mechanism through multiple iterations of crRNAs, providing a roadmap to design crRNAs for improved effectiveness, which will be important against rapidly evolving viruses.

Abstract Title: Whole genome CRISPR/Cas9 screening to understand resistance to BH3-mimetic drugs in lymphoma

Authors: Sarah Diepstraten 1 2 , John E La Marca 1 2 3 4 ,Yexuan Deng 1 2 3 4 , Andreas Strasser 1 2 , Marco Herold 1 2 3 4 , Gemma Kelly 1 2

Affiliations: 1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 2. Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia 3.Olivia Newton-John Cancer Research Institute, Heidelberg, Melbourne, Australia 4.School of Cancer Medicine, La Trobe University, Bundoora, Melbourne, Australia

Full Abstract: One hallmark of cancer is dysregulation of the intrinsic apoptosis, or cell death, pathway. This pathway is mediated by proteins of the BCL-2 family, which includes both pro-apoptotic and pro-survival factors, the levels of which are usually carefully maintained to determine whether a cell lives or dies. BH3-mimetics are exciting new anti-cancer drugs which directly bind and inhibit specific pro-survival proteins, tipping the balance of the apoptosis pathway towards favouring cell death. As testament to their efficacy, the BH3-mimetic drug venetoclax, which targets the pro-survival protein BCL-2, has been approved by many regulatory agencies worldwide for the treatment of particular blood cancers. New BH3-mimetics targeting MCL-1, an essential survival factor for a wide range of cancers, are currently in clinical trials for diverse haematological malignancies. However, emerging clinical data suggests that while BH3-mimetics are initially highly effective at killing cancer cells, drug resistance frequently develops over long-term treatment, resulting in patient relapse. We are using a systematic approach to explore how lymphoma cells can become resistant to BH3-mimetics targeting BCL-2 or MCL-1. We employ whole-genome CRISPR/Cas9 knockout and activation screens to identify factors which contribute to drug resistance in lymphoma. To enable these studies, we developed a faithful mouse model of aggressive double hit lymphoma, through use of our novel CRISPR-activation enabled mouse. From these screens, we recently characterised the tumour suppressor protein p53, among others, as an important mediator of the cellular response to BH3-mimetic drugs.

Abstract Title: Development of a diagnostic assay to detect Trichomonas tenax using CRISPR-Cas12a technology

Authors: Joshua Slattery1, Camilla Donnelly2, Anna Walduck1, Bernd Kalinna1, Martin Pal1,2,3

Affiliations: 1Rural Health and Research Institute, Charles Sturt University, Orange, NSW, Australia; Gulbali Institute, Charles Sturt University, Wagga Wagga, NSW, Australia; 3School of Dentistry and Medical Sciences, Faculty of Science and Health, Charles Sturt University, Wagga Wagga, NSW, Australia

Full Abstract: Periodontal disease is the major cause of tooth loss in adults worldwide. In Australia, a third of adults are suffering from moderate-severe periodontitis and an additional third suffering the milder gingivitis. While previously bacteria have been thought to be responsible for the onset of this disease, recently the protozoan Trichomonas Tenax has been implicated in disease progression. However, suitable point-of-care detection methods aiding a causative role for T. tenax are lacking. Here, we describe the development of a novel diagnostic assay for the detection of T. tenax using CRISPR-Cas12a technology. For this, Cas12a protein was purified in-house and a 6kb region of the T. tenax genome was screened to identify potential guide RNA sequences utilising a TTTV-PAM sequence. A total of 170 potential guide RNA sequences were analysed by developing a ranking formula calculating a specificity score for each candidate guide RNA. Each sore was based on potential off-targets in the human genome as well as sequence overlaps with the closely related species T. vaginalis. Using this ranking, 14 guide RNAs were subsequently tested using synthetic T. tenax DNA and a 5’FAM- TTTTTTTTT-3’ZEN/BHQ fluorescent reporter. 12 of 14 guide RNAs demonstrated detectable fluorescence with on-target DNA and no fluorescence in negative controls, and a 4pM target DNA concentration was determined as detection limit. In summary, we have identified multiple unique sequences within the genome of T. tenax suitable for the design of a detection assay based on CRISPR-Cas12a technology. Future experiments are warranted to move this assay into a field-based point-of-care testing regime, and the use of patient-derived clinical samples will require the development of an isothermal Recombinase Polymerase Amplification (RPA) step to improve sensitivity prior to CRISPR target recognition.

Abstract Title: Novel CRISPR-Cas9 Fusion for Safer Gene Editing

Authors:  Tahmina Tabassum1, Giovanni Pietrogrande1, Jake Bradford2, Dimitri Perrin2 & Ernst J. Wolvetang1 

Affiliations:  1Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia 2Queensland University of Technology, Brisbane, Australia 

Full Abstract: Efficiency and safety are key issues dominating the surge of new modified CRISPR tools designed to improve precise genome editing. CRISPR has been widely effective in gene disruption studies but successful application in gene correction or replacement studies is limited. Insertion of genetic material via homologous recombination (HR) is a challenge, making development of safer gene editors a growing research area especially for clinical and translational applications. DNA breaks are governed by complex DNA repair response pathways involving various regulators. Cas9 fusions with HR regulating proteins have successfully increased knock-in efficiencies in multiple studies. However, there has been no breakthrough for reducing off target effects in such editing practices. Using small molecules to inhibit the non-homologous end-joining pathway is the most common practice with the trade-off of cytotoxicity and global DNA repair dysregulation. In this study we have developed a novel CRISPR-Cas9 fusion protein for safer editing without the need for global dysregulation of repair mechanisms. Cas9 is fused to a motif which helps reduce indel formation through post-translational modifications at target site. We targeted HEK293 cells for gene correction of transgenic GFP with our fusion protein and wildtype SpCas9 where fluorescence data confirms comparable GFP correction by HR between both modalities. Interestingly, sequencing results reveal significant reduction of on-target indel mutations in cells edited with our fusion editor. This preliminary finding is promising and has guided optimisation of the editor to assess off-target implications in primary cells and apply in CAR-NK therapy.

Abstract Title: Suppression of HIV transcription via LNP-delivered mRNA-encoded CRISPR-Cas13: a novel latency promoting agent

Authors: Priyank Rawat (1), Stan Kan (1,2), Paula Cevaal (2), Ajantha Solomon (2), Kiho Tanaka (2), Joseph A Trapani (1), James McMahon (3), Sharon R Lewin (2,3,4), Michael Roche (2,5), Mohamed Fareh (1)

Affiliations: 1. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Australia 2. Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia 3. Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, Australia 4. Victorian Infectious Diseases Service, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia 5. STEM College, RMIT University, Melbourne, Australia

Full Abstract: Background: Prolonged persistence of HIV latently infected cells on antiretroviral therapy (ART) in people with HIV (PWH) serves as a roadblock towards a cure for HIV infections. CRISPR-Cas13 is a programmable RNA-nuclease that can effectively degrade viral transcripts and could be exploited to eliminate viral reactivation after cessation of ART. We hypothesised that the CRISPR-Cas13 system could knock down the essential HIV Tat protein, thus locking HIV into deep latency. Methods: We used a proprietary lipid nanoparticle (LNP) formulation to deliver a Tat-targeting Cas13 mRNA to HIV latently infected cell lines, JLat A2 and ACH2. Tat expression was measured by RT-qPCR, western blotting and viral reactivation via flow cytometry-based assessment of GFP reporter and viral p24 expression. Additionally, we assessed the ability of Tat-targeting Cas13 mRNA to suppress viral expression in human CD4+ T cells infected with a replication-competent reporter virus via flow cytometry and digital PCR-based HIV transcription profiling.Results: In JLat A2 cells, LNP-delivered Tat-targeting crRNA and RfxCas13d mRNA led to 80%±5% reduction in Tat mRNA expression and 90%±9% reduction in GFP expression indicative of viral reactivation. In ACH2 latency model, Tat-targeting RfxCas13d mRNA resulted in reduction of 65%±10% in viral p24 expression. Similar findings were observed using the pspCas13b ortholog. Additionally, we observed a significant reduction in cell viability (40%±8) with RfxCas13d Tat-targeting mRNA relative to a non-targeting control, a consequence of collateral activity of RfxCas13d. Finally, in human CD4+ T cells infected with a replication competent HIV reporter virus, Tat-specific crRNA and Cas13 mRNA resulted in 50%±10% reduction in productive infection, consistent with an overall reduction in the viral RNA and DNA expression. Conclusions: Collectively, our results demonstrate that a Tat-targeting Cas13 mRNA delivered via LNP effectively suppresses Tat expression in HIV latency cell lines and primary CD4+ T cells infected with an HIV reporter virus.

Abstract Title: Editing of HBV DNA in vitro and in vivo using a CRISPR/Cas9 Base Editor approach

Authors: Zak Janetzki1, Laura McCoullough1,2, Joan Ho3, Thomas Payne3, Stewart Fabb3, Colin Pouton3, Mohamed Farah4,5, Joe Trapani4,5, Margaret Littlejohn1,2, Peter Revill1,2

Affiliations: 1Department of Infectious Diseases, The University of Melbourne. 2Victorian Infectious Diseases Reference Laboratory (VIDRL), Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity. 3Monash Institute for Pharmaceutical Sciences, Monash University. 4Cancer Immunology Program, Peter MacCallum Centre. 5Sir Peter MacCallum Centre Department of Oncology, The University of Melbourne.

Full Abstract: Introduction: Current hepatitis B virus (HBV) treatments do not target the HBV covalently closed circular DNA (cccDNA) minichromosome reservoir, nor do they target HBV integrated DNA. There is a desperate need to develop novel therapeutics that target both cccDNA and integrated DNA; to improve HBV cure rates. CRISPR/Cas9 base editors (BEs) are a promising approach as they utilise the CRISPR/Cas9 guiding system to introduce specific C:G to T:A edits into target DNA. Edits can be predicted and there is less chance of genome instability when targeting integrated HBV DNA. The aim of this project analyse single guide RNA (sgRNAs) targeting all HBV open reading frames (ORFs) and to test the efficacy of transient expression of BEs and sgRNAs in reducing HBV replication and protein expression in vitro and in vivo. 

Methods: All sgRNAs introduce premature stop codons to reduce HBV protein expression and replication. The efficacy of sgRNAs were tested via plasmid transfection with HBV DNA and Cas9 BEs in HepG2 cells. HBV replication markers and proteins were measured and compared to a non-targeting control sgRNA. 

Results: sgRNAs with different BEs targeting HBV ORFs achieved knockdown of HBV proteins and intracellular core-associated HBV DNA with varying efficacy. Further analysis of the impact on HBV RNA, DNA and protein expression in vitro will be performed. A combination of sgRNAs will also be tested. Different models will be used including HBV stable cell lines, an HBV infection system, and a murine model. BEs will also be delivered as mRNA packaged in lipid nanoparticles in vitro and in vivo, with and without current therapeutics. 

Conclusion: These studies will determine the utility of Cas9 BEs for introducing specific base changes into the HBV cccDNA and integrated DNA, as an important first step towards developing this approach as a novel HBV therapeutic.

Abstract Title: CRISPR-activation for HIV latency reversal using lipid nanoparticle delivery

Authors: Moso MA1,2, Cevaal PM1, Tan A1, Kan S1, Ali A1, Fisher BM1, Kim Y1, Ong J1, Purcell D3, Pouton C4, Caruso F5, Fareh M6,7, Quadeer A3,8, McKay M3,8, Lewin SR1,2,9, Roche M1

Affiliations: 1Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity; 2Victorian Infectious Diseases Service, The Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity; 3Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity; 4Monash Institute of Pharmaceutical Sciences, Monash University; 5Department of Chemical Engineering, The University of Melbourne; 6Cancer Immunology Program, Peter MacCallum Cancer Centre; 7Sir Peter MacCallum Department of Oncology, The University of Melbourne; 8Department of Electrical and Electronic Engineering, The University of Melbourne; 9Department of Infectious Diseases, Alfred Hospital and Monash University.

Full Abstract: Background: The major barrier to HIV cure is the persistence of latently infected CD4+ T cells harbouring integrated HIV DNA. One major strategy for HIV cure is the ‘shock and kill’ approach, whereby latency reversal agents are used to upregulate HIV transcription and induce cell death through virus-mediated cytotoxicity or immune-mediated clearance. We sought to assess CRISPR activation (CRISPRa) for HIV latency reversal using the synergistic activation mediator (SAM) system and lipid nanoparticle (LNP) delivery. Methods: LNPs were synthesised using an optimised lipid mix (LNP-X) combined with CRISPRa mRNAs and HIV-targeting gRNAs (gRNA-L, O, 1, 2) (CRISPRa-LNPs). Potency of CRISPRa-LNPs with single or multiplexed gRNAs was assessed in the Jlat-A2 T-cell line, which contains a GFP reporter gene under the control of the HIV promoter. Mismatch tolerance of CRISPRa was evaluated using single- or double-base mutations in gRNA-L. CRISPRa-LNP activity in primary T cells was assessed using a gRNA targeting the host gene, CD25. Ex vivo CRISPRa activity was assessed in CD4+ T-cells from people with HIV on antiretroviral therapy (n=8) and measured using digital RT-PCR to quantify induction of HIV transcripts. Results: HIV-targeting CRISPRa-LNPs led to potent HIV expression in Jlat-A2 cells, with highest potency observed using multiplexed gRNAs L+O. Single base mutations from position 1-10 and sequential double mismatches up to positions 3,4 retained >50% activity, indicating a degree of mismatch tolerance. CD25-targeting CRISPRa-LNPs led to 3.2-fold increase in CD25 expression in resting CD4+ T-cells, demonstrating successful delivery of CRISPRa to primary cells. CD4+ T-cells treated ex vivo with CRISPRa-LNPs demonstrated median 2.0-fold increase in cell-associated HIV transcripts, indicating increased HIV transcription. Conclusion: LNP-X can deliver CRISPRa machinery to primary T cells and reactivate transcription of latent HIV. Further optimisation of delivery of CRISPRa is being undertaken to improve potency of this approach in resting CD4+ T-cells.