A Paradigm Shift in Infectious Diseases: Lessons from a Pandemic

Viral infections are an extremely common and predictable problem in organ and hematopoietic stem cell transplant patients. Antiviral drugs given either prophylactically or as early therapy for patients detected to be shedding virus appears to be an effective strategy for reducing herpes virus infections. However, long-term treatment with these drugs is associated with significant toxicity, expense and the appearance of drug resistance virus isolates ultimately resulting in treatment failure. Over the last few years, there is increasing evidence that cellular immune therapies can reverse the outgrowth of hematological malignancies and can also provide therapeutic benefit against lethal viral infections. While the expansion and adoptive transfer of virus-specific T-cells from the healthy original donor can be an effective strategy to control viral replication, this is not possible when donors are seronegative or are subsequently inaccessible. Studies from our group have demonstrated the successful expansion of human cytomegalovirus (CMV) and Epstein-Barr virus (EBV)-specific T-cells from solid organ and stem cell transplant recipients with active viral infection or disease and the long term reconstitution of protective anti-viral immunity following their adoptive transfer back into the patients. More recently, we have also developed a single platform technology to extend this immunotherapeutic strategy for multiple pathogens including CMV, EBV, adenovirus and BK polyoma virus. T cells directed towards multiple pathogens can be rapidly expanded and can be used for adoptive immunotherapy. Finally, in collaboration with an Australian biotech company, Cellestis Inc., we have also developed a novel T cell-based immune monitoring technology (QuantiFERON-CMVâ) which will allow us to identify high risk transplant patients who may develop virus-associated complications post-transplantation.

Novel coronavirus SARS-CoV-2 outbreaks have rapidly spread to multiple countries, highlighting the urgent necessity for fast, sensitive, and specific diagnostic tools for virus surveillance. We have previously demonstrated that Class 1 type I CRISPR, which is composed of Escherichia coli Cascade, Cas3, and programmable pre-crRNA, mediates distinct DNA cleavage activity in human cells. Here, the previously unknown collateral single-stranded DNA cleavage we observed with type I CRISPR-Cas3 highlights its potential for development as a Cas3-mediated rapid (within 40 min), low-cost, instrument-free detection method for SARS-CoV-2. This Cas3-based assay is comparable with Cas12- and real-time reverse-transcriptase PCR-based assays in its speed and sensitivity, but offers greater specificity for single-base-pair discrimination while negating the need for highly trained operators. These findings support the use of CRISPR diagnostics for point-of-care testing in patients with suspected SARS-CoV-2 infections.

This presentation introduces the process of accelerated productization, pioneered by Diagnostics Development (DxD) Hub. DxD Hub is a Singapore national platform for diagnostics development and productization, funded by the National Research Foundation and hosted by the Agency for Science, Technology and Research (A*STAR). The key to accelerated productization lies in (i) end-to-end productization: understanding user requirements, and developing the regulatory framework of minimal viable product based on format of the end product; and (ii) agile iterative process: quick succession of iteration based on evolving market and user input. DxD Hub model is based on that of a public-private partnership involving R&D entities, clinical community and commercial parties. Using the example of RESOLUTE - a direct polymerase chain reaction (PCR) test, this presentation illustrates the productisation journey of a diagnostic test. RESOLUTE received provisional authorisation from the Health Sciences Authority in April 2020, and is a collaboration between Defence Science Organisation, Advanced Remanufacturing Technology Centre, and commercial partner Advanced Medtech.

The general focus of my lab is to characterize at the molecular level, genome editing proteins derived from the bacterial CRISPR-Cas system and apply them for reversing disease causing mutations. Using biophysical tools, we dissect the mechanism of action of Cas9 proteins with respect to nucleic acid interrogation and use this knowledge to design editors with high gene targeting specificity. We combine biochemical tools for producing recombinant Cas9 proteins, induced pluripotent stem cell (iPSC) technology for studying their efficacy in patient cells and single molecule experiments for evaluating their targeting specificity. Based on this broad theme, there are two key areas where I carry out research on:

1) We have extensively characterised a TypeII Cas9 from Francisella novicida (FnCas9) and demonstrated its ability in genome editing of the sickle cell anemia mutation in patient derived iPSCs and HSCs. FnCas9 is highly specific in target interrogation and produces negligibe background insertions/deletions in patient Hematopoietic stem cells. Currently we are working towards completing pre-clinical studies on FnCas9

2) We have repurposed the high specificity of FnCas9 towards a Point of Care diagnostic assay for detecting nucleotide variants in a target. This can perform with minimum equipment and technical know-how and is being currently deployed for rapid and accurate diagnosis of COVID- 19 positive cases. By tying up with industries, we are scaling up the production of assay components to enable testing on a large scale during the ongoing pandemic.

Current bottlenecks for improving accessibility and scalability of SARS-CoV-2 testing include diagnostic assay costs, complexity, and supply chain shortages. To resolve these issues, we developed SalivaDirect. The critical component of our approach is to use saliva instead of respiratory swabs, which enables non-invasive frequent sampling and reduces the need for trained healthcare professionals during collection. Furthermore, we simplified our diagnostic test by (1) not requiring nucleic acid preservatives at sample collection, (2) replacing nucleic acid extraction with a simple proteinase K and heat treatment step, and (3) testing specimens with a dualplex quantitative reverse transcription PCR (RT-qPCR) assay. We validated SalivaDirect with reagents and instruments from multiple vendors to minimize the risk for supply chain issues. Regardless of our tested combination of reagents and instruments from different vendors, we found that SalivaDirect is highly sensitive with a limit of detection of 6-12 SARS-CoV-2 copies/μL. When comparing paired nasopharyngeal swabs and saliva specimens using the authorized ThermoFisher Scientific TaqPath COVID-19 combo kit and our SalivaDirect protocol, we found high agreement in testing outcomes (>94%). Being flexible and inexpensive ($1.29-$4.37/sample), SalivaDirect is a viable and accessible option to help alleviate SARS-CoV-2 testing demands. SalivaDirect received Emergency Use Authorization from the FDA on August 15th, 2020. Details can be found on our website: covidtrackerct.com/about-salivadirect.