Expanded point-of-care (POC) screening and monitoring options for detection of disease biomarkers from easily collectable specimens such as saliva are needed to reduce the incidence and mortality rates of debilitating diseases and enhance the resilience of the medical system, and improve care. InnoTech Precision Medicine with Massachusetts Institute of Technology is working to develop an innovative diagnostic platform that can be used for POC screening and monitoring of disease biomarkers. In our initial proof-of-concept, we are developing a POC test for detection of high-risk HPV in saliva. HPV is a viral infection that causes most Oropharyngeal squamous cell carcinoma (OPSCC) cases. Recent studies have demonstrated that HPV can be detected in saliva from patients with OPSCC. Currently, the initial screening and diagnosis of OPSCC is done at the late symptomatic stage based on symptoms that are often mild and can mimic less serious conditions such as cold or sore throat, delaying diagnosis and treatment. DNA tests for HPV are most often based on RT-PCR detection of the viral genetic material from serum or saliva that is collected and sent to central laboratories. RT-PCR-based tests rely on the amplification and detection of viral genetic material. These tests have long actionable turn- around-time, and high cost. Thus, there are no rapid, accurate and low-cost tests for routine monitoring of high- risk groups (i.e., young, sextually active, multiple partner) and monitoring of response to therapy and recurrence. Thus, a POC test for non-invasive monitoring of HPV in saliva during well-being exams can have a significant impact and save lives. Our objective is to develop a rapid, easy-to-use prototype for the detection of HPV in saliva based on the combination of biochemical signal amplification with CRISPR-Cas 12 electrochemical readout and our proprietary robust, accurate and low-cost gold-leaf electrodes. Utilizing our proprietary gold-leaf electrode along with a CRISPR-Cas12 assay for sensitive and specific electrochemical detection of target nucleic acid (NA) biomarkers, we have already demonstrated the detection of HPV 18 with plasmid DNA as well as in patient specimen. Our overall objective for this proposal is to develop a reproducible process for processing of saliva and develop a prototype electrochemical biosensor for the multiplex detection of HPV 16 and HPV 18 along with a reference gene to ensure accurate test results. To accomplish this goal, we will first develop a workflow for spatial multiplexing of our gold leaf electrodes for detection of HPV16, HPV 18 and an optimal reference gene. We will then verify this multiplexed test initially with plasmid DNA, and then with saliva specimens. We anticipate that combining easy-to-fabricate spatially multiplexed electrodes with CRISPR- Cas12a for the detection of a specific nucleic acid sequence will enable our Phase II development, regulatory approval and commercialization of a point-of-need technology that can be scaled and manufactured for accurate, easy-to-use and low-cost point-of-need testing and to reduce the burden of HPV infection and OPSCC.
Public Health Relevance Statement: Narrative This NIH SBIR Phase I project will support the development of a sensitive, easy-to-use, low-cost, multiplex POC test for detection of HPV in saliva. Robust strategy for saliva processing and novel methods for specific Cas12- based readouts of multiplex HPV using proprietary gold-leaf electrodes will be developed.
Project Terms: commercialization; high risk; response to therapy; response to treatment; therapeutic response; therapy response; treatment response; effective treatment; effective therapy; bio-markers; biologic marker; biomarker; Biological Markers; product development; manufacturing process development; screenings; screening; CRISPR; CRISPR/Cas system; Clustered Regularly Interspaced Short Palindromic Repeats; precision-based medicine; precision medicine; Sexting; amplification and detection; detection of amplification; amplification detection; diagnostic assay; invention; relapse patients; high risk group; high risk individual; high risk people; high risk population; nucleic acid delivery; point-of-care detection; detection tests; detection test; diagnostic system; diagnostic platform; detection assay; detection sensitivity; point of care testing; manufacture; technology platform; technology system; promote resilience; build resilience; build resiliency; develop resilience; develop resiliency; enhance resilience; enhance resiliency; improve resilience; improve resiliency; increase resilience; increase resiliency; promote resiliency; resilience development; Base Sequence; Nucleotide Sequence; nucleic acid sequence; Biological Assay; Assay; Bioassay; Biologic Assays; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Chemical Engineering; Dentists; Diagnosis; Disease; Disorder; DNA; Deoxyribonucleic Acid; Electrodes; endonuclease; Engineering; Enzymes; Enzyme Gene; Future; Genes; Goals; Gold; Housekeeping; Housework; Incidence; Human Papillomavirus; HPV; Human Papilloma Virus; Infectious Human Wart Virus; wart virus; Laboratories; Massachusetts; Methods; Morbidity - disease rate; Morbidity; mortality; Multiple Partners; United States National Institutes of Health; NIH; National Institutes of Health; Nucleic Acids; Patients; Personal Satisfaction; well-being; wellbeing; Program Development; Reagent; Recurrence; Recurrent; Resources; Research Resources; ROC Curve; ROC Analyses; receiver operating characteristic analyses; receiver operating characteristic curve; Saliva; Sensitivity and Specificity; Signal Transduction; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Technology; Testing; Time; United States; Virus Diseases; Viral Diseases; viral infection; virus infection; virus-induced disease; Work; gRNA; Guide RNA; Caring; Malignant Oropharyngeal Neoplasm; Malignant Oropharyngeal Tumor; Oropharnyx Cancers; Oropharyngeal Cancer; Oropharyngeal Carcinoma; Oropharynx Cancer; Oropharynx Carcinoma; malignant oropharynx tumor; malignant oropharynx neoplasm; improved; Clinical; Phase; Biochemical; Medical; Ensure; Blood Serum; Serum; Sore Throat; leaf; Plant Leaves; Oropharyngeal Epidermoid Carcinoma; oropharynx epidermoid carcinoma; oropharynx squamous cell carcinoma; Oropharyngeal Squamous Cell Carcinoma; Point of Care Technology; Pathologist; Human papilloma virus infection; HPV infection; Human papillomavirus infection; Diagnostic; Specimen; Research Specimen; programs; Genetic Materials; System; Test Result; Viral; Early Diagnosis; early detection; particle; Performance; success; virus genetics; Viral Genetics; Reverse Transcriptase Polymerase Chain Reaction; RT-PCR; RTPCR; reverse transcriptase PCR; Biosensor; biological sensor; novel; DNA amplification; Devices; Sampling; assay development; nucleic acid detection; HPV 16; HPV-16; HPV16; Human papilloma virus type 16; Human papillomavirus type 16; human papilloma virus 16; type 16 Human papilloma virus; type 16 Human papillomavirus; Human papillomavirus 16; HPV 18; HPV-18; HPV18; Human papilloma virus type 18; Human papillomavirus type 18; human papilloma virus 18; Human papillomavirus 18; µfluidic; Microfluidics; Housekeeping Gene; Microfluidic Microchips; Microfluidic Device; Microfluidic Lab-On-A-Chip; microfluidic chip; Symptoms; Data; Detection; Monitoring for Recurrence; Predictive Value; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Validation; validations; Monitor; Preparation; preparations; Development; developmental; point of care; HPV-High Risk; High Risk Oncogenic HPV; High risk HPV; High risk Human Papillomavirus; High risk Human papilloma virus; cost; multiplex detection; virus detection; viral detection; designing; design; process reproducibility; process repeatability; innovate; innovative; innovation; Cancerous; translational medicine; plasmid DNA; noninvasive monitor; non-invasive monitor; prototype
