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Principal Investigator: Harsimranjit S Sekhon
Organization: UPSTATE MEDICAL UNIVERSITY
Fiscal Year: 2024
Award: $53,974
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY
CMV disease is the largest cause of post-transplant patient mortality. After a transplant, patients have to
undergo extensive screening and prophylactic therapy in order to prevent reactivation of the virus. In many
cases, even if these precautions are taken, the virus will reactivate after the patients finish antiviral therapy. This
is due to the virus establishing latency in cells of the myeloid lineage. Because the antivirals target and inhibit
virus replication, CMV is very difficult to target in its latent stage due to low levels of replication. This proposal
aims to develop molecular tools that have the potential to be used in clinic to prevent CMV-related mortality, by
combining the fields of protein and DNA engineering.
In the first aim, a CMV detection platform will be developed that combines the techniques of rolling circle
amplification and hybridization chain reaction to then turn on a luminescent protein biosensor that can be
detected with a cell phone camera. This tool will allow identification of active CMV infection with high sensitivity
by performing the one-pot reaction at room temperature with no special equipment for detection other than a cell
phone. Patients will be able to self-screen at home using this low-cost alternative to PCR that does not require
trained personnel or expensive equipment.
In the second aim, DNA self-assembly technique of toehold-dependent strand displacement will be
utilized to trigger a toxic protein switch in response to CMV-specific RNA input. The components will be
introduced into a population of cells that may be latently infected with CMV, and the switch will turn on in response
to a latency-associated CMV RNA that is expressed in target cells. This will cause specific death of CMV-infected
cells. The potential application of this switch is its use in bone marrow transplants—the switch may be introduced
in the cells of donor bone marrow, and any infected cells can be killed before transplantation into the recipient.
Development of DNA/RNA-activated protein switches have been limited to natural cas proteins so far.
There is a strong desire for the discovery of more cas proteins to extend the toolbox of CRISPR. The techniques
developed in this proposal will circumvents that need by “CRISPR-izing” proteins of choice to perform any desired
function based on a nucleic acid input. In aim 1 of this proposal, an RNA-activated luminescent protein switch
will be constructed, and in aim 2, an RNA-activated toxic protein. These findings will give rise to a new class of
proteins to perform functions unattainable by cas proteins in nature.
Terms: <Anti-viral Agents><Anti-viral Therapy><BCAR1><BCAR1 Protein><BCAR1 gene><Bio-Informatics><Bioinformatics><Biological><Bioluminescent Proteins><Biosensor><Biotech><Biotechnology><Blood monocyte><Bone Marrow><Bone Marrow Grafting><Bone Marrow Reticuloendothelial System><Bone Marrow Transplant><Bone Marrow Transplantation><Breast Cancer Anti-Estrogen Resistance 1 Protein><CKRAS protein><CMV><CMV infection><CRISPR><CRISPR/Cas system><CRK-Associated Substrate><CRKAS><Cas protein><Cause of Death><Cell Body><Cell Death><Cell Function><Cell Phone><Cell Physiology><Cell Process><Cells><Cellular Function><Cellular Phone><Cellular Physiology><Cellular Process><Cellular Telephone><Cessation of life><Clinic><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats><Color><Cytomegalic Inclusion Disease><Cytomegalovirus><Cytomegalovirus Infections><DNA><DNA Sequence><DNA amplification><Death><Deoxyribonucleic Acid><Detection><Development><Devices><Diagnostic tests><Disease><Disorder><Elements><Engineering><Equipment><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Genes><Goals><HCMV><Herpesviridae><Herpesviruses><Home><Human Resources><Hybrids><In Vitro><Inclusion Disease><Individual><Induction of Apoptosis><Infection><Low-resource area><Low-resource community><Low-resource environment><Low-resource region><Low-resource setting><Luminescent Proteins><Mammalian Cell><Manpower><Marrow Transplantation><Marrow monocyte><Mediating><Methods><Mobile Phones><Molecular><Monitor><Myelogenous><Myeloid><Nature><Non-Polyadenylated RNA><Nucleic Acids><Oligo><Oligonucleotides><Outcome><Output><Patients><Photoproteins><Plasmids><Population><Prevalence><Process><Protein Engineering><Proteins><Quantitative RTPCR><Quantitative Reverse Transcriptase PCR><RNA><RNA Gene Products><RNA Nucleases><RNase><Rapamune><Rapamycin><Reaction><Research><Resource-constrained area><Resource-constrained community><Resource-constrained environment><Resource-constrained region><Resource-constrained setting><Resource-limited area><Resource-limited community><Resource-limited environment><Resource-limited region><Resource-limited setting><Resource-poor area><Resource-poor community><Resource-poor environment><Resource-poor region><Resource-poor setting><Ribonuclease Family Protein><Ribonucleases><Ribonucleic Acid><Salivary Gland Virus Disease><Salivary Gland Viruses><Sampling><Sirolimus><Special Equipment><Subcellular Process><System><Techniques><Technology><Temperature><Testing><Time><Training><Transplant Recipients><Transplantation><Viral><Virus><Virus Activation><Virus Induction><Virus Replication><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><aptamer><biologic><biological sensor><cell killing><combat><cost><cytomegalovirus group><design><designing><detection limit><detection platform><detection system><developmental><flexibility><flexible><flow cytophotometry><gene signatures><genetic protein engineering><genetic signature><herpes virus><home test><home-based test><homes><iPhone><inhibitor><innovate><innovation><innovative><interest><monocyte><mortality><necrocytosis><novel><oligos><p130 cas protein><p130CAS><personnel><point of care><post-transplant><post-transplantation><posttransplant><posttransplantation><prevent><preventing><prophylactic><protein design><qRTPCR><response><screening><screenings><self assembly><smart phone><smartphone><structural biology><synthetic biology><tool><transplant><transplant patient><viral RNA><viral activation><viral detection><viral induction><viral infectious disease treatment><viral multiplication><viral replication><virus RNA><virus detection><virus multiplication>