Mic-seq: single cells provirus monitoring to inform on HIV cure strategies
With at least 36.9 million people living with HIV, it is one of the foremost global challenges. Despite the success of antiretroviral therapy, reducing the viral load of infected individuals to undetectable levels, it is not a cure. A fundamental barrier to complete HIV eradication is the lack of an assay that can identify, sort, quantify and characterise the latently-infected cells that comprise the persistent reservoir of infection.
In response, researchers at Oxford University have developed a revolutionary microfluidics-based method for isolating and targeting this very rare cell population, with the capability of retaining RNA, DNA and proteins for downstream analysis. The facility to isolate individual latently infected cells, enables proviral and integration site sequencing from single cells to assess viral replication competency. As well as providing a tool to quantify the HIV reservoir, this novel technique will also allow characterisation of host factors that impact latency and enhance target discovery.
The HIV cure barrier
Currently, there are 36.9 million people worldwide living with HIV. Whilst antiretroviral therapy (ART) can reduce the viral load of infected individuals to undetectable levels, total HIV eradication remains challenging due to dormant integrated copies of the viral genome in host cells. This latent reservoir allows the virus to re-establish infection on stopping ART, and represents the key target to achieve HIV cure.
Quantifying and characterising this reservoir is critical to monitoring new therapies, defining clinical trial end-points and discovering new drug targets. Given the inability of current technologies to isolate and assess this very rare population of cells, there exists a need to address this important research gap.
Single cell microfluidics
By employing microfluidic technologies, Oxford University researchers have developed a revolutionary technique to isolate latent HIV+ cells, and crucially, to retain their associated nucleic acids for downstream analysis. The technique relies upon the generation of water-in-oil droplets (WOs) that encapsulate single cells which are accompanied by PCR reagents and a fluorescent probe that – critical to latency assays – fluoresces only in the presence of HIV proviral DNA, and not RNA.
After PCR, the WOs are either encapsulated in water-in-oil-in water droplets (WOWs) that are amenable to sorting via flow-cytometry (i.e. HIV+ and HIV- populations), or directly sorted on an additional microfluidic device developed by the group. The platform has multiple applications, and in particular, the retention of nucleic acids after lysis, allows for a variety of downstream assessments.
The benefits of this invention include:
- Providing the ability to isolate, sort, and quantify the extremely rare population of latent cells
- Quantification may allow for patient stratification for clinical trials
- To characterise the reservoir from enriched populations may assist in the development of new therapeutics targeting these rare cells
- Combining the sensitivity of PCR based methods with single cell diagnostics and isolation of associated nucleic acids
- The potential to combine the technology with commercial flow cytometry, offers the potential to combine nucleic acid and proteomic signals
- Opens up a new area of research against HIV, with options to explore remission and cure
- The ability to apply this technology to any clinical condition typified by very rare cell populations eg Oncology,Haematology, Rheumatology, Immunology, amongst others.
Oxford University Innovation Ltd. is seeking discussions with companies interested in licencing this technology.
about this technology