Nolan: Technology Development for HIV Reservoir Characterization
HIV is able to remain a chronic, life-long infection due to its ability to stay hidden within infected T cells. These cellular “reservoirs” maintain at least one copy of integrated, non-expressed HIV, and consequently remain invisible to our body’s immune defenses, and are insensitive to anti-HIV drugs. Unfortunately, in vitro experi- ments to activate such latently infected cells suggest that such activation is not 100% effective, and therefore would be ineffective as a component of an HIV cure strategy. Since hidden integrants are found at a frequency of 1 in 104 to 105, technologies must be developed to enable the total characterization of multiple protein and mRNA states in such rare cells. To date no technology exists which can approach the problem suf ciently well to claim the characterization levels and sensitivity required that would provide a solution.
The technology proposed to be developed and adapted towards the “rare cell” problem with HIV matches the
total – ‘omic solution of characterizing multiple protein epitopes and RNAs in extremely rare cells from large cell populations. We have created a technology that rapidly attaches DNA barcodes any protein or nucleic acid event, with cell-speci c barcodes for 100’s of thousands to millions of cells at a time. The technique can be completed in a 12-hour block of time (target a single 5-6 hour timeframe), hundreds of times faster than any commercial or published technology for such a scale of cellular barcoding. Adaptations of the technology have been designed that would enable rare events (such as HIV integration) to be used to lter for “cell barcodes”. Once a cell barcode has been found that is linked to such a rare event, the cell barcode can then be used to reach back into the original material to nd any other proteins, transcripts or genomic features that might be linked to that cell barcode. The investment would enable researchers to de ne the rare event at any level of complexity (single events or multiple co-existent events) in a exible manner, and then use that de nition to mine a cell population for all other events related to the cell barcode(s) that match the original de nition. Various adaptations enable the process to be carried out in an iterative process, on the same original sample, as further de nition of the problem re nes the results.
This research effort is led by Garry Nolan, PhD (Chair SAB Apprise Bio Inc., and Department of Microbiology and Immunology - Stanford School of Medicine). The award was made in November, 2014, with an original agreement length of 2 years.
1. To identify and quantify the frequency of rare cells containing HIV integrants
2. To simultaneously be able to further characterize those cells with respect to associated DNA, mRNA, or coincident protein features