The major barrier to achieving a functional cure for Human Immunodeficiency Virus (HIV) is persistence of the virus in long-lived, latent reservoirs. Unfortunately, little is known about the genetic determinants and molecular pathways underlying the establishment and maintenance of HIV latency, especially in primary human cells. Recently, we have developed a high-throughput CRISPR-Cas9 platform for gene editing in primary human T cells and have leveraged this technology to identify over 40 novel host factors controlling active HIV replication. In Aim 1, we propose to examine the role of these host factors in HIV latency by extension of this editing technology to resting memory T cells, which constitute a major component of the latent reservoir. These data will be complemented with chemical perturbation of these same genes and pathways in collaboration and consultation with the lab of Alexander Marson (UCSF), a current Gilead collaborator who is leveraging similar technology to explore druggable pathways in HIV latency. In Aim 2, we propose to advance this technology beyond population-level statistics by leveraging single-cell RNA sequencing approaches to directly link specific editing events (i.e., genotypes) to specific defects in HIV replication (i.e., phenotypes) in a heterogenous, edited populations. In collaboration and with the mentorship of Steven Wolinsky (Northwestern University), modified scRNA-Seq beads will be used to capture reads across the CRISPR target site of an edited gene while HIV reads will serve to quantify active HIV transcription. As a proof-of-principle, this technology will first be used to characterize mutagenesis of the HIV co-receptor CXCR4 using several unique guide RNA. This approach will then be used downstream of our latency assay to further characterize the influential genotypes and transcriptional co-correlates of reactivation for the most impactful genes identified above. Overall, these two aims will unite the expertise of two renowned domain experts to support the career of an early-stage investigator and ultimately advance our capability to explore the genetic determinants underlying HIV replication and latency.
|Effective start/end date||9/4/19 → 9/4/21|
- Gilead Sciences, Inc. (Agmt 10/7/19)