A study recently published in Cell Reports describes an efficient, scalable approach that allows for the multiplex editing of HIV host factors and the discovery of gene modifications that confer viral resistance.1
The “Berlin patient” is the only known individual to be cured of HIV thus far, and this was achieved through a stem cell transplant from a donor with a genetically variant CCR5 gene that blocked the virus from invading the cells.2 This case has increased research focused on engineering human immune cells that are devoid of the host factors needed for HIV development. However, such efforts have met various limitations; for example, there are concerns about viral-based delivery such as that used in current trials involving zinc-finger nucleases (ZFN) to delete HIV co-receptors CXR4 and CCR5.
In a 2015 paper, the authors of the present study described a novel method for the direct delivery of Cas9 into primary human CD4+ T cells via electroporation of clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) ribonucleoproteins (RNPs).3 “This transient delivery of editing Cas9 RNPs enables high efficiency ‘knockout’ and ‘knockin’ genome editing and could provide a high-throughput method for therapeutic engineering of HIV-resistant human T cells,” they wrote.
“This approach would have several benefits over the traditional methodologies currently in trial, as it does not rely on viral delivery, does not involve long-term expression off a nucleic acid cassette, and has low rates of off-target editing.”
In the current study, researchers from the University of California, San Francisco applied this approach to the development of a high-throughput platform for arrayed editing of host factors required for HIV infection. Next, they used the platform to assess the impact of knocking out CXCR4 and CCR5 in multiple donors.
They found a reduction in the CXCR4-positive cells from 90% to 30-40%, and the CCR5+ cells decreased by up to 60-80% compared to controls. There were no notable effects on CD4 expression or CD25 induction as a result of targeting either CXCR4 or CCR5, and both types of cells demonstrated HIV resistance in a tropism-dependent manner.
In additional experiments, the researchers used their approach to explore other host factors involved in HIV infection following entry, and they observed that targeting LEDGF or TNPO3 led to decreased infection in a tropism-independent manner. They further determined that the platform has the capacity to edit multiple genes simultaneously and to generate double-knockout cells without compromising the efficiency of the individual Cas9 RNPs.
“We believe that this approach will enable us to rapidly and reproducibly test hypotheses in primary cell models, accelerating the pace of basic and applied scientific inquiry,” said lead author Judd Hultquist, PhD, a postdoctoral scholar at the University of California, San Francisco. “Besides obvious implications for cell-based therapies, this approach should also help researchers readily validate targets and mechanism-of-action models for novel pharmaceutical therapies to not only HIV, but all diseases impacting the T cell compartment,” he told Infectious Disease Advisor.
Next steps in this line of research will focus on targeted editing to not only ablate gene sequences but to modify them. “Many studies have been performed on patient cohorts looking for unique gene signatures that may protect individuals from HIV infection. This technology holds the key to begin engineering these sequences back into human cells to test their protective potential.”
- Hultquist JF, Schumann K, Woo JM, et al. A cas9 ribonucleoprotein platform for functional genetic studies of HIV-host interactions in primary human T cells. Cell Rep. 2016; 17:1-15. doi: 10.1016/j.celrep.2016.09.080.
- Yukl SA, Boritz E, Busch M, et al. Challenges in detecting HIV persistence during potentially curative interventions: a study of the Berlin patient. PLoS Pathog. 2013;9:e1003347. doi: 10.1371/journal.ppat.1003347.
- Schumann K, Lin S, Boyer E, et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc Natl Acad Sci U S A. 2015; 112:10437-10442. doi: 10.1073/pnas.1512503112.