CRISPR Variant Provides Next Step in Search for HIV Cure

Researchers have recruited the use of a variant of CRISPR/Cas9 gene-editing technology to identify genetic mutations that cause human immune cells to be resistant to HIV infection.

This high-throughput cell-editing platform allowed researchers to test how well scores of different genetic tweaks could defend immune cells against HIV for a study published in Cell Reports.

Through this new system, researchers are able to quickly modify the genetic code of newly-donated human immune cells, and could potentially provide them with the tools to accelerate the search for an HIV cure.

“This is an ability HIV researchers have wanted for a long time,” said co-lead study author Judd F. Hultquist, PhD. “I hope this will take what seemed like an insurmountable task a year ago and make it something everyone can do.”

Over the years, there has been substantial progress in the management and treatment of HIV; however, a cure for the virus is still out of reach at this time.

Interestingly, there is a group of individuals who are not susceptible to the virus who piqued the interest of scientists. This particular group have immune cells that appear to be naturally-resistant to HIV infection, and researchers hope that one day they can edit HIV patient immune systems to mimic these HIV-resistant individuals.

“There have been lots of efforts to sequence the genomes of resistant people to discover the mutations that make them immune to the virus,” Hultquist said. “But there are many different genes that could be involved: some control the virus’s ability to enter immune cells, others control how the virus tricks cells into expressing its genes. Until now, there was no way to test which of these mutations actually confer resistance in primary human T cells.”

Although T cells are only able to survive outside the body for a couple of weeks, they are resistant to viruses that researchers use in other cell types to deliver DNA instructions about how to build the necessary machinery for CRISPR/Cas9 gene editing.

In a study last year, the study authors were able to successfully use CRISPR for the first time to perform precise DNA sequence replacements in primary human T cells. This was done by prefabricating the CRISPR machinery in test tubes, and then adding them to the freshly donated immune cells.

“It’s incredibly fast,” researcher Kathrin Schumann, PhD. “The desired editing occurs rapidly, and then the cell degrades the CRISPR machinery so it can’t go making changes. That’s really important: otherwise it’s like doing surgery and leaving in the scalpel.”

To improve this technique, researchers created an automated system for high-throughput, parallel editing of T cells. This allowed researchers to mutate different gene candidates in hundreds of thousands of T cells obtained from healthy volunteers. Then the mutant cells were exposed to the HIV virus, followed by the screening through the cells to find which mutations could prevent infection.

Authors noted that the crucial feature of the system is its speed, since the T cells cannot survive outside the body for long.

“If we want to start editing T cells and putting them back into people as a therapy,” said co-senior study author Negan J. Krogan, PhD. “I think this will be the gold standard for how to do that quickly, safely, and efficiently.”

The technique was then employed to mutate the genes CXCR4 and CCR5. These genes encode receptor molecules used by different HIV strains to sneak by and infect immune cells.

They have also been targeted in previous cell therapy trials. When either of these genes were inactivated, it successfully blocked HIV infection in human T cells by the relevant HIV strain, according to the study.

Additional experiments demonstrated that by simultaneously blocking a gene the HIV virus needs to gain cell entry and a gene the virus needs to survive and reproduce in the cell, it creates a 2-layer security system for these T cells.

In order to demonstrate the power and efficiency of the new high-throughput technology, researchers developed 146 different CRISPR-based edits that were each designed to deactivate 1 of 45 genes that are linked to HIV’s ability to integrate into host cells.

There were several absent genes identified that conferred HIV resistance, with some having been predicted in prior studies, and others that had never been directly tied to HIV before.

Researchers stated that they are planning to use the new platform to help identify additional weaknesses in the HIV life cycle that they could use for targeted drugs or cell therapy. Furthermore, they hope to insert subtler mutations that could alter cell function just enough to confer resistance without fully deactivating the gene and impeding cell function.

Overall, researchers are hopeful that the system could be used in the future for more conditions than just HIV.

“This toolkit has been a huge missing piece in infectious disease research,” said senior investigator Alexander Marson, MD, PhD. “Now we have the ability to make modifications in human immune cells and right away see the effects. The potential is immense — this is just the tip of the iceberg.”