Behind the Breakthrough: amfAR Fellow Spearheads Major HIV CRISPR Study

In 2021, Dr. Ujjwal Rathore was awarded an amfAR Mathilde Krim Fellowship in Biomedical Research for his promise as an early-career investigator. Now, he is the senior author of a seminal study published in the prestigious scientific journal Cell.

As a doctoral student in India, Dr. Rathore was initially drawn to the complexity of HIV biology and the need for effective and accessible vaccines. But when confronted with the scale of the challenge, namely, the over 40 million people currently living with HIV, he shifted focus.

Seeking to investigate how “host factors”—genes, proteins, and cellular machinery—permit HIV to infect, hijack, and persist, he moved across the world to the University of California, San Francisco, where he could learn and leverage CRISPR gene-editing, technology.

Acting as a kind of precise genetic ‘dimmer switch,’ CRISPR can be used to adjust DNA and ‘dim’ or ‘brighten’ individual genes one at a time. These dimmer switches allow scientists to finely tune gene expression and observe corresponding changes in cellular behavior.

Using CRISPR to systematically ‘turn off’ (dim) or ‘turn up’ (brighten) genes, Dr. Rathore could determine which specific genes impact how T cells respond to HIV infection. His original goal, to identify the genes involved in HIV latency, the silent reservoir of virus that persists hidden in T cells and remains one of the biggest barriers to a cure, was ambitious.

For more than three years, the approach failed. “Resting” T cells, a pool of dormant immune cells that silently retain latent HIV, are not as permissive to gene editing as “active” T cells that are rapidly replicating.

“There wasn’t technology to do large-scale gene editing in resting T cells,” he explained. “If you want to study one gene, you can do it. But if you want to study thousands at once, it just didn’t exist.”

So, he pivoted. Dr. Rathore broadened the study scope to determine how HIV infects T cells generally, including “active” T cells. That shift led to the development of a powerful CRISPR screening process in which Dr. Rathore and his team adjusted each of roughly 20,000 unique genes from human T cells, one by one, to see whether HIV infection was more or less robust in their absence.

The scale of the work was enormous, requiring intensive experiments starting before sunrise and continuing past sundown. “You basically don’t sleep for a few days,” he said.

The perseverance was worthwhile. The team identified genes that code for previously unknown antiviral proteins that naturally help defend cells against HIV. Two stood out in particular: PI16, which blocks HIV from entering cells, and PPID, which prevents HIV from replicating. They determined that targeting these proteins reduces HIV infection of human T cells in the culture dish. These findings could form the basis of new targets for HIV vaccines or therapies.

Beyond the identified proteins, the publication provides a major resource for the field: a powerful new genetic roadmap showing how human T cells interact with HIV. Other researchers can leverage this platform to better understand HIV persistence and latency, and explore new drug targets.

Dr. Rathore credits amfAR’s Krim fellowship for making the work possible. The support came during a difficult period, when many of the initial experiments were failing.

“It allowed me to take more risks,” he said. “The salary support allowed me the academic freedom to focus on difficult problems instead of worrying about timelines.”

Although, the most valuable benefits of the fellowship were not financial. To apply for the fellowship, Dr. Rathore solicited feedback on his ideas from experts in the field and created lasting connections that will advance his career.

“It boosted my confidence, and that actually contributed to the success of everything we did.”

For Dr. Rathore, the work is far from over. Now applying for faculty positions, he aims to leverage the platform to revisit HIV latency and to explore how these discoveries may apply more broadly across a spectrum of human disease, including autoimmunity and cancer.

Dr. Kelsey Hopland is the program officer of amfAR’s research department.

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