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immunology

Enlisting CRISPR in the Quest for an HIV Cure

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Today, thanks to remarkable advances in antiretroviral drugs, most people with the human immunodeficiency virus (HIV) can expect to live an almost normal lifespan. But that means staying on medications for life. If those are stopped, HIV comes roaring back in just weeks. Finding a permanent cure for HIV infection, where the virus is completely and permanently eliminated from the body, has proven much tougher. So, I’m encouraged by recent work that shows it may be possible to eliminate HIV in a mouse model, and perhaps—with continued progress—someday we will actually cure HIV in humans.

This innovative approach relies on a one-two punch: drugs and genetic editing. First, HIV-infected mice received an experimental, long-acting form of antiretroviral therapy (ART) that suppresses viral replication. This step cleared the active HIV infection. But more was needed because HIV can “hide” by inserting its DNA into its host’s chromosomes—lying dormant until conditions are right for viral replication. To get at this infectious reservoir, researchers infused the mice with a gene-editing system designed to snip out any HIV DNA still lurking in the genomes of their spleen, bone marrow, lymph nodes, and other cells. The result? Researchers detected no signs of HIV in more than one-third of mice that received the combination treatment.

The new study in Nature Communications is the product of a collaboration between the NIH-funded labs of Howard Gendelman, University of Nebraska Medical Center, Omaha, and Kamel Khalili, Temple University, Philadelphia [1]. A virologist by training, Khalili years ago realized that HIV’s ability to integrate into the genomes of its host’s cells meant that the disease couldn’t be thought of only as a typical viral infection. It had a genetic component too, suggesting that an HIV cure might require a genetic answer.

At the time, however, the tools to remove HIV DNA from human cells without harming the human genome weren’t available. That’s changed in recent years with the discovery and subsequent development of a very precise gene-editing tool known as CRISPR/Cas9.

CRISPR/Cas9 editing systems rely on a sequence-specific guide RNA to direct a scissor-like, bacterial enzyme (Cas9) to just the right spot in the genome, where it can be used to cut out, replace, or repair disease-causing mutations. Efforts are underway to apply CRISPR/Cas9 to the treatment of sickle cell disease, muscular dystrophy, and more.

Could CRISPR/Cas9 also remove HIV DNA from infected cells and eliminate the infection for good? Such an approach might be particularly helpful for people on ART who have persistent HIV DNA in the cells of their cerebrospinal fluid. A recent NIH-funded study in Journal of Clinical Investigation found that an association between this HIV reservoir and neurocognitive difficulties [2]

Earlier work by Khalili’s team showed that CRISPR could indeed remove HIV DNA from the genomes of host cells [3]. The problem was that, when delivered on its own, CRISPR couldn’t snip out every last bit of viral DNA from all cells as needed to get rid of HIV completely and permanently. It was crucial to reduce the burden of HIV genomes to the lowest possible level.

Meanwhile, Gendelman’s lab had been working to develop a new and more effective way to deliver ART. Often delivered in combinations, standard ART drugs are effective in suppressing HIV replication. However, people need to take their oral medications daily without fail. Also, most ART triple therapy drugs are water soluble, which means its cocktail of medications are swiftly processed and excreted by the body without reaching many places in the body where HIV hides.

In his quest to make ART work more effectively with fewer doses, Gendelman’s team altered the chemical composition of antiretroviral medicines, generating fat-soluble drug nanocrystals. The nanocrystals were then packaged into nanoparticles and delivered by intramuscular injection. The new drug formulation, known as long-acting slow-effective release (LASER) ART, reaches lymph nodes, spleen, gut, and brain tissues where HIV lurks [4]. Once there, it’s stored and released slowly over time. Still, like conventional ART, LASER ART can never completely cure HIV.

So, Gendelman teamed up with Khalili to ask: What would happen if LASER ART was followed by a round of CRISPR/Cas9? In a series of studies, the researchers tested LASER ART and CRISPR/Cas9, both alone and in combination. A total of 23 HIV-infected mice engineered to have some “humanized” immune features received the experimental combination therapy.

As expected, neither LASER ART nor CRISPR/Cas9 by itself proved sufficient to eradicate HIV in the mice. However, when LASER ART and CRISPR/Cas9 were delivered sequentially, the results were much different. Researchers found no evidence of HIV in the spleens or other tissues of more than one-third of the sequentially treated animals.

It’s important to note that this gene-editing approach to eradicating HIV is being applied to non-reproductive cells (somatic). The NIH does not support the use of gene-editing technologies in human embryos (germline) [5].

Of course, mice, even with humanized immune systems, are not humans. More research is needed to replicate these findings and to figure out how to make this approach to HIV treatment more effective in animal models before we can consider moving into human clinical trials. Still, these findings do provide a new reason for increased hope that an actual cure may ultimately be found for the tens of millions of people in the United States and around the globe now living with HIV.

References:

[1] Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice. Dash PK, Kaminski R, Bella R, Su H, Mathews S, Ahooyi TM, Chen C, Mancuso P, Sariyer R, Ferrante P, Donadoni M, Robinson JA, Sillman B, Lin Z, Hilaire JR, Banoub M, Elango M, Gautam N, Mosley RL, Poluektova LY, McMillan J, Bade AN, Gorantla S, Sariyer IK, Burdo TH, Young WB, Amini S, Gordon J, Jacobson JM, Edagwa B, Khalili K, Gendelman HE. Nat Commun. 2019 Jul 2;10(1):2753.

[2] Spudich S et al. Persistent HIV-infected Cells in Cerebrospinal Fluid are Associated with Poorer Neurocognitive Performance. J Clin Invest. 2019. DOI: 10.1172/JCI127413 (2019).

[3] In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models. Yin C, Zhang T, Qu X, Zhang Y, Putatunda R, Xiao X, Li F, Xiao W, Zhao H, Dai S, Qin X, Mo X, Young WB, Khalili K, Hu W. Mol Ther. 2017 May 3;25(5):1168-1186.

[4] Creation of a nanoformulated cabotegravir prodrug with improved antiretroviral profiles. Zhou T, Su H, Dash P, Lin Z, Dyavar Shetty BL, Kocher T, Szlachetka A, Lamberty B, Fox HS, Poluektova L, Gorantla S, McMillan J, Gautam N, Mosley RL, Alnouti Y, Edagwa B, Gendelman HE. Biomaterials. 2018 Jan;151:53-65.

[5] Statement on Claim of First Gene-Edited Babies by Chinese Researcher. The NIH Director, NIH. 2018 November 28.

Links:

HIV/AIDS (National Institute of Allergy and Infectious Diseases/NIH)

HIV Treatment: The Basics (U.S. Department of Health and Human Services)

Fast Facts (HIV.gov)

Global Statistics (HIV.gov)

Kamel Khalili (Temple University, Philadelphia, PA)

Howard Gendelman (University of Nebraska Medical Center, Omaha)

NIH Support: National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; National Institute of Allergy and Infectious Diseases; National Institute on Aging; National Institute on Drug Abuse; Common Fund


Thoughts from the Front Lines of Rare Disease Research

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Harper Spero and Alexandra Freeman
Harper Spero with physician-researcher Alexandra Freeman, who helps lead the Job’s syndrome research team at the NIH Clinical Center. Courtesy of Harper Spero.

There are nearly 7,000 rare diseases, some of which affect just a few dozen people. Yet, if one considers all these conditions together, about 30 million people in the United States have rare diseases. On this Rare Disease Day, I’d like to challenge each of you to think about how we can raise the visibility of individuals living with rare diseases, as well as the researchers working hard to help them.

I’d like to introduce you to Harper Spero, who is using her rare gift of storytelling to share the experiences of people with a wide variety of conditions that she likes to call “invisible illnesses.” Through her podcast series, called Made Visible, this 34-year-old New York City native is among the many people helping to spread the word that rare diseases are not rare.

Spero knows what it’s like to live with a rare disease. Shortly after she was born, it became clear that she was unusually prone to infections. But doctors had a hard time figuring out what exactly was wrong with this little girl. Finally, at the age of 10, Spero was diagnosed with Hyper-Immunoglobulin E Syndrome (HIES), also known as Job’s syndrome. There currently is no cure for this rare genetic disease, which impairs the immune system and affects multiple parts of the body. But Spero is determined to live a normal life despite her chronic “invisible illness.”

Spero also knows what it’s like to take part in biomedical research. Seven years ago, she came to the NIH Clinical Center here in Bethesda, MD, seeking help for a large cyst in her right lung. It marked the beginning of a positive partnership with a Job’s syndrome research team led by two of NIH’s many dedicated physician-scientists, Alexandra Freeman and Steven Holland. Not only did the NIH researchers work with Spero to figure out the best ways of managing her symptoms, they are using what they’ve learned from her and about 175 other Job’s syndrome patients to develop approaches for earlier diagnosis and interventions. Spero, who visits the Clinical Center annually and communicates with the NIH team on a weekly basis, has been so inspired by the experience that she even chose to feature Dr. Freeman in one of her recent podcasts.

Unlike Spero, I don’t have a podcast—at least not yet. But I do have a blog, and Spero was kind enough to respond to a few of my questions on rare diseases and medical research. So, I’m sharing her thoughts below—I hope you are inspired by them as much as I was!

Why do you feel it is important for people with rare diseases to take part in medical research?

Without research, we can’t make any improvements, changes or find cures. Participating in medical research allows researchers and doctors to learn about the trends (or lack of) between patients, and determine what’s working and what’s not.

What have your own experiences been with the health-care system and medical research?

When I was younger, I really didn’t want to be a specimen. I was going through so much trying to find answers and treatments for myself that it was hard to think about how it would help other patients down the road to be sharing my experiences. I didn’t want to add another doctor’s visit to my schedule. After coming to NIH in 2012, I recognized the importance of being part of the research because it could essentially help me, other patients and for early detection of rare diseases. I recognize that the medical researchers are often much more compassionate than many doctors who simply treat symptoms. Researchers are curious and genuinely care to understand you and your story.

Your podcast is fantastic. How has it affected you to hear and share the stories of so many people affected by rare diseases?

I was definitely aware how many people were living with rare diseases, but I was surprised by how many people were willing to share their stories on my show and how many people wanted to listen to these stories. I hadn’t heard stories being shared in this way around this topic and I wanted to be the one who brought them to life. Many of my guests haven’t publicly (let alone with friends or family) shared their stories so I’m honored that they’re willing to do it with me. They see how important it is to have these conversations and to educate people on what it’s like to have an invisible illness.

What would you tell someone who’s just learned he or she has a rare disease?

You don’t have to do this alone! Find a team of medical professionals you trust to support you. I spent most of my life without a team of doctors that I loved and truly understood me, and now I can’t imagine my life without my team at NIH. Also, talk to your loved ones—let them know what you’re feeling and discuss how they can support you. This is likely new for them too and there’s no right way of navigating and managing a rare disease.

What would you tell a young person who’s considering becoming a rare disease researcher?

Thank you for your interest in doing this! We need more compassionate, curious and passionate people doing this work and investing their time to learn more and help find answers for rare diseases. Please treat us with respect and care.

If you could change one thing in the medical care/research of rare disease, what would it be? And what about in society in general?

There’s a way to do your job without treating patients like guinea pigs. We’re humans too, and we’re humans who have likely been through the wringer in the medical world. Be kind to us. Treat us the way you’d like to be treated. Compassion seems to be a word I’m using a lot. I think society can be more compassionate towards one another especially around rare disease. You can never fully understand what someone is going through so ask questions, show you care and treat people with kindness.

What are your hopes for the future?

I’d love there to be more answers and solutions for navigating a rare disease. A lot of the treatments I do are based on trial-and-error. What works for one patient definitely doesn’t always work for me. So, we’re constantly trying to navigate what works best for me. I’d love to see a cure to be found for Hyper IgE/Job’s Syndrome, as well as other rare diseases.

Links:

Podcast Series: Made Visible

NIH Patient Shares Stories of ‘Invisible Illness,The NIH Record, February 8, 2019

Hyper-Immunoglobulin E Syndrome (HIES) (National Institute of Allergy and Infectious Diseases/NIH)

Rare Disease Day at NIH 2019 (National Center for Advancing Translational Sciences/NIH)

Rare Diseases Are Not Rare! Challenge Offers New Tools to Raise Awareness. January 2019 (NCATS)

Video: Rare Disease Patient Profiles (NCATS)

Genetic and Rare Diseases Information Center (NCATS)

Undiagnosed Diseases Network (Common Fund/NIH)

Video: One in a Million (Undiagnosed Diseases Network, University of Utah Health, Salt Lake City)


Fighting Cancer with Natural Killer Cells

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GIF of immune cells attacking

Credit: Michele Ardolino, University of Ottawa, and Brian Weist, Gilead Sciences, Foster City, CA

Cancer immunotherapies, which enlist a patient’s own immune system to attack and shrink developing tumors, have come a long way in recent years, leading in some instances to dramatic cures of widely disseminated cancers. But, as this video highlights, new insights from immunology are still being revealed that may provide even greater therapeutic potential.

Our immune system comes equipped with all kinds of specialized cells, including the infection-controlling Natural Killer (NK) cells. The video shows an army of NK cells (green) attacking a tumor in a mouse (blood vessels, blue) treated with a well-established type of cancer immunotherapy known as a checkpoint inhibitor. What makes the video so interesting is that researchers didn’t think checkpoint inhibitors could activate NK cells.


Snapshots of Life: Finding Where HIV Hides

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HIV

Credit: Nadia Roan, University of California, San Francisco

Researchers have learned a tremendous amount about how the human immunodeficiency virus (HIV),  which causes AIDS, infects immune cells. Much of that information comes from studying immune cells in the bloodstream of HIV-positive people. Less detailed is the picture of how HIV interacts with immune cells inside the lymph nodes, where the virus can hide.

In this image of lymph tissue taken from the neck of a person with uncontrolled HIV infection, you can see areas where HIV is replicating (red) amid a sea of immune cells (blue dots). Areas of greatest HIV replication are associated with a high density of a subtype of human CD4 T-cells (yellow circles) that have been found to be especially susceptible to HIV infection.


Sharing a Story of Hope

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Whether by snail mail, email, or social media, it’s the time of year for catching up with family and friends. As NIH Director, I’m also fortunate to hear from some of the amazing people who’ve been helped by NIH research. Among the greetings to arrive in my inbox this holiday season is this incredible video from a 15-year-old named Aaron, who is fortunate enough to count two states—Alabama and Colorado—as his home.

As a young boy, Aaron was naturally athletic, speeding around the baseball diamond and competing on the ski slopes in freestyle mogul. But around the age of 10, Aaron noticed something strange. He couldn’t move as fast as usual. Aaron pushed himself to get back up to speed, but his muscles grew progressively weaker.


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