CRISPR

HIV Evades Previously Successful Gene Editing Treatment, Causing Disease Resistance

On March 4th, a research team from Temple University successfully removed latent human immunodeficiency virus (HIV) from infected human cells using an emerging gene editing technology called CRISPR-Cas9 (2), a promising treatment that could improve the quality of life for millions (1). By April 19th, a competing team demonstrated that HIV could mutate to escape CRISPR-Cas9. Rather than discrediting Temple University’s findings, the competing research demonstrates process as a core tenet of science and underscores the importance of collaboration.

More than 35 million people in the world are infected with HIV and that number continues to grow (1).  Once HIV enters the body, it targets key immune cells, such as T-cells, and incorporates HIV genetic information into the host genome. At this stage, HIV can remain latent- the viral DNA exists in the host genome but is not being used to produce more HIV (4). In its active form, HIV replicates using the host cell’s own mechanisms and destroys T-cells.

HIV targets a specific subset of T-cells, called T-helper cells (TH cells), that are responsible for fighting infection.  Acquired Immunodeficiency Syndrome (AIDS), HIV’s deadly successor, occurs when HIV has drastically reduced the TH cell population to the point that the body’s immune system is ineffective (5).

Currently, HIV is managed through antiretroviral treatment that prevents viral replication within the body6. This prevents HIV from spreading to other cells and can aid in restoring the TH cell population. While antiretroviral therapy has been effective in prolonging and improving the quality of life, the drugs are expensive and must be taken vigilantly for the remainder of one’s life. Antiretroviral treatment does not eradicate HIV in its latent stage and some strains of HIV have developed resistance to the treatments6. At this point in time, HIV is impossible to cure.

CRISPR offers the unique opportunity to remove HIV in its latent stage from the genome of cells, effectively curing cells of infection and preventing reinfection by the virus. 

The advent of CRISPR-Cas gene editing technology marked a huge advance in modern science.  Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated proteins (Cas) provide immune protection to bacteria, but have recently been harnessed as a potent gene editing technology (3). CRISPR-Cas systems are capable of modifing DNA in cells, including human cells. CRISPR has the potential to edit out genes responsible for detrimental human diseases as well as making genetic modifications that prevent mushrooms from browning on the shelf of a grocery store. The applications of CRISPR are seemingly limitless.

Upon CRISPR’s release, researchers at Temple University were immediately interested in using CRISPR-Cas9 to treat and possibly cure HIV.  The Cas9 protein can be “programmed” to target removal of specific segments of a genome using a genetic template created by researchers (3).

At Temple University, Rafal Kaminski and his team used CRISPR-Cas9 to effectively remove HIV from the genome of human TH cells in a petri dish, the first step to making modifications in a living organism. The CRISPR-Cas9 treatment prevented HIV infection of treated human THcells and inhibited viral replication in peripheral blood cells from HIV+ samples. Kaminski  and his team had not only cured cells of latent HIV infection, they had prevented other cells from being infected by the virus.

The possibility of curing HIV using CRISPR-Cas9 was within reach. However, scientific breakthroughs rarely happen in one fell swoop and it was not long before a different research team found flaws in Rafal Kaminski’s model.

Six weeks later, Zhen Wang of McGill University published evidence that HIV develops evasion resistance to CRISPR-Cas9 technology. The introduction of Cas9, while fostering deletion of HIV in some THcells, caused mutations in others that promoted HIV resistance and increased viral infectivity. These mutations occurred exactly where Cas9 bound to the DNA targeted for removal, indicating that Cas9 is responsible for the mutations. The mutated HIV replicated more quickly than control HIV, increasing the potency of the virus and the potential for infection (2).

Collectively, this research suggests that CRISPR-Cas9 technology requires some fine tuning before it will be successful for treating HIV. Several labs have confirmed the findings of Kaminski, including Wang, but there is no doubt that the CRISPR treatment must undergo revision. Wang suggested that targeting multiple sites within the HIV genome with a variety of templates may prevent the dangerous mutations that currently occur.

A room full of scientists will earnestly declare that science is a process, not simply a collection of facts. Science is driven by the kind of discussion demonstrated by Rafal Kaminski and Zhen Wang; no one is expected to have the answer right on the first try. Wang’s findings do not invalidate CRISPR-Cas use in HIV treatment, but advise caution. As is true with most things in science, harnessing CRISPR-Cas technology to treat HIV will be a process.

 

References

1.     Kaminski, R., Chen, Y., Fischer, T., Tedaldi, E., Napoli, A., Zhang, Y., Karn, J., Hu, W., and Kahili, K. (2016). Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Scientific Reports. 6.

2.     Wang, Z., Pan, Q., Gendron, P., Cen, S., Wainberg, M., and Liang, C. (2016). CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape. Cell Reports. 15. 481-489.

3.     Jink, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J., and Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 333. 816-821.

4.     Douek, D. C., Roederer, M., and Koup, R. (2009). Emerging Concepts in the Immunopathogenesis of AIDS. Annu Rev Med. 60. 471-484

5.     Okoye, A.A., and Picker, L.J. (2013). CD4+ T cell Depletion in HIV Infection: Mechanisms of Immunological Failure. Immunolo Rev. 254. 54-64.

6.   National Institute of Health AIDSinfo. (2016). HIV Treatment: The Basics.

Showdown Between Two Groundbreaking Scientists Determines the Future of Genome Editing Technology

The United States Patent and Trademark Office (USPTO) currently has to resolve a billion dollar patent battle over a genome editing tool called CRISPR/Cas9. Jennifer Doudna of University of California, Berkley and Feng Zhang of the Broad Institute both claimed they made the major discoveries to the CRISPR system. With the potential to edit human genomes and cure genetic diseases at stake, the patent battle over CRISPR has become the center of attention for scientists worldwide.

CRISPR- or clustered regularly interspaced short palindromic repeats- is a genome editing technology that allows biologists and medical researchers to precisely edit parts of the genome along with a protein called Cas9, which acts as molecular scissors to cut DNA at specified locations [1]. Other genetic editing techniques such as transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZNF) have been around since the early 2000s, but the simplicity and efficiency of CRISPR has led it to become the superior genome editing technology.

CRISPR’s genomic editing capability has a wide range of potential functions. One major application is genetically modifying foods and crops; CRISPR can be used to genetically modify the common white button mushroom to gain resistance to browning [2]. Additionally, CRISPR has the potential to cure genetic diseases and be used as a cancer treatment. While much research must be done before this is possible, many scientists think CRISPR can revolutionize therapeutics.

The CRISPR legal battle arose when the Broad Institute paid extra to expedite the patent process, giving them several patents that cover the use of CRISPR in eukaryotic organisms. This would allow them to use the technology in all multicellular organisms, ranging from mice to humans. Dr. Doudna and UC Berkeley filed an earlier patent application, but the Broad Institute argued that the application of CRISPR in eukaryotes was strictly speculation in Dr. Doudna’s work.

While the Broad Institute was the first to receive the patent, lawyers from UC Berkeley argued that Dr. Zhang and the Broad Institute overstated their discoveries in the patent application and never demonstrated or documented such genetic capabilities, specifically the use of tracrRNA- an RNA molecule that activates the DNA cutting segment of the CRISPR/Cas9 system. In addition, UC Berkeley lawyers pointed out that Dr. Zhang omitted several co-inventors from the patent application, which they argue was “deceitful intent”.

Both Dr. Doudna and Dr. Zhang were groundbreaking scientists even before the CRISPR discovery. Dr Doudna was awarded the prestigious Alan T. Waterman Award for her work in RNA crystallization and function while working at Yale University. Comparatively, Feng Zhang was featured in 35 Innovators Under 35 in 2013 for his work with optogenetics- a technique which uses light intensities to control light-sensitive cells- to cure psychiatric diseases in mouse models.

Despite their accolades, the influence of money cannot be overlooked, especially in a case this monumental. A 2015 report from the renowned global market research firm MarketsandMarkets projected the global genome editing market to be worth $3.5B by 2019 [3], a $2.7B increase from 2014. Additionally, the report stated, “By technology, the market is divided into CRISPR, TALEN, ZNF, antisense technology, and other technologies. Of these, CRISPR will account for the largest and the fastest-growing segment of the global genome editing market by 2019.”

Due to the financial potential, many scientists are investing in CRISPR-focused biotechnology companies. Dr. Doudna and Dr. Zhang, along with leading geneticist George Church, cofounded a biotechnology startup called Editas, which plans to use the CRISPR system to create personalized human therapeutics. The patent battle led to Dr. Doudna leaving this company and starting a rival biotech startup called Caribou Biosciences, Inc.

While the financial implications of the CRISPR breakthrough are immense, many people around the world are more concerned about the ethics behind the new technology. Since her CRISPR paper was published, Dr. Doudna has traveled all around the country discussing the issues of human modification with fellow scientists, congressmen and women, and even White House Officials. The major concerns are the implications of editing human eggs, sperm, and embryos.

 The Napa Bioethics Forum, a small consortium of scientists including Dr. Doudna, met in the spring of 2015 and have since urged researchers to avoid using CRISPR in human research until the precision and capabilities of the technology have been further explored [4].

Despite the recommendation by Dr. Doudna and the Napa Bioethics Forum, Chinese researchers led by Junjiu Huang, a genomic engineer at Sun Yat-sen University, have studied the use of CRISPR in nonviable embryos, or embryos that cannot result in live births. Only a fraction of the embryos maintained the genetic manipulation, while others showed incorrect cuts within the DNA[5]. These results point to the serious challenges with CRISPR and demonstrate that more research must be done before it is applied to human therapeutics.

The potential of CRISPR is promising, but no one knows how far the technology will progress. With the potential to make billions of dollars in human therapeutics on the line, the patent battle over CRISPR will only become more heated.

 

Bibliography:

Baltimore, D. et al. (2015). A prudent path forward for genomic engineering and germline gene modification. Science Perspective. http://science.sciencemag.org/content/348/6230/36.full

Begley, S. (2016). Clash of scientific titans: CRISPR hits the courts, with money and prestige at stake. STAT. https://www.statnews.com/2016/03/08/crispr-patent-fight/

Begley, S. (2016). In the CRISPR patent fight, the Broad Institute gains edge in early rulings. STAT. https://www.statnews.com/2016/03/18/crispr-patent-dispute/

Doudna, J. (2015). Genome-editing revolution: My whirlwind year with CRISPR. Nature Comment. http://www.nature.com/news/genome-editing-revolution-my-whirlwind-year-with-crispr-1.19063

Gill C. (2016). Gene-edited mushroom created by Penn State researcher is changing GMO dialogue. Penn State News. http://news.psu.edu/story/405406/2016/04/19/research/gene-edited-mushroom-created-penn-state-researcher-changing-gmo

Jinek, M., et al. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816-820.

Liang, P. et al. (2015). CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & Cell 6. 363-372.

Marketsandmarkets.com (2015). Genome Editing / Genome Engineering Market by Application (Cell Line Engineering, Animal & Plant Genetic Engineering), Technology (CRISPR, Antisense, TALEN, Zinc Finger Nuclease) & End User (Biotechnology & Pharmaceutical, CRO) - Global Forecast to 2019.

Regalado, A. (2015). CRISPR Patent Fight Now A Winner-Take-All Match. MIT Technology Review. https://www.technologyreview.com/s/536736/crispr-patent-fight-now-a-winner-take-all-match/

Servick, K. (2016). Accusation of errors and deception fly in CRISPR patent fight. Science Community.

http://www.sciencemag.org/news/2016/03/accusations-errors-and-deception-fly-crispr-patent-fight

Yeadon, J., (2014). Pros and Cons of ZNFS,TALENS and CRISPR/CAS. The Jackson Laboratory Blog Post.  https://www.jax.org/news-and-insights/jax-blog/2014/march/pros-and-cons-of-znfs-talens-and-crispr-cas