January 10

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A Look at CRISPR and the First Genetically-Modified Humans

She's not actually genetically modified. Just a big fan of GMOs.

Cracking Pandora’s Box – New Tools and New Frontiers

In November of 2018, Chinese scientist He Jiankui revealed to the world that he had orchestrated the genetic modification and birth of two twin girls in China[1]. Psuedonymed Nana and Lulu, these newborns are the first known human babies to be born following modification of their genomes with a new, powerful and controversial technology – CRISPR.

Neuwrite has discussed CRISPR previously in a three part series by Caroline Sferrazza in 2017, giving a brief introduction and history as well as exploring the ethical and legal landscape of CRISPR technology.

CRISPR systems are a tool for modifying the genome of an organism, composed simply of a designer-made “guide” RNA and a protein called Cas9. In nature they were discovered in bacteria, where CRISPR/Cas9 function as a sort of immune system that protects bacteria from viral invaders. It was discovered that many bacteria have DNA regions which code for special RNAs that can recognize invading viruses’ genomes. When these CRISPR RNAs recognize an invading virus they will bind to it, and a bound CRISPR RNA tells Cas9 it is time to start cutting up DNA. In bacteria, this is a great way to find and chop up the DNA of a would-be invader, rendering it harmless. In the lab, this same system can be harnessed to target DNA sequences of anything, and with unprecedented specificity[2].  And what this means for the future of genetically modifying humans is proving to be a source of great excitement and uncertainty.

Maybe not true.
An instant classic, id software’s 1993 dungeon crawler DOOM takes players on an interactive tour through He Jiankui’s lab

Before doomsday preparations get too far along, it is important to note that CRISPR’s incredible precision has its functional limitations. To affect the entire body, CRISPR gene modifications must be made before birth – during the short few moments following fertilization of an egg. This is an increasingly common procedure in life science research, permitting the rapid generation of genetically modified laboratory animals and genetic manipulations of practically any cell that can be grown in a dish. Researchers have shown that monkeys modified with CRISPR can be born and lead normal, healthy lives, an important step in demonstrating the efficacy of genetic manipulation on behaviorally complex species closely related to humans[3].   

CRISPR experiments had even been carried out, to some objection, on fertilized human eggs[4-7]. During the normal process of in vitro fertilization (IVF), a prospective mother donates her eggs to a team of physicians and scientists, who will fertilize the eggs in a dish (“in vitro”), then take these healthy samples and implant them back into the mother to induce pregnancy and eventual birth. Researchers showed that while the eggs are growing in a dish, before implantation, they can have their genomes manipulated by CRISPR and continue to replicate in vitro without apparent detriment. But these experiments ended in the dish; fertilized eggs subjected to CRISPR modification were not implanted and thus never initiated a pregnancy. 

Research on genetic modification in developing humans had halted at the point of fertilization in vitro, and what knowledge we have gained modifying bacteria, yeast, cell cultures, or even laboratory animals may not directly translate to human biology. Understanding of the long-term effects of CRISPR modification in non-human primates progresses slowly due to necessary time and resource burdens, while studies on safety and efficacy in human embryos are limited and only be performed by few universities in the world (many countries worldwide have banned or restricted CRISPR treatment of human embryos[7]). So with where our knowledge and control of CRISPR currently stands, the scientific community generally[a] supported a sort of ethical moratorium: Genetically modifying viable human embryos, then implanting them into a woman and carrying to term, is impermissible. He Jiankui and his colleagues broke this moratorium, and in irresponsible, amateurish fashion.

CCR5 – Nana and Lulu

Many biologists and ethicists do argue that the use of CRISPR in humans could be permissible to cure a disease for which there is no alternative treatment. Some diseases could be avoided altogether, especially those with strict, defined genetic causes (like Huntington’s disease). He Jiankui and his team chose a gene that partially fits the above criteria: a specific, small deletion in the CCR5 gene can provide dramatic resistance to infection by HIV/AIDS. And this deletion is a well-studied genetic variant, as it is already present today in many healthy people of European descent who are demonstrably less likely to contract HIV even when exposed[8].

He’s team designed a guide RNA for use with CRISPR/Cas9 that would target the CCR5 gene and introduce a deletion in embryos. Embryos that received the treatment could then have their DNA sequenced to check if the CCR5 gene was successfully modified, and those that were successful could be implanted via in vitro fertilization back into donating mothers.

But alas, things got messy.

The known modification of CCR5 already present in some people (called CCR5Δ32) provides incredible resistance to HIV infection. But it also carries with it an increased risk[a] of dying from the flu, a virus every person is far more likely to encounter than HIV: worldwide there are an average 600,000,000 cases of influenza each year, causing severe illness in 3,000,000 people and resulting in 500,000 deaths[9]. Furthermore, HIV is preventable and even treatable without gene therapy. Aside from (strong but steadily decreasing) social stigma, persons infected with HIV can expect to enjoy long, normal, healthy lives with the medications now readily available in first world countries[10]. Their partners can even expect to avoid contracting the virus themselves without the use of a condom[11] – and condoms already prevent as many as 60-95% of cases when used correctly[12]. It is unclear then if a pressing medical need is met by mutating CCR5, with the introduction of compromised viral immunity and existent, efficacious HIV prevention and treatment tactics[c] that do not require genetic engineering.

In any case, the CRISPR didn’t work as intended.

Taken from his November 28th presentation, the first time he shared his actual data with experts.
He Jiankui earning a B- on his lab report. Professors noted his considerable understanding of CRISPR theory, but were forced to deduct points for wanton moral oversights.

He Jiankui’s team did cause deletion of portions of the CCR5 gene, but not deletions that have been studied before[13]. Humans have two copies of every gene – one from their mother and one from their father – and both copies of CCR5 need to be of the Δ32 variety to provide dramatic HIV resistance. Lulu has one unperturbed copy[d] of CCR5, and thus will have only partial HIV protection if any at all[14]. And of the three actually mutated CCR5 copies – one in Lulu and two in Nana – none have been vetted in animals for potential pitfalls, or even efficacy in providing HIV resistance.

He Jiankui did one of two things: He either failed to screen for a successful CRISPR mutation before implanting the embryos, which is a simple, obvious, and easy procedure for scientists to conduct. Or more baffling still, He did screen the embryos, saw mutations with unknown effects in the embryos, and decided to implant regardless. Either way this experiment was carried out with glaring technical disregard. Lulu and Nana were implanted and carried to term with mutated genes that confer no known benefit to HIV resistance and without empirical demonstration of many real or potential safety considerations.

The birth of these twins has not been independently verified so we are taking He’s word that the twins, who he is understandably (and thankfully) keeping anonymous to the public, have been born. But the scientific community generally believes He’s claims – indeed, upon conclusion of He’s presentation on the twins, one geneticist remarked of the data shown in the image above: “I can believe that he did it because it’s so bad.”[15] He Jiankui is adamant and “proud”[16] that he conducted the work – apparently ignorant to its technical sloppiness and ethical infirmity.

Chocko is my dog.
Another lab, known publicly as Chocko “The Dog” Nelson, is being introduced here to lighten the mood after reading and writing the section above bummed me out. Chocko is not, however, without controversy. Accused by at least one biologist (pictured mid-interrogation bottom right) of being genetically enhanced to produce “a SUPER good boy,” Chocko has responded only with his signature tail wag; arousing further suspicions.

Prevention and Enhancement

Modifying CCR5 was theoretically intended to provide resistance to a potential threat before the twins could even encounter it – a category of treatment known broadly as prevention. This is similar to single or seasonal vaccinations, wearing gloves when handling food, or washing hands after working outside: each serves to stop illness before it even occurs. And as CRISPR technology is further developed – those safety precautions and unintended consequences checked and understood – not only will the moratorium on genetically modifying and implanting embryos likely be lifted, it may arguably become morally necessary to genetically modify embryos and obliterate genetic disease from the human race wherever we are able.[17] In the coming decades we may see the beginning of the end of Huntington’s, sickle cell disease, and cystic fibrosis plaguing our species, expedited by the advent of CRISPR.[18]

Another category of “treatment” draws different criticisms than prevention – the potential enhancement of the human mind and physique. And unlike modern enhancements, like steroids for muscle gain, plastic surgery for cosmetics, or Vitamin C supplements for immune health, enhancements made to embryos’ DNA by CRISPR will be inheritable by future offspring through regular procreation.

Steroids (Hugh Jackman) are mentioned below.
The CRISPR techniques being tested don’t really bring us any closer to superhuman (or supervillain) mutant powers, and are really limited to one or a few small changes at a time. Our set of matching leather full-length body suits will have to stay hanging in the closet for a little while longer.

With the development of CRISPR, there are some difficult questions we will soon need to ask and address, especially with the social implications of choosing certain genes as “desirable.”. Maybe the idea of adding and removing traits from our gene pool reeks of dystopic Brave New Worlds; maybe we stand just to fall like Icarus, fooling with tools outside of our comprehension and control. It is uncomfortable to imagine the biological future of our species could be swayed by arbitrarily popular functional or cosmetic genetic fads. All the while, CRISPR technology is not available everywhere nor to everyone – do we risk even further socioeconomic divide as people with money and access to genetic technologies have advantages literally built into their families?

And as we dive deeper yet into the realm of human cognition, a recent study identified hundreds of gene loci (areas on or near genes) that correlate positively with increased IQ, narrowing the search for genetic determinants of intelligence[19]. But noted in that same study, psychiatric disorders share a high co-occurrence with elevated intelligence – reminiscent of how many of history’s most influential artists and thinkers were characteristically unwell. While a smarter species on a smarter planet sounds like the guarantee of a better future, the potential pitfalls of mucking with genes controlling the brain and its development forebode a dance with Pandora’s box that must be played out with great care.

I prefer Spotify - they give a student discount.
Dancing with a box? Does anyone do that?

Finally, there is one gene worth noting here that has been shown to predispose some immunological deficiencies while providing cognitive enhancement when knocked out in mice – CCR5. [20,21]

The study describing this enhancement did not use the CCR5Δ32 mutant that provides HIV resistance, but instead a CCR5 that was “effectively null,” or did not work at all in its usual role. It is possible (I would venture to say even likely) that the genetic mutations carried by Nana and Lulu will more closely replicate an “effectively null” CCR5 than HIV protection – the type of deletions shown in He Jiankui’s data may very well render their CCR5 genes to be inefficient or completely inactive. While He Jiankui’s experiments did not give rise to CCR5Δ32, intellectual acumen may indeed be engineered into humanity’s first CRISPR’d children.

Scientists, ethicists, and politicians have been thus far limited in creating laws pertaining to ethical CRISPR use. He Jiankui’s experiments will, at the least, provoke stricter oversight for human genetic modification.  With luck it will prompt deeper discussion and an overall greater societal understanding of the ethics of CRISPR use and the future of human genome engineering. In CRISPR we have a burgeoning new technology demanding respect and patience; inspiring in the same breath excitement and intrigue. Wielded with caution and compassion, it is quite likely to enrich the health of our planet and people significantly in years to come.

Footnotes:

[a]  Not everyone agrees. One of the most prominent biologists alive today, George Church, provides somewhat of a dissenting opinion to the overwhelming criticism He Jiankui is receiving in [22]

[b]  This study is statistically significant and the data compiled from human cases of influenza, but it does have a small sample size.

[c]  I am of the opinion that CCR5 does not meet the requirements for beginning CRISPR on human embryos. I also do NOT want to downplay the horrific nature of HIV. But the criteria for CRISPR modification should be held to a very high burden, which the existing treatment for HIV, I think, make HIV no longer a candidate for the first CRISPR modifications on human embryos.
  Worth note – CCR5 is a popular, promising target for modification in adult humans, which has its own set of difficulties and limitations but without many of the ethical quandaries. An article on therapeutic adult CCR5 genetic modifications is given in reference [23]

[d]  It is also possible only some of Lulu’s cells have the one mutation while others have none at all – called genetic mosaicism. From what He Jiankui has shown we cannot conclude either way. Dr. Sean Ryder’s lab twitter discusses this in various tweets in [24]

References:

[1] The He Lab. About Lulu and Nana: Twin Girls Born Healthy After Gene Surgery As Single-Cell Embryos. https://www.youtube.com/watch?v=th0vnOmFltc
[2] Adli M. The CRISPR tool kit for genome editing and beyond. Nat Commun. 2018;9(1):1911. doi:10.1038/s41467-018-04252-2
[3] Shen H. First monkeys with customized mutations born. Nature. January 2014. doi:10.1038/nature.2014.14611
[4] Liang P, Xu Y, Zhang X, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363-372. doi:10.1007/s13238-015-0153-5
[5] Ma H, Marti-Gutierrez N, Park S-W, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017;548(7668):413-419. doi:10.1038/nature23305
[6] Zeng Y, Li J, Li G, et al. Correction of the Marfan Syndrome Pathogenic FBN1 Mutation by Base Editing in Human Cells and Heterozygous Embryos. Mol Ther. 2018;26(11):2631-2637. doi:10.1016/j.ymthe.2018.08.007
[7] Ledford H. Where in the world could the first CRISPR baby be born? Nature. 2015;526(7573):310-311. doi:10.1038/526310a
[8] Novembre J, Galvani AP, Slatkin M. The Geographic Spread of the CCR5 Δ32 HIV-Resistance Allele. Clark A, ed. PLoS Biol. 2005;3(11):e339. doi:10.1371/journal.pbio.0030339
[9] Pozo F, Moreno S, Casas I, et al. CCR5 deficiency predisposes to fatal outcome in influenza virus infection. J Gen Virol. 2015;96(8):2074-2078. doi:10.1099/vir.0.000165
[10] WebMD. AIDS/HIV Treatments: ART (Antiretroviral Therapy) Tests, Side Effects, and More. https://www.webmd.com/hiv-aids/understanding-aids-hiv-treatment#1. Accessed January 8, 2019.
[11] US CDC. Pre-Exposure Prophylaxis (PrEP) | HIV Risk and Prevention | HIV/AIDS | CDC. https://www.cdc.gov/hiv/risk/prep/index.html. Accessed January 10, 2019.
[12] Pinkerton SD, Abramson PR. Effectiveness of condoms in preventing HIV transmission. Soc Sci Med. 1997;44(9):1303-1312. http://www.ncbi.nlm.nih.gov/pubmed/9141163. Accessed January 10, 2019.
[13] He Jiankui; International Summit on Human Genome Editing. 28 Nov 2018 – International Summit on Human Genome Editing – He Jiankui presentation – YouTube. https://www.youtube.com/watch?v=tLZufCrjrN0.
[14] Trecarichi EM, Tumbarello M, de Gaetano Donati K, et al. Partial protective effect of CCR5-Delta 32 heterozygosity in a cohort of heterosexual Italian HIV-1 exposed uninfected individuals. AIDS Res Ther. 2006;3:22. doi:10.1186/1742-6405-3-22
[15] Chen A. Flawed DNA editing of alleged ‘designer babies’ may have put their health at risk – The Verge. https://www.theverge.com/2018/11/29/18116830/crispr-baby-he-jiankui-genetics-ethics-science-health-mutation. Accessed January 3, 2019.
[16] Alex Lash. Xconomy: Under Fire, He Jiankui Says He’s “Proud” to Help Make CRISPR’d Babies. https://xconomy.com/national/2018/11/28/under-fire-he-jiankui-says-hes-proud-to-help-make-crisprd-babies/. Accessed January 8, 2019.
[17] Hesman Saey T. Parents may one day be morally obligated to edit their baby’s genes | Science News. https://www.sciencenews.org/blog/science-public/ethics-gene-editing-babies-crispr. Accessed January 3, 2019.
[18] Brokowski C. CRISPR Ethics: Moral Considerations for Applications of a Powerful Tool. J Mol Biol. 2019;431(1):88-101. doi:10.1016/J.JMB.2018.05.044
[19] Coleman JRI, Bryois J, Gaspar HA, et al. Biological annotation of genetic loci associated with intelligence in a meta-analysis of 87,740 individuals. Mol Psychiatry. March 2018:1. doi:10.1038/s41380-018-0040-6
[20]Zhou M, Greenhill S, Huang S, et al. CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory. Elife. 2016;5. doi:10.7554/eLife.20985 14611
[21] Maung R, Hoefer MM, Sanchez AB, et al. CCR5 knockout prevents neuronal injury and behavioral impairment induced in a transgenic mouse model by a CXCR4-using HIV-1 glycoprotein 120. J Immunol. 2014;193(4):1895-1910. doi:10.4049/jimmunol.1302915
[22] Cohen J. ‘I feel an obligation to be balanced.’ Noted biologist comes to defense of gene editing babies. Science (80- ). November 2018. doi:10.1126/science.aaw2120
[23] Sean Ryder @ryderlab https://twitter.com/RyderLab
[24] Le Page M. CRISPR babies: more details on the experiment that shocked the world | New Scientist. https://www.newscientist.com/article/2186911-crispr-babies-more-details-on-the-experiment-that-shocked-the-world/. Accessed January 3, 2019.
[25] Clayton E, Hysolli E, Smith C. Reddit –  Science Discussion… Let’s discuss the technology and ethics of this development! https://www.reddit.com/r/science/comments/a2obkm/science_discussion_last_week_a_team_of_scientists/
[26] Ihry RJ, Worringer KA, Salick MR, et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat Med. 2018;24(7):939-946. doi:10.1038/s41591-018-0050-6
[27] Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. 2018;24(7):927-930. doi:10.1038/s41591-018-0049-z

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