By this point my interest in CRISPR research is apparently well known to the people around me: a friend shared a research article with me on Facebook by Kaminski et al. (2016) published earlier this month in the journal Scientific Reports. In this paper the researchers used the CRISPR/Cas9 genome-editing technology to eliminate the human immunodeficiency virus (HIV) genome from infected cells.
HIV is the causative agent of acquired immune deficiency syndrome (AIDS): the virus infects some of the so-called T cells in our immune system and integrates its genetic material into the DNA of the host cell. The infection causes a lot of T cells to die and eventually – over a period of years or often even decades – this makes the immune system less and less efficient. Therefore the causes of death of AIDS patients are usually infections, which would normally be fought off by a healthy immune system, or rare types of tumours. Currently there are efficient retroviral therapies available to treat HIV infection. However, these therapies do not remove the virus DNA from the patient, rather they keep the virus at bay (it is said to be “latent”). Therefore the treatment is usually lifelong and expensive.
Kaminski et al. mainly used a human cell line to test whether they could design guide RNAs that would specifically guide the Cas9 protein to the DNA sequences at either end of the HIV genome. The cell line is called 2D10 and has been well characterised and has a single HIV genome inserted at a known location, making it a good model to test their experimental tools. Since CRISPR is such a ubiquitous tool in the lab already a lot of the paper actually focusses on making sure that cutting out the HIV genome – which they manage successfully – does not have any unintended consequences. In particular, the researchers checked that Cas9 does not introduce mutations elsewhere in the host genome.
Having established these controls Kaminski et al. then go on to show that 2D10 cells with Cas9 and the guide RNAs (gRNAs) are less susceptible to a new HIV infection. To further test their system the researchers used human T cells from healthy individuals to show that these cells can also be made more resistant to infection when given the Cas9/gRNAs. Lastly, the paper shows that the technique can also target HIV DNA in human T cells from infected patients. However, here the Cas9 was not able to entirely excise the HIV genomes. Partly this can be attributed to the fact that human cells are much more heterogeneous than the 2D10 cell line: the virus will have integrated at different sites in the host genome in different T cells and there may also be several integration sites per cell.
This is an impressive study and a good step towards being able to treat patients with HIV using genome-editing technology, but there are still some shortcomings. To me one of the main problems seems to be the way in which the Cas9 and guide RNAs are delivered into the infected T cells: often this is done by putting the DNA that codes for the Cas9 protein and the gRNAs into a lentivirus, which belongs to the same group of viruses as HIV itself. The lentivirus would therefore itself integrate into the genome of the host cell and this might cause problems in itself, for example, by disrupting important genes. Furthermore, and the authors allude to this, not all HIV genomes are exactly the same and so for each patient one might have to design individual gRNAs.
Since we are on the topic of HIV/AIDS I would like to mention something another friend has brought to my attention. Some countries, such as the USA, Canada and France, have programmes to make so-called pre-exposure prophylaxis (PrEP) available to people who are HIV-negative but at high risk of contracting the infection. The National Health Service in England has recently released a statement explaining that it will no longer pursue this avenue although the once-daily pill has been shown to decrease the relative risk of becoming infected by over 90% in men who have sex with men (see, for example, Grant et al., 2010). The National AIDS Trust has therefore started a campaign for people to write to their local MPs so that this issue can be raised in parliament. And I did just that right now and realised that my MP is none other than Jeremy Corbyn.
On a slightly more upbeat note, here is a wallpaper design by Nature for their special CRISPR issue (downloaded directly from their website):
And regarding the CRISPR patent dispute, there was a good News & Views article on the topic a few weeks ago and the take-home message is that it will probably take several years for it to be decided.
Lastly, happy Easter to all those who celebrate it in one way or another. Instead of (chocolate) eggs I will share with you a slightly abstract art image that I inadvertently took on the microscope a couple of months ago. With a little bit of imagination the organoids could be mistaken for Easter eggs.
Grant RM, Lama JR, Anderson PL, McMahan V, Liu AY, Vargas L, Goicochea P, Casapía M, Guanira-Carranza JV, Ramirez-Cardich ME, Montoya-Herrera O, Fernández T, Veloso VG, Buchbinder SP, Chariyalertsak S, Schechter M, Bekker L-G, Mayer KH, Kallás EG, Amico KR, Mulligan K, Bushman LR, Hance RJ, Ganoza C, Defechereux P, Postle B, Wang F, McConnell JJ, Zheng J-H, Lee J, Rooney JF, Jaffe HS, Martinez AI, Burns DN, Glidden DV (2010) Preexposure Chemoprophylaxis for HIV Prevention in Men Who Have Sex with Men. New England Journal of Medicine 363: 2587-2599
Kaminski R, Chen Y, Fischer T, Tedaldi E, Napoli A, Zhang Y, Karn J, Hu W, Khalili K (2016) Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Scientific Reports 6: 22555