Dear RG friends,
I have been looking at the recent failed attempts to remove HIV using CRISPR/Cas9, and I have been thinking about potential ways to solve the current problems with the technique. In its current form, Cas9 will make a nick at the spot targeted by the guide RNA, and the fate of what happens to the nick is left to random chance. In the ideal case, a deletion, frameshift, or insertion occurs and renders the gene non-functional. However, it is also possible for the DNA to stick back together unchanged, a neutral mutation may occur (which now renders the sequence un-targetable by this specific guide RNA), or a mutation could even end up making the HIV more aggressive. [1,2]
I was thinking about ways to overcome this problem as a fun exercise, and I have come up with a potential solution. I wanted to share, get some feedback about the feasibility, and hear some better ideas.
The core of the idea is the following: Modify Cas9 so that, once it identifies the target sequence, it kills the cell – rather than merely making the nicks. Then, provide a cocktail of guide RNAs that target many different sequences from the HIV genome. Kill all infected cells.
There is a protein called “Tyrosyl-DNA-phosphodiesterase”, which is responsible for breaking off the covalent bond that forms between DNA and topoisomerase. The active site of this protein contains two histidines – one histidine nucleophilically attacks the phosphate to break the tyrosine-DNA bond, and the second histidine deprotonates a water molecule so that it can attack the phosphate and displace the TDP. If the histidine responsible for proton abstraction is mutated into an arginine, then the first histidine remains stuck to the DNA. A protein-DNA complex with a lifetime of 13 minutes is formed. Here is an image of the mechanism. [3,4]
The catalytic mechanism of Cas9 is similar to that of TDP. In Cas9, His 840 is responsible for deprotonating the water molecule. [5] It should be possible to generate Cas9-DNAcovalent complexes through a His480Arg mutation.
If it can be experimentally shown that such complexes can be formed, the next question is: will these complexes lead to cell death?
If you look at [4], they mention that cell lines were able to grow even with the TDP mutants present. However, I do think it is possible to send an aggressive enough attack to kill the cells.
The attack would be the following: once we have the modified Cas9 available, we mix it with many guide RNA sequences that target specifically different spots of the HIV genome. The Cas9 should leave healthy cells intact, but in the cells in which the HIV genome is present we should see a lot of DNA-protein complexes forming, which would ideally result in irreparable damage. Since there are so many targets, it is unlikely that enough mutations would accumulate to make the cells immune.
I am not sure about what percentage of white blood cells tends to contain the viral DNA. If the percentage is too high, the treatment could be delivered slowly to apply a small selective pressure against the HIV while providing antivirals to prevent the spread of the virus into more cells, until the population of healthy cells is enough to be able to survive a high dose.
What do you think?
Thank you!
Sources
[1] Chen Liang, Mark A Wainberg, Atze T Das, and Ben Berkhout. 2016. CRISPR/Cas9: a double-edged sword when used to combat HIV infection. Retrovirology 13:37.
[2] Zhen Wang, Qinghua Pan, Patrick Gendron, Weijun Zhu, Fei Guo, Shan Cen, Mark A Wainberg and Chen Liang. 2016. CRISPR/Cas9-derived mutations both inhibit HIV-1 replication and accelerate viral escape. Cell Reports 15:1-9.
[3] Pommier, Yves, et al. "Tyrosyl-DNA-phosphodiesterases (tdp1 and tdp2)." DNA repair 19 (2014): 114-129.
[4] Interthal, Heidrun, et al. "SCAN1 mutant Tdp1 accumulates the enzyme–DNA intermediate and causes camptothecin hypersensitivity." The EMBO journal 24.12 (2005): 2224-2233.
[5] Nishimasu, Hiroshi, et al. "Crystal structure of Cas9 in complex with guide RNA and target DNA." Cell 156.5 (2014): 935-949.
http://i.imgur.com/XgadA6a.jpg
I think that approaches aiming at HIV-induced cell death can be very dangerous and therefore need very careful consideration. HIV can persist in important cells that are long-lived and not easily replaced after death. For example, high proportions of astrocytes in brains of HIV-infected individual were shown to contain HIV genomes. Astrocytes can efficiently restrict productive infection, so that infection is often non-productive or latent [1,2]. Deliberately inducing the death of these HIV-carriers in the brain bears a high risk of harming the patient.
[1] Gray LR, Roche M, Flynn JK, Wesselingh SL, Gorry PR, Churchill MJ. Is the central nervous system a reservoir of HIV-1? Curr Opin HIV AIDS. 2014 Nov;9(6):552-8.
[2] Schneider M, Tigges B, Meggendorfer M, Helfer M, Ziegenhain C, Brack-Werner R. 2015. A new model for post-integration latency in macroglial cells to study HIV-1 reservoirs of the brain. AIDS. 2015 Jun 19;29(10):1147-59.
Furthermore, it has been shown by two different labs that HIV-1 can easily evolve escape mutation against the CRISPR sequence in vitro, which means CRISPR will always play catch-up to the virus (Wang et al., 2016b, Wang et al., 2016a)
WANG, G., ZHAO, N., BERKHOUT, B. & DAS, A. T. 2016a. CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape. Mol Ther, 24, 522-6.
WANG, Z., PAN, Q., GENDRON, P., ZHU, W., GUO, F., CEN, S., WAINBERG, M. A. & LIANG, C. 2016b. CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape. Cell Rep, 15, 481-9.
Difficult to say and judge. It must be examined for many aspects before any therapy judgement!
I would like to suggest review of the following paper that presents a review of, and methods to, address treatment of HIV using existing and novel approaches usind CRISP/Cas.
Article The therapeutic application of CRISPR/Cas9 technologies for HIV
Kind regards
stuart
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