How can we use Gene Editing to protect or cure us from viral infections?
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Manel Lladó SantaeulariaDec 06, 2020
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The gene editing revolution, spearheaded by the easy-to-use and flexible CRISPR/Cas9 system, is changing the paradigm of therapy. The ability to easily and efficiently modify the genes in our cells has created a landscape of possibilities to develop cures for different diseases.
Gene editing approaches have especially focused on inherited diseases and cancer, with very promising results . However, a field that is still in its infancy is gene editing to treat viral infections.
CRISPR/Cas9 is the adaptive immune system of bacteria, which allows them to protect themselves from viruses. Is it possible for us to do something similar? Researchers have shown that this is indeed the case. CRISPR/Cas9 (as well as other endonucleases like TALENs) has been used to generate CD4+ lymphocytes lacking the CCR5 receptor, which avoids infection by the human immunodeficiency virus (HIV) .These lymphocytes could be transplanted to patients to substitute the function of HIV-infected lymphocytes. Similarly, other publications have shown that in vitro targeting of CRISPR to essential viral genes can lead to inactivation of integrated and episomal viral genomes and thus elimination of infections by HIV, herpesviruses, hepatitis B virus and human papilloma virus (HPV) and polyoma JC virus . Some of these therapies have already reached the clinical trial stage .
However, several issues still need to be addressed. Especially for in vivoapproaches, delivery systems to target the cells that are infected by the virus, as well as achieving high editing efficiency, are points or concern . Additionally, the capacity of viruses to mutate could lead to positive selection of mutant viruses not carrying the Cas9 target sites. Last but not least: could we cause chromosomal rearrangements by generating double-strand breaks in viral genomes integrated in different chromosomes?
Some questions that need to be addressed are:
What are the risks of eliminating our natural cell receptors to avoid viral infections through them?
What gene editing designs would be more efficient and safer to target latent infections? Would knock-out be enough or should we aim for targeted deletion of viral sequences?
What are the best ways to deliver the treatment to the infected area? (topic, systemic?)
How can we make sure that we have edited all cells containing the virus?
How can we be sure that this approach is safe? How can we avoid generation of Cas9-resistant viruses and of chromosomal rearrangements?
How can we quantify the benefit generated? Is it enough to justify the risks? Should we limit it to life-threatening conditions?
Would it be ethical to use gene editing to make us immune to other viruses apart from HIV? Should we wait until infections occur before treating? Is it worth the risks doing it directly in tissues, or should we limit it to ex-vivo approaches?
Could we potentially adapt the bacterial immune system to be active in all cells of our organism, generating superhumans with resistance to a library of viruses?
[1]Ernst, M.P.T., et al., Ready for Repair? Gene Editing Enters the Clinic for the Treatment of Human Disease. Mol Ther Methods Clin Dev, 2020. 18: p. 532-557.
[2]Lee, C., CRISPR/Cas9-Based Antiviral Strategy: Current Status and the Potential Challenge. Molecules, 2019. 24(7).
Using Recombinase to achieve targeted HIV excision
Antonio CarusilloDec 27, 2020
A novel help from an old friend!
I wanted to start my first contribution to this interesting topic by going back to a more “vintage” technology compared to Zinc Finger Nucleases (ZFNs), Transcription-Activator Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).
Let me here introduce the Recombinase.
Recombinases are a class of enzyme which mediate genetic recombination.
These enzymes are found in bacteriophages and fungi catalyze DNA exchange reactions between short nucleotide target site sequences specific for each recombinase.
With recombinases is possible to trigger specific excision, insertion, inversion and translocation of DNA sequences.
Source: Wikipedia
Recombinases are mostly known for their application in basic research and we can acknowledge the Cre recombinase and the Flp-FRT Recombinase.
Why am I bringing this up?
Let’s take the example of the HIV virus. Upon infection of its target cells ( mainly the CD4+ T lymphocytes), the virus will stably integrate itself within the genome of the cells. This makes it hard to eradicate the virus from the host.
The most common genetic engineering strategies aim to prevent virus enter in the target cells by abrogating the expression of its main entry point, the CCR5 receptor.
While this can prevent the entry of the virus, it can’t do much about the virus which is already there. In fact, for such approach, the HIV+ patients' hematopoietic stem cells (HSCs) are collected from the bone marrow of the patient, edited via CRISPR/TALENs/ZFNs and infused back to the patients . This approach relies on the expectation that the CD4+ CCR5 negative cells will be shielded from the CCR5+ HIV tropic virus ( we don’t have to forget that HIV virus can also enter – if “forced” too- via the CXCR4 receptor too ) and will repopulate the patient where the cells already infected will be eliminated by the virus.
This on the long run will result in the virus depletion which can’t find a suitable host cell.
However, what is known is that:
CCR5 is not the only entry point of the virus
Transcriptionally silent ( and still able to replicate) virus reservoir can still be present in the organism, for example in resting memory CD4+ T cells . This may result in the rebound of the virus for example at the moment in which it becomes able to infect via the CXCR4 receptor .
For these reasons, a possibility may be to Knock Out both CCR5 and CXCR4 at the same time, a practice known as multiplexing.
However, this approach while possible with CRISPR may be tricky to achieve with ZFNs and TALENs, due to the way it works. Moreover, all the platforms, even though to different extents are poisoned by off-targets. Increasing the numbers of DNA double-stranded breaks performed at the same time can increase on the one hand the chances of off-targets and also the chances of gross chromosomic rearrangements as multiple breakpoints in the genome may recombine together giving for example head to head chromosomic fusions .
This poses concerns to the safety and the overall efficacy of the approach.
At this point, the Recombinase comes in!
If you recall what I said at the beginning, recombinases can be used to achieve excision.
So what if we could excise the virus from the host genome once and for all?
This would “clean” the host cells of the patient from the virus, eradicating also the reservoir which may still infect the patient in a later time point.
This was the question posed by a German group which using direct devolution engineered an HIV-specific recombinase . In short, direct evolution consists of iterative rounds of mutagenesis ( via different means), selection and characterization till the desired feature are achieved.
In fact, the major drawbacks of the Recombinase is that you can’t reprogram them easily as you would do with CRISPR, for example, were just changing the guide RNA you can repurpose the Cas9 to cleave another target.
In this case, you have to engineer the protein itself so that it will recognize that precise nucleotide pattern of the target of interest.
In the case of the HIV therapy, the Long terminal repeats (LTRs) of the virus which are needed for the virus to integrate into the host genome.
By using several databases they identified a 34 base pairs region within the LTR which is common in all the HIV-subtypes (A, B and C). Their starting point was the CRE recombinase, due to the slight similarity between the 34 bp target site and the CRE target, the LoxP sequence. After more than 140 evolution cycles they could engineer the Brec1 recombinase which features the highest specificity and the highest efficacy. Later characterization identified 26 specific mutations to be present at the same time in order to obtain these features.
A gigantic effort!
With this Brec1 they could show that it was possible to target and excise with success the HIV-pro virus ( the integrated one) from HIV+ patient cells both in vitro and in a mice models. Under the safety profile, no adverse effects were observed.
Contrary to a CRISPR ( and previous similar technologies ) such approach:
can eradicate the virus, instead of preventing its entry
no off-targets were detected due to the high specificity of the engineering of the protein
it doesn’t trigger a DNA repair response which in some papers has been linked to p53 pathway activation and toxic responses like apoptosis
to date, no “anti-recombinase” immune response ( like for CRISPR) has been reported
Of course, the main drawback is that engineering a target-specific Recombinase, to date, is still a massive effort. Which probably is worth taking for cases like HIV therapy due to the number of patients across the globe ( 38 million to date!).
Is this approach feasible?
It is! In fact, the same group who developed the Brec1, under the umbrella of the German Provirex Company it is now completing the recruitment and the first tests in 8 HIV patients.
It will be interesting to see the response of the patient to the therapy!
So far I could not get my hands on their protocol, in their paper the Brec1 was delivered via a lentiviral vector to the cells of the mice models so to consistently express the Brec1. This may be an option as without the target sequence the Brec1 won’t do anything to the organism. It may be also üackaged in a no integrating adeno-associated virus (AAV) as it is small enough to fit into, but it this case the protection won’t be “permanent” but you should administrate the Brec1 each time you suspect to have been in contact with an HIV infected patient.
Nevertheless, such an approach is amazing and promising!
On this line other therapies may be devised, where off-targets can be a problem and targeting the virus reservoir a challenge.
[1]Falkenhagen A, Joshi S. Genetic Strategies for HIV Treatment and Prevention. Mol Ther Nucleic Acids. 2018;13:514-533. doi:10.1016/j.omtn.2018.09.018
[2]Alkhatib G. The biology of CCR5 and CXCR4. Curr Opin HIV AIDS. 2009;4(2):96-103. doi:10.1097/COH.0b013e328324bbec
[3]Battistini, A. & Sgarbanti, M. HIV-1 latency: an update of molecular mechanisms and therapeutic strategies. Viruses 6, 1715–1758 (2014).
[4]Verheyen J, Thielen A, Lübke N, Dirks M, Widera M, Dittmer U, Kordelas L, Däumer M, de Jong DCM, Wensing AMJ, Kaiser R, Nijhuis M, Esser S. Rapid Rebound of a Preexisting CXCR4-tropic Human Immunodeficiency Virus Variant After Allogeneic Transplantation With CCR5 Δ32 Homozygous Stem Cells. Clin Infect Dis. 2019 Feb 1;68(4):684-687. doi: 10.1093/cid/ciy565. PMID: 30020413.
[5]Yu S, Yao Y, Xiao H, Li J, Liu Q, Yang Y, Adah D, Lu J, Zhao S, Qin L, Chen X. Simultaneous Knockout of CXCR4 and CCR5 Genes in CD4+ T Cells via CRISPR/Cas9 Confers Resistance to Both X4- and R5-Tropic Human Immunodeficiency Virus Type 1 Infection. Hum Gene Ther. 2018 Jan;29(1):51-67. doi: 10.1089/hum.2017.032. Epub 2017 Jun 9. PMID: 28599597.
[6]Lekomtsev, S., Aligianni, S., Lapao, A. et al. Efficient generation and reversion of chromosomal translocations using CRISPR/Cas technology. BMC Genomics 17, 739 (2016). https://doi.org/10.1186/s12864-016-3084-5
[7]Karpinski, J., Hauber, I., Chemnitz, J. et al. Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nat Biotechnol 34, 401–409 (2016). https://doi.org/10.1038/nbt.3467
Hi Antonio! Great contribution! I find recombinases to be a very interesting and often forgotten tool, although as you mentioned, design is not easy and a lot of protein evolution efforts may be needed to target them to a particular sequence. I was wondering wether it could be possible to exploit Cas9 RNA-based specificity and design flexibility together with the specific recombination ability of recombinases. And this is indeed possible, and was actually done all the way back in 2016 , although with low efficiency. However, it was reported last year that RNA-Guided Recombinase-Cas9 fusion proteins can target genomic DNA achieving both deletions and integrations. This was achieved by targeting the recombinase-Cas9 to two target sites with PAM-distal orientations (meaning the first PAM is in the 5' strand and the second one in the 3') and having the two hyperactive recombinase domains be in the middle so they can recognize each other and excise the DNA fragment between the two targeted sites. [1]
Although this kind of strategy is still in relatively early development, I'm sure if they achieve high efficiencies we will definitely see them being applied to viral infections.
1. Standage-Beier K, Brookhouser N, Balachandran P, Zhang Q, Brafman DA, Wang X. RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and Integration. CRISPR J. 2019 Aug;2(4):209-222. doi: 10.1089/crispr.2019.0013. PMID: 31436506; PMCID: PMC6707420.
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Multiplex targeting of highly conserved regions to minimize mutational escape
Manel Lladó SantaeulariaOct 20, 2021
One of the main limitations of gene editing against viruses is the capability of viruses to mutate and thus change the Cas9-targeted region, thus being able to escape cleavage by Cas9. This is an important risk because it could render any targeting approach useless and even lead to the development of stronger, more dangerous viruses.
A research group suggested that targeting highly conserved regions in the viral genome could lead to reduced mutational escape . This is because highly conserved regions are conserved for a reason, which is normally the fact that they code for essential parts of proteins, or have a crucial structural of regulatory function. In those cases, small variations can greatly affect the function of those genomic regions, which is why they remain highly conserved. Targeting these regions does not only reduce the risk of mutational escape, but could also be more efficient at eliminating the virus from the cells or render it unable to operate.
An additional method proposed by the same authors is the multiplex targeting of different regions of the virus at the same time. More cuts in the genome mean a lower possibility that all cuts are "escaped", but raise the question of whether that will generate too much cytotoxicity, as well as potential genomic rearrangements leading to potential new virus genomes. Additionally, the phenom of chromothrypsis, which could be related to several double-strand breaks in the same genomic region, also should be considered.
Future research will establish whether this approach is the most desirable one, or whether other approaches like targeted deletion of the viral genome or Cas9-mediated repression of viral protein expression present more advantages.
Manel Lladó Santaeularia Dec 06, 2020
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