Targeting Conserved Sequences Circumvents the Evolution of Resistance in a Viral Gene Drive against Human Cytomegalovirus
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Evaluation Summary:
This paper will be of interest to experimental virologists and others following the development of "gene drive" technology to promote the rapid spread of specific mutations through a population. The authors first nicely confirm their prior finding that a gene drive virus can be used to transfer mutations into a normal virus when both are infecting the same cell. They then evaluate a strategy with potential to ameliorate the undesirable but expected emergence of viruses that acquire resistance to the gene transfer. Although the experiments are well done, the data are mostly convincing and accurately interpreted, and the presentation is clear, the studies provide a relatively minor advance in fundamental understanding of this potentially innovative therapy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The use of defective viruses that interfere with the replication of their infectious parent after coinfecting the same cells—a therapeutic strategy known as viral interference—has recently generated a lot of interest. The CRISPR-based system that we recently reported for herpesviruses represents a novel interfering strategy that causes the conversion of wild-type viruses into new recombinant viruses and drives the native viral population to extinction.
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Evaluation Summary:
This paper will be of interest to experimental virologists and others following the development of "gene drive" technology to promote the rapid spread of specific mutations through a population. The authors first nicely confirm their prior finding that a gene drive virus can be used to transfer mutations into a normal virus when both are infecting the same cell. They then evaluate a strategy with potential to ameliorate the undesirable but expected emergence of viruses that acquire resistance to the gene transfer. Although the experiments are well done, the data are mostly convincing and accurately interpreted, and the presentation is clear, the studies provide a relatively minor advance in fundamental understanding of this potentially innovative therapy.
(This preprint has been reviewed by eLife. We include the …
Evaluation Summary:
This paper will be of interest to experimental virologists and others following the development of "gene drive" technology to promote the rapid spread of specific mutations through a population. The authors first nicely confirm their prior finding that a gene drive virus can be used to transfer mutations into a normal virus when both are infecting the same cell. They then evaluate a strategy with potential to ameliorate the undesirable but expected emergence of viruses that acquire resistance to the gene transfer. Although the experiments are well done, the data are mostly convincing and accurately interpreted, and the presentation is clear, the studies provide a relatively minor advance in fundamental understanding of this potentially innovative therapy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Gene drive is a process by which variant genes spread through a population at a higher rate than is typical for normal inheritance patterns. There is considerable current interest in applying CRISPR technology to achieve gene drive in sexually-reproducing organisms. In this paper, Walter et al. report their studies of gene drive applied to the very different setting of infections by human cytomegalovirus (HCMV), a medically important cause of disease and death in congenitally infected newborns and in patients with weakened immune systems. The long-term goal of this work is to develop gene drive technology for use in treating viral infections.This research builds on a recent publication in which these authors demonstrated that a gene drive cassette inserted into HCMV can promote specific recombination …
Reviewer #1 (Public Review):
Gene drive is a process by which variant genes spread through a population at a higher rate than is typical for normal inheritance patterns. There is considerable current interest in applying CRISPR technology to achieve gene drive in sexually-reproducing organisms. In this paper, Walter et al. report their studies of gene drive applied to the very different setting of infections by human cytomegalovirus (HCMV), a medically important cause of disease and death in congenitally infected newborns and in patients with weakened immune systems. The long-term goal of this work is to develop gene drive technology for use in treating viral infections.This research builds on a recent publication in which these authors demonstrated that a gene drive cassette inserted into HCMV can promote specific recombination from this "gene drive virus" into another, recipient normal virus that is present and replicating in the same co-infected cell. The components of the gene drive cassette result in cutting of the normal virus genome at a specific site which is then repaired either by transfer of the gene drive cassette into that site, creating a new, "recombinant gene drive virus" or by mutations of the site that creates a "drive-resistant virus." A common obstacle in this kind of method is that it tends to select gene drive-resistant mutants over time. In this case, the data show nicely that the drive-resistant mutants have mutations at the expected site and thus are immune to the further recombination events and have the potential to replicate and cause disease. The authors test whether designing the guide RNA to target a presumably critical site within a viral gene that is needed for efficient viral replication will lessen the chances that a drive-resistant mutant virus will be able to replicate efficiently.
In the first part of the paper, the authors confirm and extend their prior work. Their mathematical modeling predicts that viruses resistant to gene drive will be selected and come to predominate over time. They test this idea using a gene drive system targeting the same non-essential virus gene, UL23 as they used previously. The results (Fig. 2) nicely confirm their prediction and show that even starting with only a small fraction of the gene drive virus results in spread of the gene drive cassette through the population. A striking demonstration of how robust the gene drive method is shown(in Supplementary Fig. 2. Introduction of the gene drive cassette into fibroblasts by transfection of a plasmid, a notoriously inefficient process, is nonetheless sufficient to generate enough recombinant gene drive virus to enable gene drive through the population over 50-70 days.
One observation in the data seems puzzling and requires clarification. Since the wild type virus, Towne-GFP, replicates 15-times more efficiently than the gene drive virus (Fig. 2B) and the experiment in Figs 2C-D was performed by starting at a low moi (0.1), most cells would be infected with only one virus. The cells infected with Towne-GFP would be expected to produce abundant virus, at least for most of the first week and so there would an increase relative and actual Towne-eGFP until there is enough virus so that a substantial population of cells are infected with both viruses.
The author then tried to modify gene drive viruses to target genes that are needed for efficient replication. The idea here is that the original and recombinant gene drive viruses would replicate poorly because of insertional mutation of the important viral gene. Importantly, any drive-resistant viruses that emerged would likely also replicate poorly due to mutations at the cut site, which they designed to be at specific positions within the genes that seemed likely to be very sensitive to mutation. Among the 8 other genes they targeted with this approach, only two worked. In 4 of the other cases, they unable to make the recombinant viruses, which is not really surprising since these genes are known to be essential for the virus to replicate at all. In two other cases, they observed only very limited drive, for unknown, though potentially interesting reasons. In the two cases that did work to promote gene drive, they were able to isolate drive-resistant mutants at a late time point. Their analyses nicely confirmed the presence of mutations at the expected sites and, importantly, that mutations caused these viruses to replicate somewhat less well than the wild type virus. Thus, the author showed that their strategy of targeting important loci can work, but much more needs to do to (i) understand the design rules (i.e. why did 6 or the 8 versions not work) and (ii) to understand how robust the system really is. In one of the two genes that seemed to work (UL35), the gene drive-resistant mutants did not replicate significantly less than wild type virus. In the other case (UL26), the drive-resistant viruses were isolated at a time when the titers of this population were still increasing (Fig 4D) so it is not clear if virus that replicates as well as wild type virus would emerge later.
Overall, these studies are well done and interesting The ultimate goal of applying gene drive methods to treat viral infection has many obstacles to overcome; these data are a small step forward.
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Reviewer #2 (Public Review):
This study describes an interesting attempt to engineer suppression gene drives for human cytomegalovirus (hCMV, herpesvirus 5). hCMV is a nuclear replicating dsDNA virus and is implicated in multiple human diseases. M. Walter and E. Verdin previously described a CRISPR/Cas9 Gene Drive (GD) targeted at hCMV's UL23 (aka. GD-UL23) that spread via recombination. Although the function of the UL23 tegument gene is dispensable for virus propagation in cell culture, it is required for evasion of immune response in vivo. Notably, loss-of-function (LOF) UL23 allele and GD-UL23 still incurred fitness cost, and the brief spread of GD-UL23 correlated with the total virus load reduction. However, viruses harboring UL23 alleles resistant to GD-UL23 rapidly evolved and blocked the further spread of GD-UL23. The goal of a …
Reviewer #2 (Public Review):
This study describes an interesting attempt to engineer suppression gene drives for human cytomegalovirus (hCMV, herpesvirus 5). hCMV is a nuclear replicating dsDNA virus and is implicated in multiple human diseases. M. Walter and E. Verdin previously described a CRISPR/Cas9 Gene Drive (GD) targeted at hCMV's UL23 (aka. GD-UL23) that spread via recombination. Although the function of the UL23 tegument gene is dispensable for virus propagation in cell culture, it is required for evasion of immune response in vivo. Notably, loss-of-function (LOF) UL23 allele and GD-UL23 still incurred fitness cost, and the brief spread of GD-UL23 correlated with the total virus load reduction. However, viruses harboring UL23 alleles resistant to GD-UL23 rapidly evolved and blocked the further spread of GD-UL23. The goal of a current work was to engineer a next generation suppression GD in hCMV, which would be immune to resistant alleles and therefore, would spread better and cause a long-term reduction of viral levels. M. Walter et al. targeted GD into genes essential for hCMV propagation to ensure that their LOF resistant alleles could not propagate. Two GDs targeting UL26 and UL35 genes were developed and analyzed in the study. Both GDs (GD-UL26 and GD-UL35) induced a transient reduction in the total viral titer (up to 50% and 80%, respectively) before induced resistant viruses spread at the expense of each GD and brought the viral levels up. In conclusion, this study failed to develop a suppression GD that could overcome the evolution and spread of induced resistance. Nevertheless, naturally occurring or induced resistant alleles are Achilles' heel of any gene drive especially in rapidly evolving and recombinogenic viruses. Therefore, I think claims that are not supported by the data should be tempered.
Strengths:
The manuscript describes a promising approach for developing a suppression gene drive in a virus. hCMV is a good model for this type of research, since it is a nuclear replicating dsDNA virus.
The scale of work presented in the Table 1 is impressive. Eight essential genes were targeted by GD plasmids. It would be interesting to know some details of this massive work. Why recombinant viruses were not generated for three genes? Did each gRNA direct its target cleavage efficiently? Could Towne-GFP virus rescue these recombinant viruses?
I admire the choice of gRNA for GD-UL26. It looks truly conserved at both DNA and amino acid levels; and yet UL26 resistant alleles were induced that were only marginally unfit in comparison to GD-UL26.
Weaknesses:
I think that the presentation of findings can be improved.
Claims are overstated throughout the manuscript, including its title.
Notably, GD-UL36 works better than GD-UL26 however the GD-UL36 gRNA target contains multiple SNPs at the PAM region making it less stable in a long run for any real world application.
Points of potential interest:
Both GD-UL26 and GD-UL35 may not spread catalytically via recombination with Towne-GFP. Instead they can spread at the expense of Towne-GFP viruses (i.e. by destroying them), similarly to toxin / antidote drives in insects.
It would be useful for non-specialists to describe how fibroblast cell numbers were controlled during long cell culture experiments. I can image that selection between fibroblast cells can happen during 70 days, e.g. virus load may affect the probability of cells death or the speed of cell proliferation. In turn, this cell selection affects quantification of viral load and composition.
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