Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9

Oh, Hyung Suk; Neuhausser, Werner M.; Eggan, Pierce; Angelova, Magdalena; Kirchner, Rory; Eggan, Kevin; Knipe, David M.

doi:10.7554/elife.51662

Cited by 35 publications

(46 citation statements)

References 72 publications

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“…One limitation to our study is that we cannot distinguish whether the observed reduction in de novo production of viral genomes resulted from a reduction in the number of reactivation events, or instead from a decrease in virus production after a reactivation event has initiated. Our previous results suggest that newly synthesized HSV genomes are efficiently targeted by nucleases 13 , while results from our group and others demonstrate that chromatin modification of latent HSV can reduce the efficacy of gene editing 13,20,25,36 . This has important implications for the ultimate embodiment of gene editing for HSV, as it will determine whether sustained long-term expression of nuclease (as achieved using AAV vectors 22 ) is required for therapeutic benefit, or whether alternative transient delivery approaches might be sufficient.…”

Section: Discussionmentioning

confidence: 90%

Gene editing and elimination of latent herpes simplex virus in vivo

et al. 2020

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We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.

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Section: Discussionmentioning

confidence: 90%

Gene editing and elimination of latent herpes simplex virus in vivo

et al. 2020

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“…By using different cell models, these screens identified involvement of the cellular gene products MCLI and XPOI in KSHV-related tumorigenesis, as well as host-dependency factors that are critical for EBV-transformed B cells (33)(34)(35). In addition, direct manipulation of several herpesvirus genomes using the CRISPR-Cas9 platform was reported (7,32,(36)(37)(38). Furthermore, the effectiveness of CRISPR-Cas9 in eliminating latent EBV episomes in cell culture, by targeting key factors for latent genome replication, has been described (36,39).…”

Section: Discussionmentioning

confidence: 99%

Targeting the Kaposi’s Sarcoma-associated Herpesvirus Genome With the CRISPR-Cas9 Platform in Latently Infected Cells

Haddad

Kalt

Shovman

et al. 2020

Preprint

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Background: Kaposi’s sarcoma-associated herpesvirus (KSHV) is a transforming gammaherpes. Like other herpesviruses, KSHV infection is for life long and there is no treatment that can cure of patients from the virus. In addition, there is urgent need to target viral genes to study their role during the infection cycle. The CRISPR-Cas9 technology offers a means to target viral genomes and thus may offer a novel strategy for viral cure as well for better understanding of the infection process. We evaluated the suitability of this platform for the targeting of KSHV. Methods: We have used BAC16 genome, which contains an expression cassette encoding hygromycin-resistance and a GFP marker gene. Three genes were targeted: gfp which serves as a marker for infection; orf45 encoding a lytic viral protein; and orf73, encoding LANA which is crucial for latent infection. The fraction of cells expressing GFP as well as viral DNA levels and LANA expression were monitored and viral genomes were sequenced. Results: We found that KSHV episomes can be targeted by CRISPR-Cas9. Interestingly, the quantity of KSHV DNA declined, even when target sites were not functionally important for latency. In addition, we show that antibiotic selection, used to maintain infection, interferes with the outcome of targeting.Conclusions: Our study provides insights to the use of this fundamental approach for the study and manipulation of KSHV. It provides guidelines for the targeting CRISPR-Cas9 to the viral genome and for outcomes interpretation.

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“…Delivery of scAAV2-LAT-targeting ribozyme in the eyes of rabbits with latent HSV-1 infection blocked viral reactivation in more than 60% of the infected animals [ 80 ]. Furthermore, in recent years, multiple studies have demonstrated that the lytic and quiescent HSV genome can be targeted using the CRISPR-Cas9 system in vitro [ 81 , 82 , 83 ]. It will be interesting to see if these effects can be recapitulated through in vivo studies.…”

Section: Corneal Disordersmentioning

confidence: 99%

Adeno-Associated Virus Mediated Gene Therapy for Corneal Diseases

et al. 2020

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According to the World Health Organization, corneal diseases are the fourth leading cause of blindness worldwide accounting for 5.1% of all ocular deficiencies. Current therapies for corneal diseases, which include eye drops, oral medications, corrective surgeries, and corneal transplantation are largely inadequate, have undesirable side effects including blindness, and can require life-long applications. Adeno-associated virus (AAV) mediated gene therapy is an optimistic strategy that involves the delivery of genetic material to target human diseases through gene augmentation, gene deletion, and/or gene editing. With two therapies already approved by the United States Food and Drug Administration and 200 ongoing clinical trials, recombinant AAV (rAAV) has emerged as the in vivo viral vector-of-choice to deliver genetic material to target human diseases. Likewise, the relative ease of applications through targeted delivery and its compartmental nature makes the cornea an enticing tissue for AAV mediated gene therapy applications. This current review seeks to summarize the development of AAV gene therapy, highlight preclinical efficacy studies, and discuss potential applications and challenges of this technology for targeting corneal diseases.

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Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9

Cited by 35 publications

References 72 publications

Gene editing and elimination of latent herpes simplex virus in vivo

Gene editing and elimination of latent herpes simplex virus in vivo

Targeting the Kaposi’s Sarcoma-associated Herpesvirus Genome With the CRISPR-Cas9 Platform in Latently Infected Cells

Adeno-Associated Virus Mediated Gene Therapy for Corneal Diseases

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