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Critical Analysis Essay (5 Pages)

Open Posted By: highheaven1 Date: 29/04/2021 High School Rewriting & Paraphrasing

5 pages analyzing the attached research article.

In the analysis of this research article answer the following questions:

- What did they do?

- What you like?

- What you didn't like?

- What you think they did incorrectly? How could it have been done better?

- What you would do in addition to what they did?

- What future experiments should be done in this area/to further this research?

- Any other additional insights?

Category: Business & Management Subjects: Auditing Deadline: 12 Hours Budget: $150 - $300 Pages: 3-6 Pages (Medium Assignment)

Attachment 1

Articles https://doi.org/10.1038/s41587-020-00781-8

1Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China. 2National Research Center for Translational Medicine, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China. 3Department of Ophthalmology and Vision Science, Shanghai Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, China. 4Department of Ophthalmology, The Affiliated Hospital of Guizhou Medical University, Guiyang, China. 5Department of Biomedicine, Aarhus University, Aarhus, Denmark. 6These authors contributed equally: Di Yin, Sikai Ling, Dawei Wang. ✉e-mail: [email protected]; [email protected]

HSV-1 is among the most common human viruses, with 50–80% of the world population being seropositive1. It belongs to the alpha subfamily of herpesviruses, which are enveloped viruses carrying double-stranded DNA and are capable of establishing latent infections in sensory neurons2. HSV-1 infec- tion can cause a wide variety of diseases, including herpes simplex encephalitis, which has a high mortality rate if untreated3. HSV-1 infection in the cornea can cause HSK, which is the leading factor for infectious blindness4. After primary infection and productive replication in corneal epithelium, HSV-1 is transported through ophthalmic nerves in a retrograde direction to the trigeminal gan- glia (TG), where the virus establishes a latent reservoir that persists throughout an individual’s lifetime5. Under certain conditions, including immunosuppression, the latent viruses in the TG can be reactivated, leading to recurrence and aggravation of disease. Typical blinding HSK develops subsequently to infection in the eye, at which point virus can often not be detected6. Most of the tissue damage occurring in human corneas during HSK is immune medi- ated rather than a direct viral cytopathic effect7. Globally, it is esti- mated that 1.5 million episodes of ocular HSV occur each year and 40,000 people develop visual disability4.

Despite the high prevalence, there is no vaccine currently avail- able for HSV infection8,9. The first-line treatment option for HSV-1 infection is acyclovir (ACV). This compound was developed nearly half a century ago and analogs have subsequently been made, all tar- geting the viral DNA polymerase. In specific patient groups, includ- ing immunocompromised individuals and individuals receiving chronic antiviral prophylaxis, drug resistance occurs frequently10–12. Alternative strategies, including small molecules that inhibit the viral helicase–primase complex, antibodies and peptides, are still under development13–15. Recently, Jaishankar et al. reported that BX795, a commonly used inhibitor of TANK-binding kinase 1, blocks HSV-1 infection in vivo by targeting Akt phosphorylation in infected cells16. However, none of these strategies can remove the

existing virus and modulate its reservoir in the TG, and they are therefore incapable of preventing recurrence.

CRISPR targets genomes directly and has been very success- ful in treating genetic diseases in preclinical studies17–22. About 2 years ago, the US Food and Drug Administration approved CRISPR for phase I/II trials to treat β-thalassemia, sickle cell dis- ease and Leber congenital amaurosis type 10 (ClinicalTials.gov: NCT04208529, NCT03745287 and NCT03872479). Its therapeu- tic potential on infectious diseases is promising. Dash et al. dem- onstrated viral clearance in latent infectious reservoirs in human immunodeficiency virus type 1 (HIV-1)-infected humanized mice by combining antiviral prodrugs and CRISPR23. However, to the best of our knowledge, no investigational new drug applica- tion has been registered for infectious diseases. This reflects the challenge of delivering CRISPR to infection sites and especially to viral reservoirs24. One study delivered an HSV-1-targeting endonuclease using adeno-associated virus (AAV) in a mouse model of latent HSV infection; however, this study revealed nei- ther a detectable loss of viral genome nor therapeutic efficacy25. Recently, the same group showed detectable elimination of latent genomes and therapeutic efficacy by using an improved AAV vector and two meganucleases targeting the HSV genome26. So far, the anti-HSV activity of CRISPR has only been characterized in vitro, and no studies have shown the therapeutic efficacy of CRISPR against HSK in vivo27,28.

In this study, we developed HELP and showed its therapeutic efficacy in three different HSK models and in human-derived cor- neas. Furthermore, we found that HELP was capable of modulat- ing the HSV-1 reservoir in the TG. Corneas maintained a healthy status after intracorneal injection of HELP, as shown by a variety of clinically relevant assays. Cas9 expression from HELP only lasted for 3 d in vivo, and no off-target effects were detected in the cod- ing regions of the mouse and human genomes. Taken together, our study supports the clinical translation of HELP for treating

Targeting herpes simplex virus with CRISPR–Cas9 cures herpetic stromal keratitis in mice Di Yin1,6, Sikai Ling1,6, Dawei Wang   2,6, Yao Dai1, Hao Jiang3,4, Xujiao Zhou3, Soren R. Paludan   5, Jiaxu Hong   3,4 ✉ and Yujia Cai   1 ✉

Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus from the site of infection or latent reservoirs in the trigeminal ganglia. Here, we target HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (desig- nated HSV-1-erasing lentiviral particles, termed HELP). We show that HELP efficiently blocks HSV-1 replication and the occur- rence of herpetic stromal keratitis (HSK) in three different infection models. HELP was capable of eliminating the viral reservoir via retrograde transport from corneas to trigeminal ganglia. Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing. These results support the potential clinical utility of HELP for treating refractory HSK.

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Articles NATuRE BioTECHNoLogy refractory HSK, which has been resistant to conventional drugs and corneal transplantation.

Results HELP blocks HSV-1 replication in vitro. In this study, we designed a guide RNA (gRNA) expression cassette simultaneously target- ing two essential genes of HSV-1, UL8 and UL29 (refs. 29,30), and co-packaged it with SpCas9 mRNA in an mRNA-carrying lentiviral particle (mLP) via the specific binding of pac site-containing SpCas9 mRNA to bacteriophage-derived MS2 coat protein located at the N terminus of lentiviral Gag and GagPol polyproteins (Fig. 1a–c). The MS2 coat protein specifically recognizes and interacts with the pac site-containing SpCas9 mRNA and co-packages it into the lentivi- ral particle during viral assembly. The gRNA expression cassette is reverse transcribed and maintained as circular episomal DNA, cor- responding to an integration-defective lentiviral vector (Fig. 1b). As the UL8 gRNA is cloned into the ∆U3 region of the long termi- nal repeat (LTR), it is copied from the 3′ LTR to the 5′ LTR during reverse transcription (Fig. 1b). We produced HELP by cotransfec- tion of six plasmids into 293T cells and harvested the particles by ultracentrifugation (Fig. 1c). As controls, we also produced mLPs with a single-gRNA expression cassette, for UL8, UL29 or a scram- bled sequence (non-targeting gRNA). To verify whether HELP was indeed capable of inhibiting HSV-1, 293T cells were transduced with HELP for 24 h and infected with HSV-1 (HSV-1–GFP). The super- natants were harvested 1 d and 2 d after HSV-1 infection and sub- jected to a virus yield assay. We found inhibitory effects for all viral gene-targeting mLPs, with the UL8/UL29 co-targeting HELP being the most efficient (Fig. 1d and Supplementary Fig. 1). The aver- age copy number of Cas9 mRNA in HELP was 3.5 (Supplementary Fig. 2). Additionally, we conducted a dose–response experiment for HELP, which showed an increasing level of virus inhibition that reached saturation at 400 ng of p24 (Fig. 1e). We therefore chose HELP in all the subsequent experiments.

HSV-1 infection is sensitive to type I interferons (IFNs) induced by pathogen-associated molecular patterns, even in the absence of gene editing (Supplementary Fig. 3; ref. 31). To exclude a necessity for type I IFNs here, we evaluated the antiviral activity of HELP in both wild-type and interferon alpha and beta receptor subunit 2 (IFNAR2)-knockout HaCaT cells. We found that HELP, but not the scrambled control, significantly inhibited HSV-1 replication in both cell lines (Fig. 1f,g). Furthermore, we analyzed the UL8 and UL29 loci and found that, on average, the indel frequency was about 40% for UL8 while only 7% for UL29 (Fig. 1h). The indel rate in UL29 was relatively low. As ICP8 (encoded by UL29) plays multifunc- tional roles in the viral life cycle, including in viral DNA synthesis, we reasoned that mutations in UL29 make the virus unable to repli- cate and tend to be underestimated30. Indeed, when using plasmids containing UL8 and UL29 sequences as the targets, we obtained even higher indel rates with UL29 gRNA than with UL8 gRNA (Fig. 1i). Notably, the antiviral activity of HELP is underestimated using PCR-based indel analysis, as not all the cleavage outcomes, for example, unrepaired double-strand breaks or large deletions, can be amplified (Supplementary Fig. 4). Also, we found that HELP did not provoke innate immune sensing, in contrast to HSV-1 strains, which were all sensed by THP-1-derived macrophages at a multiplicity of infection (MOI) of 1 and induced a moderate but significant IFN response (Supplementary Fig. 5). Together, these data suggest that HELP inhibits HSV-1 through DNA disruption but not through a type I IFN-dependent innate immune response.

The corneal stroma is highly linked to keratitis recurrence32. The stroma is rich with nerve trunks that originate from the TG where HSV-1 maintains latency33. Therefore, we explored whether HELP was functional in primary corneal stromal cells from mice. Primary stromal cells were transduced with non-GFP HELP for 24 h and then infected with HSV-1–GFP. We found that HELP potently

suppressed GFP expression as well as viral replication using both low and high MOIs at either 24 h or 48 h after infection, while the scrambled control did not show any protective effects (Fig. 1j–m).

HELP blocks HSV-1 infection of corneas and neurons in the prevention model. Persisting nuclease expression may bring addi- tional risks. From a safety perspective, transient nuclease exposure is desired for CRISPR therapeutics. However, it is unclear whether transient Cas9 expression can control HSK, as HSV-1 propagates quickly (about 18 h for the lytic replication cycle). It is difficult for the CRISPR machinery to remove every HSV-1 genome. On the other hand, HSV-1 encounters harsh antiviral responses in vivo. Here, we hypothesize that reducing the viral load to a certain level is sufficient to control the virus in vivo. To verify this, we performed dose–response experiments of HSV-1 infection on scarified cor- neas of mice. Indeed, only when the HSV-1 load was over 2 × 104 plaque-forming units (p.f.u.) did the decreased viability, weight loss and symptoms of keratitis develop (Supplementary Fig. 6).

We then set out to investigate the potential of HELP as a new HSK therapeutic in vivo. To identify the kinetics of HSV-1 infec- tion in our HSK model, we visualized HSV-1 using confocal imag- ing and found that the virus progressively disseminated from the superficial side to the deeper side of corneal stroma during the time course from 12 h to 8 d post-infection (d.p.i.; Supplementary Fig. 7). The experimental setup is illustrated in Fig. 2a. HELP was adminis- trated by intrastromal injection to corneas 1 d before infection with HSV-1 strain 17syn+ (Supplementary Fig. 8). We first performed deep sequencing to determine the on-target activity of HELP on the HSV-1 genome and the off-target effects on the mouse genome. The indels induced by HELP occurred at rates of approximately 7% for the UL8 locus and 5% for the UL29 locus, while no off-target sites were found for either gRNA (Fig. 2b,c). Notably, Cas9 expres- sion only lasted for 3 d both in vitro and in vivo, which might mini- mize the off-target activity of HELP (Supplementary Fig. 9). Next, we performed confocal imaging to assess HSV-1 replication and HELP distribution in the corneas of mice, which were indicated by the viral capsid protein VP5 and GFP, respectively. We found that HSV-1 was actively replicating in the corneal stroma in mock- and scrambled control-treated mice, while it was barely detectable after HELP treatment (Fig. 2d). Accordingly, HELP were evenly distrib- uted in all corneal structures from the epithelium and stroma to the endothelium (Fig. 2d). To assess whether HELP treatment blocks the transmission of HSV-1 from corneal epithelium to the periph- eral and central nervous system, eye, TG and brain samples from all infected mice were collected and examined for HSV-1 genome copy number and infectious virus. In all samples, the viral load was sig- nificantly reduced after HELP treatment (Fig. 2e–j). Additionally, we performed confocal imaging of the whole brain and TG. In agreement with the quantitative PCR (qPCR) and p.f.u. analyses, we found that HELP diminished HSV-1 viral load to an almost unde- tectable level in both the brain and TG (Fig. 2k,l). Tissue distribu- tion is an important safety index for in vivo gene therapy. Therefore, we evaluated the dissemination of HELP in the whole body, find- ing that HELP were highly restricted to the eyes and did not local- ize to other organs, including reproductive organs (Supplementary Fig. 10). Interestingly, although they were injected in the corneas, we also detected HELP in the TG, supporting the concept of ret- rograde delivery of CRISPR machinery from neuronal termini in the corneas to the neuronal cell body in the TG (Supplementary Fig. 10). This finding was further supported by detection of HELP in the TG by confocal imaging (Fig. 2m).

HELP suppresses HSV-1-associated disease pathologies in the prevention model. To determine disease development and thera- peutic efficacy, we monitored the clinical signs of acute ocular herpes infection and scored them in a blinded fashion (Fig. 3a).

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ArticlesNATuRE BioTECHNoLogy

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Fig. 1 | HeLP blocks HSV-1 replication in vitro. a, Schematic representation of the HSV-1 genome and gRNA loci. TRL, terminal repeat long; IRL, internal repeat long; UL, unique long; IRS, internal repeat short; TRS, terminal repeat short; US, unique short. b, The gRNA sequences and expression cassettes for HELP. c, Schematic illustration of HELP production. Colored dots represent the main components of lentiviral Gag and GagPol polyproteins. Gag is composed of matrix (MA), capsid (CA) and nucleocapsid (NC), whereas Pol consists of protease (PR), reverse transcriptase (RT) and integrase (IN). d–g, The antiviral activity of HELP in different cell lines. In d, mock versus scramble, UL8, UL29 and HELP, P = 0.0220, 0.0003, 0.0003 and 0.0002, respectively, on day 1; P < 0.0001 on day 2. In e, mock versus 50 ng p24, P = 0.0011; P = 0.0009 for all other comparisons. In f, HELP versus mock and scramble, P = 0.0003 and 0.0019, respectively, on day 1; P < 0.0001 and P = 0.0011, respectively, on day 2. Mock versus scramble, P = 0.0420 on day 1. In g, HELP versus mock and scramble, P = 0.0013 and P < 0.0001, respectively, on day 1; P = 0.0002 and 0.0001, respectively, on day 2. h, TIDE analysis of indels in the HSV-1 genome. Viral DNA was from day 2 samples in f and g. i, TIDE analysis of indels in plasmids containing UL8 and UL29 target sequence, respectively. j–m, Antiviral activity in primary mouse corneal stromal cells as measured by confocal microscopy (j), flow cytometry (k,l) and p.f.u. analysis (m). In k, mock versus HELP, P = 0.0001 at 24 h; P < 0.0001 for all other comparisons. In l, *P = 0.0380, ***P = 0.0005, ***P = 0.0004, ***P < 0.0001 and ***P = 0.0003, left to right. In m, ***P < 0.0001, ***P = 0.0010, ***P = 0.0002, ***P < 0.0001, *P = 0.0236, ***P < 0.0001, ***P = 0.0006, ***P < 0.0001 and ***P < 0.0001, left to right. In j, images are representative of three independent biological replicates in one experiment. The gating strategy is provided in Supplementary Fig. 20. Data and error bars represent mean ± s.e.m. from three biologically independent replicates. Unpaired two-tailed Student’s t tests. NS, not significant; WT, wild type; KO, knockout.

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Fig. 2 | HeLP blocks HSV-1 infection of corneas and neurons in a prevention model. a, Flowchart for evaluating the antiviral effects of HELP in vivo. p24 HELP (100 ng), scrambled control mLP or 2 μl PBS (mock) was injected into the corneas of mice by intrastromal injection. After 24 h, the mice were infected with HSV-1 17syn+ (2 × 106 p.f.u. per eye). b, Deep sequencing analysis of on-target effects in HSV-1 and off-target effects in the mouse genome for UL8 gRNA; n = 4 mice. c, Deep-sequencing analysis of on-target effects in HSV-1 and off-target effects in the mouse genome for UL29 gRNA; n = 4 mice. d, Confocal imaging of HSV-1 and HELP in corneas. Mouse corneal sections were incubated with both anti-GFP (HELP) and anti-HSV-1 (VP5) antibodies. e, qPCR analysis of HSV-1 dissemination in the eye. f, p.f.u. analysis of HSV-1 dissemination in the eye. g, qPCR analysis of HSV-1 dissemination in the TG. h, P.f.u. analysis of HSV-1 dissemination in the TG. i, qPCR analysis of HSV-1 dissemination in the brain. j, P.f.u. analysis of HSV-1 dissemination in the brain. In e–j, the abundance of HSV-1 is shown as the number of viral genomes (VG) per diploid genome (DG); n = 4 mice; *P = 0.0286. k,l, Confocal analysis of HSV-1 in the whole brain (k) and TG (l). m, Confocal analysis of HELP in the TG after intracorneal injection. Data and error bars represent mean ± s.e.m.; unpaired two-tailed Mann–Whitney tests. The experiments were repeated twice with similar results.

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Importantly, mice treated with HELP did not show any disease progression (n = 6 mice), while the mock-treated and scrambled gRNA-treated eyes developed severe signs of ocular infection (Fig. 3b,c). Next, we performed histological staining to examine the pathology of the eye. The mock- and scrambled gRNA-treated eyes presented with irregular stromal matrix and increased corneal thickness, typical signs of acute infection (Fig. 3d,e). We further found that HSV-1 infection in the corneas induced a significant type I IFN response, while HELP transduction did not elicit such a response (Supplementary Fig. 11). Clinical HSK is the result of excessive virus-induced corneal inflammation mediated by the infiltration of inflammatory cells, including T cells (both CD4+ and CD8+), polymorphonuclear leukocytes and macrophages34,35. Indeed, HSV-1 infection provoked corneal expression of the inflam- matory molecules IL-6, CCL2 and CXCL10, which was blocked after HELP treatment (Supplementary Fig. 12). Using immunohis- tochemistry, we showed that HSV-1 infection led to infiltration of CD4+ and CD8+ T cells in the corneal stroma for the mock- and scrambled control-treated groups, but HELP treatment prevented T cell infiltration (Supplementary Fig. 13). Additionally, we stained corneal sections for two additional markers, CD11b and F4/80, to visualize myeloid-derived cells and macrophages, respectively. We observed CD11b+ and F4/80+ cells in non-therapeutic groups

in contrast to mice treated with HELP and non-infected controls (Supplementary Fig. 13). We also noted that PD-L1 was upregulated in the epithelium and stroma of untreated mice after HSV-1 infec- tion, consistent with previous observations (Supplementary Fig. 13; ref. 36). Increased local PD-L1 expression may inhibit viral clearance by immune cells, highlighting the importance of direct DNA degra- dation by CRISPR. To assess the presence of secreted virus, the viral titer of eye swabs was determined every other day after infection. HELP treatments significantly reduced viral presence in the eyes (Fig. 3f ). In addition, body weights were recorded every other day. No loss of body weight was observed for HELP-treated mice, while it was evident for the mock- and scrambled control-treated mice (Fig. 3g). Notably, all mice survived in the HELP-treated groups and no relapse of HSK for the HELP-treated mice was found during the 3-month follow-up (Fig. 3h and Supplementary Fig. 14).

Eye health after HELP treatment in the prevention model. Subsequently, we thoroughly analyzed corneal health using clini- cally relevant indices (Fig. 4a). To determine lesion formation, we assessed the epithelial layers of corneas using sodium fluorescein, which stains damaged epithelial cells. We found that HELP-treated corneas were significantly protected from HSV-1 infection (Fig. 4b). As reduced tear production has been shown in HSK, we assessed

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Fig. 3 | HeLP suppresses HSV-1-associated disease pathologies in the prevention model. a, Flowchart for evaluating the antiviral effects of HELP in vivo. p24 HELP (100 ng), scramble mLP or 2 μl PBS (mock) was injected into corneas. After 24 h, the mice were infected with HSV-1 17syn+ (2 × 106 p.f.u. per eye). b, Ocular disease scores (0 to 4, with 4 being severe) in mice; n = 6 mice. c, Photographs of the right eyes of mice from the different treatment groups 6 d.p.i. and 9 d.p.i. Each image is representative of three mice in one experiment. NC, non-treated control. d, Corneal histology of eyes 14 d.p.i. Each image is representative of three mice in two independent experiments. e, Thickness of the cornea as assessed by histology; n = 3 mice. HELP versus mock and scramble, P = 0.0168 and 0.0006, respectively. f, Secreted HSV-1 as assessed by eye swabs. Tear swabs from each mouse were collected at 1, 3, 5 and 7 d.p.i. The percentage of HSV-1-positive swabs was recorded; n = 6 mice. Mock versus HELP, P = 0.0056; scramble versus HELP, P = 0.0072. g, Change in body weight; n = 6 mice. h, Kaplan–Meier survival curves; n = 6 mice. Data and error bars represent mean ± s.e.m.; unpaired two-tailed Student’s t tests; NS, not significant.

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the tear secretion levels of mice using the phenol red thread test. We found that HELP treatment significantly protected the infected corneas from desiccation (Fig. 4c). HSV-1 infection often causes

denervation of the cornea with a substantial loss of sensory fibers. Next, we measured the mechanosensory function of the corneas …