Gene Editing Approaches Research Articles

Is It Ever Wise to Edit Wild-Type Alleles? Engineered CRISPR Alleles Versus Millions of Years of Human Evolution.

The tremendous burden of lipid metabolism diseases, coupled with recent developments in human somatic gene editing, has motivated researchers to propose population-wide somatic gene editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) within the livers of otherwise healthy humans. The best-characterized molecular function of PCSK9 is its ability to regulate plasma LDL (low-density lipoprotein) levels through promoting LDL receptor degradation. Individuals with loss-of-function PCSK9 variants have lower levels of plasma LDL and reduced cardiovascular disease. Gain-of-function variants of PCSK9 are strongly associated with familial hypercholesterolemia. A new therapeutic strategy delivers CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR-associated protein 9) specifically to liver cells to edit the wild-type alleles of PCSK9 with the goal of producing a loss-of-function allele. This direct somatic gene editing approach is being pursued despite the availability of US Food and Drug Administration-approved PCSK9 inhibitors that lower plasma LDL levels. Here, we discuss other characterized functions of PCSK9 including its role in infection and host immunity. We explore important factors that may have contributed to the evolutionary selection of PCSK9 in several vertebrates, including humans. Until such time that more fully understand the multiple biological roles of PCSK9, the ethics of permanently editing the gene locus in healthy, wild-type populations remains highly questionable.

Gene-Based Therapy: A New Approach to Feline Induced Sterilization?

Feline population control remains a concern as to whether it is intended for the short- or long-term. Induced sterilization of felids is critical in the case of feral, free-roaming cats, or the management of wild populations in Zoos or sanctuaries. This narrative review explores the shifting paradigm in induced sterilization methods, driven by the development of gene editing approaches recently applied to control felid reproductive activity. Although gene therapy approaches have gained attention as alternatives to more traditional methods, their clinical applications remain in the realm of thought. The objective of this study was to provide a comprehensive overview of the current state and most recent advances in gene-based contraception options, consolidate current research and evidence, and share some considerations on its potential effectiveness, advantages or limitations, and implications for animal welfare and population control strategies. Gene-based contraception therapy tested in felines, targeting the AMH pathway, was unable to suppress the estrous cycle and follicular development. However, at an experimental level, preliminary results hint at the need to change towards different molecular targets. Moreover, their side effects remain largely unknown, and several questions remain unanswered, such as the regularity of treatment applications or cost.

OVERVIEW OF PEST STATUS, CONTROL STRATEGIES FOR SPODOPTERA LITURA (FAB.): A REVIEW

The Spodoptera litura (Tobacco Caterpillar) has significantly damaged various cultivated crops, primarily affecting Solanaceous crops. It voraciously consumes the crop leaves, giving the appearance of animal grazing. It can cause extensive damage in its later stages, ultimately leading to crops decay. Commercial farmers typically depend on chemical pesticides for control. However, the overreliance on chemical insecticides to combat S. litura has led to the development of resistance over time. Various strategies have been explored in pursuit of environmentally friendly pest control measures. These include natural predators, employing techniques like sex pheromones and genetically modified crops, and utilizing RNA interference tools. While these methods have encountered implementation challenges, they are noteworthy for being safe, sustainable, and tailored to specific species. In this comprehensive review, we have delved into historical and current practices for pest management, covering cultural techniques, chemical controls, and biological methods. In addition, we have examined emerging technologies like the gene editing approach, nano-insecticides, neuropeptides and seminal fluid proteins that are promising tools in the ongoing efforts to manage S. litura.

Developing Topics.

Goal 1 of the National Plan to Address Alzheimer's Disease (AD) is to prevent and effectively treat AD and AD-related dementias (ADRD) by 2025. Toward this goal, NINDS plans 14 ADRD funding opportunities for 2024 to support training, basic mechanisms, translational, and clinical research for ADRD. Here, we describe ADRD translational therapy development initiatives, which include 1) multi-target validation, 2) early-stage therapy development, 3) genome editing and 4) animal model development and validation. Note, ADRD specific funding opportunities are not required. ADRD researchers can also take advantage of NINDS existing programs such as the NIH Blueprint for Neurotherapeutics (BPN) for Small Molecule or Biologic therapy development. These programs support optimization, manufacture, IND-enabling studies and first in human clinical trials through a combination of grants and complementary access to NIH-provided CROs and consultants. All concepts discussed here are approved, however NIH cannot guarantee future funding opportunities, award mechanisms, or funding allocation. A funding opportunity is only official when published in the NIH Guide. Multi-Target Validation for ADRD concept has the goal to support research that would validate multiple targets for future therapy development that better reflect the complexity of disease. Early-Stage Therapy Development concept would support the development of small molecule or biologic therapeutics and would be a reissue of RFA-NS-22-059, which supported assay development, therapeutic screening, optimization, and in vivo efficacy work in preparation for later-stage programs. Genome Editing for ADRD concept would support early translational research focused on somatic cell gene editing with the expectation that studies will address feasibility of the gene editing approach and would be a reissue of RFA-NS-23-017. PAR-23-154 "Development and Validation of Models for Alzheimer's Disease-Related Dementias (ADRD)" supports the development and validation of clinically- and pathophysiologically-relevant models of ADRD that can be used for future therapy development. Models should reflect multiple aspects of the human condition and include internal, face, construct, and predictive validation to the extent possible. Through these initiatives, NINDS hopes to provide a comprehensive pipeline for ADRD therapy development from target validation to the clinic.

Epidermal growth factor potentiates EGFR(Y992/1173)-mediated therapeutic response of triple negative breast cancer cells to cold atmospheric plasma-activated medium

Cold atmospheric plasma (CAP) holds promise as a cancer-specific treatment that selectively kills various types of malignant cells. We used CAP-activated media (PAM) to utilize a range of the generated short- and long-lived reactive species. Specific antibodies, small molecule inhibitors and CRISPR/Cas9 gene-editing approaches showed an essential role for receptor tyrosine kinases, especially epidermal growth factor (EGF) receptor, in mediating triple negative breast cancer (TNBC) cell responses to PAM. EGF also dramatically enhanced the sensitivity and specificity of PAM against TNBC cells. Site-specific phospho-EGFR analysis, signal transduction inhibitors and reconstitution of EGFR-depleted cells with EGFR-mutants confirmed the role of phospho-tyrosines 992/1173 and phospholipase C gamma signaling in up-regulating levels of reactive oxygen species above the apoptotic threshold. EGF-triggered EGFR activation enhanced the sensitivity and selectivity of PAM effects on TNBC cells. The proposed approach based on the synergy of CAP and EGFR-targeted therapy may provide new opportunities to improve the clinical management of TNBC.

Non-Viral RNA-Lipid Nanoparticles for High Efficiency Genome Editing of CD34+ Hematopoietic Stem and Progenitor Cells for Advanced Cell and Gene Therapies

Background and Aims: The application of CRISPR-Cas9 gene editing in hematopoietic stem (HSC) and progenitor cells (HSPCs) has the potential to revolutionize the treatment of genetic blood disorders by correcting disease-causing mutations. Previously, we demonstrated the utility of a novel lipid nanoparticle (LNP) reagent for the multiplexed engineering of gene-edited CAR T cells, showing high cell viabilities, rapid onset editing, and potent CAR-mediated killing. In this current work, we demonstrate the capability of LNPs for the efficient and gentle delivery of genetic material to CD34+ HSPCs. The benefits of a non-viral LNP-mediated approach paves the way for the development of next generation gene therapy products in the HSC field. Methods: LNPs encapsulating S.p. Cas9 mRNA and CD33 or CD45 targeted guide RNA (sgRNA) were produced using the scalable NanoAssemblr® NxGen™ microfluidic platform technology ( Fig. 1A). The LNP composition was optimized for the gentle and efficient cargo delivery to HSC/HSPCs. Purified human CD34+ cells were cultured, stimulated, and treated by the direct addition of the RNA-LNPs. Various commercial cell sources were tested including mobilized peripheral blood and cord blood. Gene knockout and viability were assessed using flow cytometry, cell proliferation using an automated cell counter, and differentiation capacity by colony-forming unit (CFU) assays. Results: One-step addition of LNPs to CD34+ HSPCs resulted in 84 ± 6% CD33 and 81 ± 2% CD45 knockout efficiencies (n=6 CD34+ donors, Fig. 1B). After treatment, cells maintained on average 95 ± 3% absolute cell viability, compared to 99% viability of the untreated cells, Fig. 1C. Furthermore, LNP treated HSCs maintained excellent cell proliferation, with over >90% relative cell proliferation to the untreated controls. The CFU assays showed no significant change in relative lineage formation for both the RNA-LNPs and the empty LNP vehicle control ( Fig. 1D). Finally, LNP production was successfully scaled-up using microfluidics from discovery to pre-clinical scales. Conclusions: LNP-mediated CRISPR-Cas9 mRNA delivery is a promising approach for gene editing in HSPCs. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins for novel gene therapies. Furthermore, LNPs can be easily manufactured using microfluidics, enabling small-scale screening of RNA libraries and rapid scale-up for clinical translation.

Defining curative endpoints for sickle cell disease in the era of gene therapy and gene editing.

A growing number of gene therapy- and gene editing-based treatments for patients with sickle cell disease (SCD) are entering clinical trials. These treatments, designed to target the underlying cause of SCD, have the potential to provide functional cures, which until now were possible only through allogeneic hematopoietic stem cell transplant. However, as these novel approaches advance from early- to late-stage clinical trials, it is essential to identify physiologically and clinically relevant endpoints that can demonstrate the achievement of a functional cure for SCD. Here, we present an overview of the pathophysiology of SCD and current treatment options, review ongoing SCD clinical trials using gene therapy or gene editing approaches, and identify the most relevant endpoints for demonstrating the attainment of a functional cure for SCD.

Current Status and Challenges in Anti-Hepatitis B Virus Agents Based on Inactivation/Inhibition or Elimination of Hepatitis B Virus Covalently Closed Circular DNA.

There has been over half a century since the discovery of hepatitis B virus (HBV) to now, but approximately 300 million patients with chronic hepatitis B (CHB) still live in the world, resulting in about one million deaths every year. Although currently approved antivirals (e.g., nucleoside analogues) are effective at reducing HBV replication, they have almost no impact on the existing HBV covalently closed circular DNA (cccDNA) reservoir. HBV cccDNA is a critical obstacle to the complete elimination of the virus via antiviral therapy. The true cure of HBV infection requires the eradication of viral cccDNA from HBV-infected cells; thus, the development of new agents directly or indirectly targeting HBV cccDNA is urgently needed due to the limitations of current available drugs against HBV infection. In this regard, it is the major focus of current anti-HBV research worldwide via different mechanisms to either inactivate/inhibit (functional cure) or eliminate (complete cure) HBV cccDNA. Therefore, this review discussed and summarized recent advances and challenges in efforts to inactivate/silence or eliminate viral cccDNA using anti-HBV agents from different sources, such as small molecules (including epigenetic drugs) and polypeptides/proteins, and siRNA or gene-editing approaches targeting/attenuating HBV cccDNA via different mechanisms, as well as future directions that may be considered in efforts to truly cure chronic HBV infection. In conclusion, no breakthrough has been made yet in attenuating HBV cccDNA, although a number of candidates have advanced into the phase of clinical trials. Furthermore, the overwhelming majority of the candidates function to indirectly target HBV cccDNA. No outstanding candidate directly targets HBV cccDNA. Relatively speaking, CCC_R08 and nitazoxanide may be some of the most promising agents to clear HBV infection in small molecule compounds. Additionally, CRISPR-Cas9 systems can directly target HBV cccDNA for decay and demonstrate significant anti-HBV activity. Consequently, gene-editing approaches targeting HBV cccDNA may be one of the most promising means to achieve the core goal of anti-HBV therapeutic strategies. In short, more basic studies on HBV infection need to be carried out to overcome these challenges.

CRISPR-Cas attack of HIV-1 proviral DNA can cause unintended deletion of surrounding cellular DNA.

Although HIV replication can be effectively inhibited by antiretroviral therapy, this does not result in a cure as the available drugs do not inactivate the integrated HIV-1 DNA in infected cells. Consequently, HIV-infected individuals need lifelong therapy to prevent viral rebound. Several preclinical studies indicate that CRISPR-Cas gene-editing systems can be used to achieve permanent inactivation of the viral DNA. It was previously shown that this inactivation was due to small inactivating mutations at the targeted sites in the HIV genome and to excision or inversion of the viral DNA fragment between two target sites. We, here, demonstrate that CRISPR-Cas treatment also causes large unintended deletions, which can include surrounding chromosomal sequences. As the loss of chromosomal sequences may cause oncogenic transformation of the cell, such unintended large deletions form a potential safety risk in clinical application of this antiviral application and possibly all CRISPR-Cas gene-editing approaches.

Engineering a biomimetic system for hepatocyte-specific RNAi treatment of non-alcoholic fatty liver disease

RNA interference (RNAi) presents great potential against intractable liver diseases. However, the establishment of specific, efficient, and safe delivery systems targeting hepatocytes remains a great challenge. Herein, we described a promising hepatocytes-targeting system through integrating triantennary N-acetylgalactosamine (GalNAc)-engineered cell membrane with biodegradable mesoporous silica nanoparticles, which efficiently and safely delivered siRNA to hepatocytes and silenced the target PCSK9 gene expression for the treatment of non-alcoholic fatty liver disease. Having optimized the GalNAc-engineering strategy, insertion orders, and cell membrane source, we obtained the best-performing GalNAc-formulations allowing strong hepatocyte-specific internalization with reduced Kupffer cell capture, resulting in robust gene silencing and less hepatotoxicity when compared with cationic lipid-based GalNAc-formulations. Consequently, a durable reduction of lipid accumulation and damage was achieved by systemic administering siRNAs targeting PCSK9 in high-fat diet-fed mice, accompanied by displaying desirable safety profiles. Taken together, this GalNAc-engineering biomimetics represented versatile, efficient, and safe carriers for the development of hepatocyte-specific gene therapeutics, and prevention of metabolic diseases. Statement of SignificanceCompared to MSN@LP-GN3 (MC3-LNP), MSN@CM-GN3 exhibited strong hepatocyte targeting and Kupffer cell escaping, as well as good biocompatibility for safe and efficient siRNA delivery. Furthermore, siPCSK9 delivered by MSN@CM-GN3 reduced both serum and liver LDL-C, TG, TC levels and lipid droplets in HFD-induced mice, resulting in better performance than MSN/siPCSK9@LP-GN3 in terms of lipid-lowering effect and safety profiles. These findings indicated promising advantages of our biomimetic GN3-based systems for hepatocyte-specific gene delivery in chronic liver diseases. Our work addressed the challenges associated with the lower targeting efficiency of cell membrane-mimetic drug delivery systems and the immunogenicity of traditional GalNAc delivery systems. In conclusion, this study provided an effective and versatile approach for efficient and safe gene editing using ligand-integrated biomimetic nanoplatforms.

Cas-Clover Editing Efficiency and Off-Target Activity in Human Hepatocytes at the KLKB1 Locus

Cutting-edge gene editing holds enormous promise for tackling devastating genetic diseases like hereditary angioedema (HAE). Here, we describe the efficient inactivation of the gene encoding pre-kallikrein, KLKB1, using our proprietary Cas-CLOVER™ high-fidelity nuclease with our non-viral, lipid nanoparticle (LNP) delivery system. Genetic inactivation of KLKB1 is an alternative clinical approach that provides durable relief to both Type I and II HAE. HAE is a rare genetic disease characterized by subcutaneous and submucosal edema, with swelling of the upper respiratory tract posing a life-threatening situation. Type I and II HAE are the most common types and are caused by mutations in the SERPING1 gene, which leads to compromised production or function of the C1 protease inhibitor. Strategies for treatment and prophylaxis include the restoration of C1 inhibitor function, or downstream antagonism of active plasma kallikrein. Safe and effective gene editing of KLKB1 could be a viable alternative for patients not adequately responding to the current standard of care. However, gene editing approaches must demonstrate an exquisitely high level of fidelity for optimal safety. To demonstrate such an approach with Cas-CLOVER, multiple guide RNAs (gRNA) targeting the human KLKB1 gene were screened in human hepatoma cell lines to identify gRNA pairs with optimal editing. Next, we evaluated KLKB1 protein reduction in primary human hepatocytes (PHH) that were incubated with LNPs encapsulating Cas-CLOVER mRNA along with each gRNA pair. Lead candidate gRNAs showed robust KLKB1 editing in a dose-responsive manner, achieving >65% editing and >85% reduction in KLKB1 protein secreted into culture medium at 0.5 ug/mL (EC90). To evaluate Cas-CLOVER off-target activity, oligo incorporation by iGUIDE was carried out by a licensed contract research organization. In this assay, double-stranded oligodeoxyribonucleotides (dsODNs) were co-electroporated with Cas-CLOVER mRNA, along with our lead KLKB1 gRNA pair, in the Huh7 cell line, and candidate off-target sites were identified by Illumina next-generation sequencing. Off-target activity was assessed by amplicon-seq at the eight top sites nominated by iGUIDE. In PHHs treated with 0.5 ug/mL of Cas-CLOVER LNPs, off-target editing was detected in 3/8 sites at very low levels (<0.25%). Remarkably, this low level of off-target editing remained unchanged when PHHs were treated with 10-fold higher concentrations of Cas-CLOVER LNP. For further evaluation of our platform, we sought to determine KLKB1 editing efficiency and fidelity in a mouse model of liver humanization. TK-Nog mice engrafted with PHHs were treated with a single intravenous injection of an LNP formulation co-encapsulating Cas-CLOVER mRNA and our lead KLKB1 gRNA pair. Amplicon-seq analysis demonstrated that 60% of KLKB1 alleles in the liver were edited. Importantly, no off-target editing was detected among the top eight sites identified by iGUIDE, including the three off-target sites validated in cultured PHHs. Next, we evaluated efficacy and tissue specificity of our platform in wild type mice. C57BL/6 male and female mice were dosed with LNP encapsulating Cas-CLOVER mRNA and mouse Klkb1-targeting gRNAs. A single intravenous LNP injection achieved high Klkb1 editing (>50% of haploid genomes) in the liver and >80% reduction in serum pre-kallikrein levels. No Klkb1 editing was detected in gonads. In summary, these results highlight the efficacy and specificity of our high-fidelity Cas-CLOVER gene editing platform that enables targeted and therapeutically relevant kallikrein reduction in a fully non-viral manner. These data provide a promising foundation for the development of a highly specific gene editing therapy for HAE.

Donor Hemoglobin Genotype Does Not Impact Outcomes Following Matched Related Donor Hematopoietic Cell Transplantation for Sickle Cell Disease: A STAR Study

Sickle cell disease (SCD) is an inherited hemoglobin (Hb) disorder which leads to significant morbidity and early mortality. Hematopoietic cell transplantation (HCT) is the only curative treatment with long-term outcome data, demonstrating excellent symptom-free survival particularly when a matched related donor (MRD) is available. Most MRD have sickle cell trait (Hb AS), but trait donor HCT is considered acceptable by the FDA and without negative impact. Further, initial gene addition and editing approaches for SCD have resulted in a trait-like status with benefit. Sickle trait, however, is associated with complications such as papillary necrosis, bacteriuria, splenic infarction, and exercise-induced death. The primary aim of this study was to compare long-term outcomes following MRD HCT for SCD based on donor Hb genotype, to objectively describe any impact on outcomes. Retrospective data was available for this analysis on 182 SCD patients ≥1 year post MRD HCT at 10 participating Sickle Transplant Advocacy and Research (STAR) Alliance centers. Summary statistics were presented as mean (standard deviation) for continuous variables and count (%) for categorical variables. Comparisons were made between Hb AA and Hb AS donor groups using both parametric and non-parametric tests for continuous variables and Chi-square test/exact test for categorical variables, with p-value <0.05 set as significant. Amongst 182 patients undergoing MRD HCT, 67% (n=122) had a Hb AS donor ( Table). In the overall cohort, median age at HCT was 8.7 (4.8) years and most patients had HbSS (n=174; 96%) with a severe clinical phenotype (n=97; 54%). HCT was performed using bone marrow (n=166; 92%) with myeloablative conditioning (n=156; 86%) in the majority. Patients with a severe disease phenotype and Hb AS donor were significantly more likely to have recurrent vaso-occlusive pain crises as an indication for HCT (p=0.033), otherwise there were no differences in baseline patient or HCT characteristics between cohorts. With mean post-HCT follow up of 4.5 (3.9) and 5.1 (4.1) years in Hb AA and Hb AS donor cohorts (p=0.145), no significant differences were seen in recipient outcomes including time to neutrophil and platelet engraftment, number of or time to last platelet transfusion, myeloid or unsorted % donor chimerism, number with acute (day 100) or chronic GVHD (1 year), or time to discontinue immunosuppression (data not shown). Disease recurrence and need for 2 nd HCT were rare in both cohorts (AA: 2 [3%] and 2 [3%]; AS: 3 [3%] and 4 [3%]; p=0.665 and 1.0, respectively). Two (3%) and 5 (4%) patients died in the AA and AS cohorts, respectively (p=1.0). Aside from a statistically (but not clinically) significant difference in Hb at day 30 (p=0.044), no difference was seen in longitudinal Hb or markers of hemolysis (LDH and ARC; Figure). No significant difference was seen in median number of RBC units transfused (AA: 4.0 [2.0-6.0]; AS: 3.0 [2.0-6.0]; p=0.454) or days to last transfusion (AA: 23.0 [14.0-77.0]; AS: 22.0 [13.0-75.0]; p=0.86). SCD complications were rare post-HCT, with no difference seen between cohorts in number (%) with VOC (AA: 1 [2%; had 2 separate VOC events]; AS: 3 [3%]; p=1.0), ACS (none reported), chronic pain (AA: 0 [0%]; AS: 1 [<1%]; p=1.0), or other SCD-related complications (AA: 2 [3%]; AS: 9 [7%; had 11 separate other events]; p=0.456). Stroke occurred in a significantly higher proportion with an AA versus AS donor (5 [8%] and 2 [2%], respectively; p=0.04). No significant differences were seen in longitudinal (1 and 2 years and at last follow up) post-HCT cardiac (EF, SF, TRJ velocity), pulmonary (FEV1, FVC, DLCO), or renal (GFR) function between cohorts (data not shown). Our results indicate that in MRD HCT for SCD, AS and AA donors yield comparable long-term outcomes. In this retrospective and registry-based study design, detailed analyses did not show differences between the two cohorts at baseline or at any timepoint post-HCT. While there were some outcome differences between the cohorts, they were either not clinically significant or did not have a scientific rationale, and thus were likely due to chance alone (denoted by bolding in Table). Given the young age of our cohort at HCT and thus a smaller proportion with SCD-related organ dysfunction, our results may not be applicable to an older cohort with significant organ dysfunction which will require a separate analysis.

Developing a multiplex gene editing approach targeting viral infection

Developing a multiplex gene editing approach targeting viral infection

Control of social status by sex steroids: insights from teleost fishes

Many animals live in highly social environments, in which individuals must behave in a way that enables them to survive and live harmoniously among conspecifics. Dominance hierarchies are typical among social species and are essential for determining and preserving stability within social groups. Although there is considerable evidence that sex steroid hormones regulate behaviors associated with dominance, such as aggression and mating, fewer studies have examined the role of these hormones in controlling social status, especially in species that exhibit social hierarchies. Furthermore, despite this research, we know remarkably little about the precise neural and molecular mechanisms through which sex steroids modulate traits associated with social rank. Here, we review the neuroendocrine regulation of social status by sex steroids in teleost fishes, the largest and most diverse vertebrate group that shows extensive variation in reproductive systems and social structures between species. First, we describe the function of sex steroids and novel steroid-related genes that teleost fishes possess due to a lineage-specific whole-genome duplication event. Then, we discuss correlational, pharmacological, and molecular genetic studies on the control of social status by sex steroids in teleost fishes, including recent studies that have implemented gene editing technologies, such as CRISPR/Cas9. Finally, we argue that gene editing approaches in teleost studies, within both integrative and comparative frameworks, will be vital for elucidating the role of sex steroids in controlling social rank and characterizing their neural and molecular mechanisms of action. Collectively, ongoing and future research in these species will provide novel insight into the evolution of the regulation of social status by sex steroids and other neuroendocrine substrates across vertebrates.

Novel Approaches to Inhibition of HBsAg Expression from cccDNA and Chromosomal Integrants: A Review.

Hepatitis B virus (HBV) is a widely prevalent liver infection that can cause acute or chronic hepatitis. Although current treatment modalities are highly effective in the suppression of viral levels, they cannot eliminate the virus or achieve definitive cure. This is a consequence of the complex nature of HBV-host interactions. Major challenges to achieving sustained viral suppression include the presence of a high viral burden from the HBV DNA and hepatitis B surface antigen (HBsAg), the presence of reservoirs for HBV replication and antigen production, and the HBV-impaired innate and adaptive immune response of the host. Those therapeutic methods include cell entry inhibitors, HBsAg inhibitors, gene editing approaches, immune-targeting therapies and direct inhibitors of covalently closed circular DNA (cccDNA). Novel approaches that target these key mechanisms are now being studied in preclinical and clinical phases. In this review article, we provide a comprehensive review on mechanisms by which HBV escapes elimination from current treatments, and highlight new agents to achieve a definitive HBV cure.

Discrepancies in indel software resolution with somatic CRISPR/Cas9 tumorigenesis models

CRISPR/Cas9 gene editing has evolved from a simple laboratory tool to a powerful method of in vivo genomic engineering. As the applications of CRISPR/Cas9 technology have grown, the need to characterize the breadth and depth of indels generated by editing has expanded. Traditionally, investigators use one of several publicly-available platforms to determine CRISPR/Cas9-induced indels in an edited sample. However, to our knowledge, there has not been a cross-platform comparison of available indel analysis software in samples generated from somatic in vivo mouse models. Our group has pioneered using CRISPR/Cas9 to generate somatic primary mouse models of malignant peripheral nerve sheath tumors (MPNSTs) through genetic editing of Nf1. Here, we used sequencing data from the in vivo editing of the Nf1 gene in our CRISPR/Cas9 tumorigenesis model to directly compare results across four different software platforms. By analyzing the same genetic target across a wide panel of cell lines with the same sequence file, we are able to draw systematic conclusions about the differences in these software programs for analysis of in vivo-generated indels. Surprisingly, we report high variability in the reported number, size, and frequency of indels across each software platform. These data highlight the importance of selecting indel analysis platforms specific to the context that the gene editing approach is being applied. Taken together, this analysis shows that different software platforms can report widely divergent indel data from the same sample, particularly if larger indels are present, which are common in somatic, in vivo CRISPR/Cas9 tumor models.

Transcriptomic and genetic approaches reveal that low-light-induced disease susceptibility is related to cellular oxidative stress in tomato.

The impact of low light intensities on plant disease outbreaks represents a major challenge for global crop security, as it frequently results in significant yield losses. However, the underlying mechanisms of the effect of low light on plant defense are still poorly understood. Here, using an RNA-seq approach, we found that the susceptibility of tomato to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) under low light was associated with the oxidation-reduction process. Low light conditions exacerbated Pst DC3000-induced reactive oxygen species (ROS) accumulation and protein oxidation. Analysis of gene expression and enzyme activity of ascorbate peroxidase 2 (APX2) and other antioxidant enzymes revealed that these defense responses were significantly induced by Pst DC3000 inoculation under normal light, whereas these genes and their associated enzyme activities were not responsive to pathogen inoculation under low light. Additionally, the reduced ascorbate to dehydroascorbate (AsA/DHA) ratio was lower under low light compared with normal light conditions upon Pst DC3000 inoculation. Furthermore, the apx2 mutants generated by a CRISPR-Cas9 gene-editing approach were more susceptible to Pst DC3000 under low light conditions. Notably, this increased susceptibility could be significantly reduced by exogenous AsA treatment. Collectively, our findings suggest that low-light-induced disease susceptibility is associated with increased cellular oxidative stress in tomato plants. This study sheds light on the intricate relationship between light conditions, oxidative stress, and plant defense responses, and may pave the way for improved crop protection strategies in low light environments.

CUB domains are not required for OVCH2 function in sperm maturation in the mouse epididymis.

Ovochymase 2 (Ovch2) is an epididymis-specific gene that is required for male fertility. While a multitude of reproductive tract-specific genes required for male fertility have been identified, OVCH2 is thus far the first protein required for male fertility that contains Complement C1r/C1s, Uegf, Bmp1 (CUB) domains located in tandem in the C-terminus of the protein. Identifying the functional significance of this unique domain has implications in better understanding fertility and infertility and as a potential contraceptive target. The goals of these studies were to understand the influence and requirement of OVCH2 CUB domains in the localization and functional requirement of OVCH2 in sperm maturation and function. To this end, we performed in vivo localization analysis of OVCH2 and reproductive phenotype analysis of mice containing C-terminal FLAG tag on OVCH2, with either the entire protein intact, or CUB2 or both CUB1 and CUB2 genetically ablated. All mice were generated through the CRISPR/Cas9 gene editing approach. We found that OVCH2 is specifically expressed in the proximal caput epididymidis, and the absence of CUB2 did not affect this localization pattern. Although the absence of both CUB domains significantly reduced sperm motility and progressive motility, this effect was not manifested in a reduction in fertility over a 6-month period mating trial, which showed no significant differences between control and CUB deletant mice. Further, the absence of one or both CUB domains did not affect reproductive organ structure or sperm morphology. Our studies demonstrate that the CUB domains are not required for fertility in male mice, at least under the normal animal housing conditions our mice were tested in, and suggest that the enzymatic activity of the OVCH2 protease, in the absence of its CUB domains, is sufficient for normal sperm processing in the epididymis. Although our findings do not preclude the possibility that OVCH2 CUB domains are required under a yet-identified stress condition, our findings demonstrate that the most likely region for deleterious mutations in men with idiopathic infertility and the most vulnerable site for inhibition of OVCH2 protein function is in its protease domain, and not its CUB domains. Our findings have implications in the genetic screening of infertile men and the development of a novel non-hormonal male contraceptive by honing in on the more critical region of a functionally required protein.

Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma.

LRP1B remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, such as LRP1B. Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/Cas9 PX459 vectors. To assess LRP1B-gene-induced alterations and expression, PCR, Sanger DNA sequencing, and qRT-PCR were carried out. Three clones (clones B9, E6, and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size, and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones' secretome identified differentially expressed proteins that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.

CRISPR/Cas9-induced asap1a and asap1b co-knockout mutant zebrafish displayed abnormal embryonic development and impaired neutrophil migration

ASAP1 (Arf-GAP with SH3 domain, the ankyrin repeat and the PH domain) is the GTPase activating protein of the small G protein Arf. To understand more about the physiological functions of ASAP1 in vivo, we chose to use the zebrafish as an animal model, and analyzed the characterization of asap1 using loss-of-function studies. Here, two isoforms in zebrafish, asap1a and asap1b, were found to be homologous to human ASAP1, and the gene knockout zebrafish lines for asap1a and asap1b were established using the CRISPR/Cas9 technique with different insertions and deletions of bases. Zebrafish with asap1a and asap1b co-knockout showed a significant reduction in survival and hatching rates, as well as an increase in malformation rates during the early stages of development, while the asap1a or asap1b single knockout mutants did not affect the growth and development of individual zebrafish. Exploring the gene expression compensation between asap1a and asap1b using qRT-PCR, we found that asap1b had increased expression when asap1a was knocked out, showing a clear compensatory effect against asap1a knockout; In turn, asap1a did not have detectable compensating expression after asap1b knockout. Furthermore, the co-knockout homozygous mutants displayed impaired neutrophil migration to Mycobacterium marinum infection, and showed an increased bacterial load. Together, these are the first inherited asap1a and/or asap1b mutant zebrafish lines by the CRISPR/Cas9 gene editing approach, and by serving as useful models, they can significantly contribute to better annotation and follow-up physiological studies of human ASAP1.