External Guide Sequence Effectively Suppresses the Gene Expression and Replication of Herpes Simplex Virus 2.

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications.

Point-of-Care Testing for Infectious Diseases Based on Class 2 CRISPR/Cas Technology.

The early detection of infectious diseases and microorganisms is critical for effective disease treatment, control, and prevention. Currently, nucleic acid testing and antigen-antibody serum reaction are the two methods most commonly used for the detection of infectious diseases. The former is highly accurate, specific, and sensitive, but it is time-consuming, expensive, and has special technician and instrument requirements. The latter is rapid and economical, but it may not be accurate and sensitive enough. Therefore, it is necessary to develop a quick and on-site diagnostic test for point-of-care testing (POCT) to enable the clinical detection of infectious diseases that is accurate, sensitive, convenient, cheap, and portable. Here, CRISPR/Cas-based detection methods are detailed and discussed in depth. The powerful capacity of these methods will facilitate the development of diagnostic tools for POCT, though they still have some limitations. This review explores and highlights POCT based on the class 2 CRISPR/Cas assay, such as Cas12 and Cas13 proteins, for the detection of infectious diseases. We also provide an outlook on perspectives, multi-application scenarios, clinical applications, and limitations for POCT based on class 2 CRISPR/Cas technology.

Suppressing Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression and Replication by RNase P Ribozyme.

Kaposi's sarcoma, an AIDS-defining illness, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus. In this study, we engineered ribozymes derived from ribonuclease P (RNase P) catalytic RNA with targeting against the mRNA encoding KSHV immediate early replication and transcription activator (RTA), which is vital for KSHV gene expression. The functional ribozyme F-RTA efficiently sliced the RTA mRNA sequence in vitro. In cells, KSHV production was suppressed with ribozyme F-RTA expression by 250-fold, and RTA expression was suppressed by 92-94%. In contrast, expression of control ribozymes hardly affected RTA expression or viral production. Further studies revealed both overall KSHV early and late gene expression and viral growth decreased because of F-RTA-facilitated suppression of RTA expression. Our results indicate the first instance of RNase P ribozymes having potential for use in anti-KSHV therapy.

A RNase P Ribozyme Inhibits Gene Expression and Replication of Hepatitis B Virus in Cultured Cells.

Hepatitis B virus (HBV), an international public health concern, is a leading viral cause of liver disease, such as hepatocellular carcinoma. Sequence-specific ribozymes derived from ribonuclease P (RNase P) catalytic RNA are being explored for gene targeting applications. In this study, we engineered an active RNase P ribozyme, M1-S-A, targeting the overlapping region of HBV S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA), all deemed essential for viral infection. Ribozyme M1-S-A cleaved the S mRNA sequence efficiently in vitro. We studied the effect of RNase P ribozyme on HBV gene expression and replication using the human hepatocyte HepG2.2.15 culture model that harbors an HBV genome and supports HBV replication. In these cultured cells, the expression of M1-S-A resulted in a reduction of more than 80% in both HBV RNA and protein levels and an inhibition of about 300-fold in the capsid-associated HBV DNA levels when compared to the cells that did not express any ribozymes. In control experiments, cells expressing an inactive control ribozyme displayed little impact on HBV RNA and protein levels, and on capsid-associated viral DNA levels. Our study signifies that RNase P ribozyme can suppress HBV gene expression and replication, implying the promise of RNase P ribozymes for anti-HBV therapy.

Challenges and Recent Advancements in COVID-19 Vaccines.

Vaccination is the most effective method for the prevention of COVID-19 caused by SARS-CoV-2, which is still a global epidemic. However, the evolution of SARS-CoV-2 is so rapid that various variants, including the Alpha, Beta, Gamma, Delta, and Omicron variants, have emerged, lowering the protection rate of vaccines and even resulting in breakthrough infections. Additionally, some rare but severe adverse reactions induced by COVID-19 vaccines may raise safety concerns and hinder vaccine promotion; however, clinical studies have shown that the benefits of vaccination outweigh the risks caused by adverse reactions. Current vaccines approved with emergency use authorization (EUA) were originally adaptive for adults only, and infants, children, and adolescents are not included. New-generation vaccines are needed to overcome the challenges of limited adaptive age population, breakthrough infection (mainly due to virus variant emergencies), and critical adverse reactions. Fortunately, some advances in COVID-19 vaccines have been obtained regarding enlarged adaptive populations for clinical applications, such as the Pfizer/BioNTech vaccine and the Moderna vaccine. In this article, we provide a review on the challenges and recent advancements in COVID-19 vaccines. The development of next-generation COVID-19 vaccines should lay emphasis on the expansion of adaptive age populations in all individuals, the induction of immune responses to viral variants, the avoidance or alleviation of rare but potentially critical adverse reactions, and the discovery of subunit vaccines with adjuvants encapsulated in nanoparticles.

Exploration of Correlations between COVID-19 Vaccination Choice and Public Mental Health Using Google Trend Search.

Since the global COVID-19 pandemic has great impact on human health and life style, the vaccination is the most effective method for disease control and prevention. However, not all people are willing to be vaccinated because some critical factors affect vaccination aspiration and vaccine choice of the public population. Among these factors, public mental health belongs to a political issue. In this study, Google Trend Search was used to explore the correlation between COVID-19 vaccination choice and public mental health during the period from August/2020 to December/2021. The results suggested that the main public concerns of COVID-19-related mental illnesses are positively correlated with the new cases amount but are negatively correlated with total cases and vaccinated cases amount. Moreover, the results support that the public population took more interest in the Pfizer/BNT COVID vaccine and Moderna COVID vaccine than the AstraZeneca COVID vaccine. Our study shows that investigations of the public mental health should be set up and conducted widely. A complete vaccination program combined with a policy for the improvement of public mental health are very effective for the control and prevention of COVID-19.

Challenges, Recent Advances and Perspectives in the Treatment of Human Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) is ubiquitous worldwide and elicits global health problems. The diseases associated with HCMV are a serious threat to humans, especially for the sick, infant, elderly and immunocompromised/immunodeficient individuals. Although traditional antiviral drugs (e.g., ganciclovir, valganciclovir, cidofovir, foscarnet) can be used to treat or prevent acute HCMV infections, their efficacy is limited because of toxicity, resistance issues, side effects and other problems. Fortunately, novel drugs (e.g., letermovir and maribavir) with less toxicity and drug/cross-resistance have been approved and put on the market in recent years. The nucleic acid-based gene-targeting approaches including the external guide sequences (EGSs)-RNase, the clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPRs-associated protein 9 (Cas9) system and transcription activator-like effector nucleases (TALENs) have been investigated to remove both lytic and latent CMV in vitro and/or in vivo. Cell therapy including the adoptive T cell therapy (ACT) and immunotherapy have been tried against drug-resistant and recurrent HCMV in patients receiving hematopoietic stem cell transplantation (HSCT) or solid organ transplant (SOT), and they have also been used to treat glioblastoma (GBM) associated with HCMV infections. These newly developed antiviral strategies are expected to yield fruitful results and make a significant contribution to the treatment of HCMV infections. Despite this progress, the nucleic acid-based gene-targeting approaches are still under study for basic research, and cell therapy is adopted in a small study population size or only successful in case reports. Additionally, no current drugs have been approved to be indicated for latent infections. Therefore, the next strategy is to develop antiviral strategies to elevate efficacy against acute and/or latent infections and overcome challenges such as toxicity, resistance issues, and side effects. In this review, we would explore the challenges, recent advances and perspectives in the treatment of HCMV infections. Furthermore, the suitable therapeutic strategies as well as the possibility for compassionate use would be evaluated.

[Manual therapy combined with posterior percutaneous endoscopic cervical decompression for the treatment of cervical spondylotic radiculopathy].

OBJECTIVE: To explore the clinical efficacy and safety of manual therapy combined with posterior percutaneous endoscopic cervical decompression(PECD) in the treatment of intractable cervical spondylotic radiculopathy. METHODS: From May 2016 to May 2018, 23 CSR patients who responded poorly to conservative treatment for at least 6 weeks underwent the combination management. Firstly, the patients received the posterior percutaneous endoscopic cervical decompression routine care for the following 4 weeks and manual therapy for another 4 weeks. A total of 23 patients were followed up, including 14 males and 9 females, the age ranged from 29 to 78 years old with an average of (50.30±12.28) years, the course of disease was 3 to 24 months with an average of (9.74±5.76) months. The lesion segment involved C4,5 in 4 cases, C5,6 in 13 cases, C6,7 in 6 cases. The visual analogue scale (VAS), neck disability index (NDI), changes of cervical physiological curvature and interbody stability, adverse events were observed before and after operation. The follow-up time points were before operation, 1 day after operation and 1, 3 and 6 months after operation. RESULTS: All patients successfully completed the operation and manual treatment for 4 to 8 times. Among the 29 cases, 23 patients were followed up for more than 6 months. There was no spinal cord and nerve root injury during the treatment and follow-up. Operation time was from 80 to 120 min with a median of 90 min;intraoperative blood loss was from 35 to 80 ml with a median of 50 ml. NDI, VAS of neck, shoulder and arm each period after operation were significantly lower than those before PECD(P<0.05), while there were no significant improvement in cervical physiological curvature and target segment intervertebral space height(P>0.05);there was no significant change in interbody stability (P>0.05). After received the manual therapy, NDI significantly decreased (P<0.05), however, there was no significant difference in VAS of neck, shoulder and arm, physiological curvature of cervical spine and intervertebral space height of target segment compared with that before manual treatment (P>0.05);there was no significant change in interbody stability (P>0.05). CONCLUSION: Manual therapy combined with PECD in the treatment of intractable cervical spondylotic radiculopathy can not only quickly improve the symptoms, but also alleviate the residual symptoms after PECD safely and effectively, and can not cause obvious signs of accelerated instability of cervical adjacent segments in the short term.

The Immunotherapy for Colorectal Cancer, Lung Cancer and Pancreatic Cancer.

Immunotherapy is a novel anti-cancer method which employs a different mechanism to conventional treatment. It has become a significant strategy because it provides a better or an alternative option for cancer patients. Recently, immunotherapy has been increasingly approved for the treatment of cancer; however, it has various limitations; for instance, it is only suitable for specific patients, the response rate is still low in most cases, etc. Colorectal cancer, lung cancer and pancreatic cancer are known as three major death-causing cancers in most countries. In this review, we discuss immunotherapeutic treatment for these three cancers, and consider the option, prospects and limitations of immunotherapy. The development of immunotherapy should focus on the discovery of biomarkers to screen suitable patients, new targets on tumors, neoadjuvant immunotherapy and the combination of immunotherapy with conventional therapeutic methods. We can expect that immunotherapy potentially will develop as one of the best therapies for patients with advanced cancer or poor responses to traditional methods.

CRISPR based genome editing and removal of human viruses.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins 9 (Cas9), a gene-editing technology, has been extensively applied as a tool for genetic engineering in basic research. Efficient genome engineering has been performed in viruses, human cells, bacteria, fungi, plants and animals, etc. Currently, it has been employed to edit human viruses for studying viral molecular biology, pathogenesis and oncogenesis, and facilitate the development of antiviral agents and vaccine. The virus is ubiquitous worldwide and elicits global health problems, many human diseases are associated with virus infections. Although traditional drugs can be used to treat or prevent productive viral infections, their efficacy is limited because of toxicity, side effects and other problems. Additionally, no current drugs are approved to be indicated for latent infections. Therefore, the next highlight is to develop antiviral approaches to against both productive and latent infections. Fortunately, CRISPR has been successfully applied in the removal of human viruses ex vivo and/or in vivo, and has the potential to be used to manufacture antiviral agents for clinical application. CRISPR/Cas9 is promising in applications, even though some technical challenges, safety concerns, ethic concerns need to be improved. In this article, the discovery and application of genome editing and removal of human viruses based on CRISPR are explored. Additionally, we evaluate the prospects and limitations of this novel antiviral strategies.

Alcohol Addiction, Gut Microbiota, and Alcoholism Treatment: A Review.

Alcohol addiction is a leading risk factor for personal death and disability. In 2016, alcohol use caused 2.2% of female deaths and 6.8% of male deaths, and disability-adjusted life years (DALYs) were 2.3% in female and 8.9% in male. Individuals with alcohol use disorder are at high risk of anxiety, depression, impaired cognition performance, and illicit drug use and are comorbid with liver disease, such as alcoholic hepatitis and liver cirrhosis, which is a major cause of personal death and disability worldwide. Psychological interventions, such as cognitive behavior therapy and motivational interviewing, as well as medical treatments, such as disulfiram, naltrexone, acamprosate, and nalmefene, are used for the treatment of alcohol addiction in Europe and the United States. However, the effect of current interventions is limited, and the need for additional interventions is substantial. Alcohol use impairs the intestinal barrier and causes changes to the intestinal permeability as well as the gut microbiota composition. Emerging studies have tried to reveal the role of the gut-brain axis among individuals with alcohol use disorder with or without alcohol liver disease. Bacterial products penetrate the impaired intestinal barrier and cause central inflammation; changes to the gut microbiota impair enterohepatic circulation of bile acids; alcohol abuse causes shortage of vital nutrients such as thiamine. Several studies have suggested that probiotics, through either oral administration or fecal microbiota transplantation, increased intestinal levels of potentially beneficial bacteria such as bifidobacteria and lactobacilli, improving the levels of liver-associated enzymes in patients with mild alcoholic hepatitis, and demonstrating beneficial psychotropic effects on anxiety and depression. In addition to medications for alcohol addiction, gene editing therapy such as clustered regularly interspaced short palindromic repeats (CRISPRs) may be another potential research target. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which are associated with ADH and ALDH genes, are major enzymes involved in alcohol metabolism, and gene editing approaches may have the potential to directly modify specific genes to treat alcoholism caused by genetic defects. Further research is needed to study the effect of the combined treatment for alcohol addiction.

Novel Antiviral Strategies in the Treatment of COVID-19: A Review.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is still a global public health problem for humans. It has caused more than 10,000,000 infections and more than 500,000 deaths in the world so far. Many scientists have tried their best to discover safe and effective drugs for the treatment of this disease; however, there are still no approved standard therapeutics or effective antiviral drugs on the market. Many new drugs are being developed, and several traditional drugs that were originally indicated or proposed for other diseases are likely to be effective in treating COVID-19, but their safety and efficacy are controversial, under study, or in clinical trial phases. Fortunately, some novel antiviral strategies, such as convalescent plasma, clustered regularly interspaced short palindromic repeats (CRISPR), and mesenchymal stem cell (MSC) therapy, potentially offer an additional or alternative option or compassionate use for the people suffering from COVID-19, especially for critically ill patients, although their safety and efficacy are also under study. In this review, we explore the applications, possible mechanisms, and efficacy in successful cases using convalescent plasma, CRISPR, and MSC therapy for COVID-19 treatment, respectively. Furthermore, the perspectives and limitations of these novel antiviral strategies are evaluated.

Potential Therapeutic Approaches Against Brain Diseases Associated with Cytomegalovirus Infections.

Cytomegalovirus (CMV) is one of the major human health threats worldwide, especially for immunologically comprised patients. CMV may cause opportunistic infections, congenital infections, and brain diseases (e.g., mental retardation and glioblastoma). The etiology of brain diseases associated with human CMV (HCMV) infections is usually complex and it is particularly difficult to treat because HCMV has a life-long infection in its hosts, high mutation rate, and latent infections. Moreover, it is almost impossible to eradicate latent viruses in humans. Although there has been progress in drug discovery recently, current drugs used for treating active CMV infections are still limited in efficacy due to side effects, toxicity, and viral resistance. Fortunately, letermovir which targets the HCMV terminase complex rather than DNA polymerase with fewer adverse reactions has been approved to treat CMV infections in humans. The researchers are focusing on developing approaches against both productive and latent infections of CMV. The gene or RNA targeting approaches including the external guide sequences (EGSs)-RNase, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and transcription activator-like effector nucleases (TALENs) are being investigated to remove acute and/or latent CMV infections. For the treatment of glioblastoma, vaccine therapy through targeting specific CMV antigens has improved patients' survival outcomes significantly and immunotherapy has also emerged as an alternative modality. The advanced research for developing anti-CMV agents and approaches is promising to obtain significant outcomes and expecting to have a great impact on the therapy of brain diseases associated with CMV infections.

Antiviral Agents as Therapeutic Strategies Against Cytomegalovirus Infections.

Cytomegalovirus (CMV) is a threat to human health in the world, particularly for immunologically weak patients. CMV may cause opportunistic infections, congenital infections and central nervous system infections. CMV infections are difficult to treat due to their specific life cycles, mutation, and latency characteristic. Despite recent advances, current drugs used for treating active CMV infections are limited in their efficacy, and the eradication of latent infections is impossible. Current antiviral agents which target the UL54 DNA polymerase are restricted because of nephrotoxicity and viral resistance. CMV also cannot be prevented or eliminated with a vaccine. Fortunately, letermovir which targets the human CMV (HCMV) terminase complex has been recently approved to treat CMV infections in humans. The growing point is developing antiviral agents against both lytically and latently infected cells. The nucleic acid-based therapeutic approaches including the external guide sequences (EGSs)-RNase, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and transcription activator-like effector nucleases (TALENs) are being explored to remove acute and/or latent CMV infections. HCMV vaccine is being developed for prophylaxis. Additionally, adoptive T cell therapy (ACT) has been experimentally used to combate drug-resistant and recurrent CMV in patients after cell and/or organ transplantation. Developing antiviral agents is promising in this area to obtain fruitful outcomes and to have a great impact on humans for the therapy of CMV infections.

Opioid Addiction, Genetic Susceptibility, and Medical Treatments: A Review.

Opioid addiction is a chronic and complex disease characterized by relapse and remission. In the past decade, the opioid epidemic or opioid crisis in the United States has raised public awareness. Methadone, buprenorphine, and naloxone have proven their effectiveness in treating addicted individuals, and each of them has different effects on different opioid receptors. Classic and molecular genetic research has provided valuable information and revealed the possible mechanism of individual differences in vulnerability for opioid addiction. The polygenic risk score based on the results of a genome-wide association study (GWAS) may be a promising tool to evaluate the association between phenotypes and genetic markers across the entire genome. A novel gene editing approach, clustered, regularly-interspaced short palindromic repeats (CRISPR), has been widely used in basic research and potentially applied to human therapeutics such as mental illness; many applications against addiction based on CRISPR are currently under research, and some are successful in animal studies. In this article, we summarized the biological mechanisms of opioid addiction and medical treatments, and we reviewed articles about the genetics of opioid addiction, the promising approach to predict the risk of opioid addiction, and a novel gene editing approach. Further research on medical treatments based on individual vulnerability is needed.

Potential Application of TALENs against Murine Cytomegalovirus Latent Infections.

Cytomegalovirus (CMV) infections are still a global health problem, because the latent viruses persist in humans and cause recurring disease. Currently, there are no therapies for CMV latent infections and the therapies for active infections are limited by side effects and other problems. It is impossible to eradicate latent viruses in animals. HCMV (human CMV) is specific to human diseases; however, it is difficult to study HCMV due to its host specificity and long life cycle. Fortunately, MCMV (murine CMV) provides an excellent animal model. Here, three specific pairs of transcription activator-like effector nuclease (TALEN) plasmids (MCMV1-2, 3-4, and 5-6) were constructed to target the MCMV M80/80.5 sequence in order to test their efficacy in blocking MCMV lytic replication in NIH3T3 cell culture. The preliminary data showed that TALEN plasmids demonstrate specific targeting and cleavage in the MCMV M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the viral infection. The most specific pairs of TALEN plasmids (MCMV3-4) were further used to confirm the negative regulation of latent MCMV replication and gene expression in Balb/c mice. The injection of specific TALEN plasmids caused significant inhibition in the copy number level of immediately early gene (ie-1) DNA in five organs of mice, when compared with the controls. The result demonstrated that TALENs potentially provide an effective strategy to remove latent MCMV in animals.

Narcotic Addiction in Failed Back Surgery Syndrome.

Back pain is a common health problem that reduces the quality of life for human beings worldwide. Several treatment modalities have been reported as effective for pain relief. Generally, patients often undergo surgical interventions as pain becomes intractable, after conservative treatment. With advances in surgical techniques, those choosing spinal surgery as an option have increased over time, and instrumentation is more popular than it was years ago. However, some patients still have back pain after spinal operations. The number of patients classified as having failed back surgery syndrome (FBSS) has increased over time as has the requirement for patients receiving long-term analgesics. Because pain relief is regarded as a human right, narcotics were prescribed more frequently than before. Narcotic addiction in patients with FBSS has become an important issue. Here, we review the prevalence of FBSS, the mechanism of narcotic addiction, and their correlations. Additionally, several potentially effective strategies for the prevention and treatment of narcotic addiction in FBSS patients are evaluated and discussed.

Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections.

The CRISPR/Cas9 system has been applied in the genome editing and disruption of latent infections for herpesviruses such as the herpes simplex virus, Epstein⁻Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus. CRISPR/Cas9-directed mutagenesis can introduce similar types of mutations to the viral genome as can bacterial artificial chromosome recombination engineering, which maintains and reconstitutes the viral genome successfully. The cleavage mediated by CRISPR/Cas9 enables the manipulation of disease-associated viral strains with unprecedented efficiency and precision. Additionally, current therapies for herpesvirus productive and latent infections are limited in efficacy and cannot eradicate viruses. CRISPR/Cas9 is potentially adapted for antiviral treatment by specifically targeting viral genomes during latent infections. This review, which focuses on recently published progress, suggests that the CRISPR/Cas9 system is not only a useful tool for basic virology research, but also a promising strategy for the control and prevention of herpesvirus latent infections.

Cell Therapy Regulation in Taiwan.

Cell therapy is not only a novel medical practice but also a medicinal product [cell therapy product (CTP)]. More and more CTPs are being approved for marketing globally because of the rapid development of biomedicine in cell culture, preservation, and preparation. However, regulation is the most important criterion for the development of CTPs. Regulations must be flexible to expedite the process of marketing for new CTPs. Recently, the Taiwan Food and Drug Administration (TFDA) updated the related regulations such as regulation of development, current regulatory framework and process, and the application and evaluation processes. When the quality of CTPs has been improved significantly, their safety and efficacy are further ensured. The treatment protocol, a new design for adaptive licensing to current clinical practice, is a rapid process for patients with life-threatening diseases or serious conditions for which there are no suitable drugs, medical devices, or other therapeutic methods available. The hospital can submit the treatment protocol to apply for cell therapy as a medical practice, which may result in easier and faster cell therapy development, and personalized treatment for individual patients will evolve quickly.