The menace of severe adverse events and deaths associated with viral gene therapy and its potential solution.

Over the past decade, in vivo gene replacement therapy has significantly advanced, resulting in market approval of numerous therapeutics predominantly relying on adeno-associated viral vectors (AAV). While viral vectors have undeniably addressed several critical healthcare challenges, their clinical application has unveiled a range of limitations and safety concerns. This review highlights the emerging challenges in the field of gene therapy. At first, we discuss both the role of biological barriers in viral gene therapy with a focus on AAVs, and review current landscape of in vivo human gene therapy. We delineate advantages and disadvantages of AAVs as gene delivery vehicles, mostly from the safety perspective (hepatotoxicity, cardiotoxicity, neurotoxicity, inflammatory responses etc.), and outline the mechanisms of adverse events in response to AAV. Contribution of every aspect of AAV vectors (genomic structure, capsid proteins) and host responses to injected AAV is considered and substantiated by basic, translational and clinical studies. The updated evaluation of recent AAV clinical trials and current medical experience clearly shows the risks of AAVs that sometimes overshadow the hopes for curing a hereditary disease. At last, a set of established and new molecular and nanotechnology tools and approaches are provided as potential solutions for mitigating or eliminating side effects. The increasing number of severe adverse reactions and, sadly deaths, demands decisive actions to resolve the issue of immune responses and extremely high doses of viral vectors used for gene therapy. In response to these challenges, various strategies are under development, including approaches aimed at augmenting characteristics of viral vectors and others focused on creating secure and efficacious non-viral vectors. This comprehensive review offers an overarching perspective on the present state of gene therapy utilizing both viral and non-viral vectors.

Biomimetic approaches for targeting tumor-promoting inflammation.

With the ultimate goal of increasing tumor accumulation of therapeutics, various nanocarriers have been designed to overcome biological barriers encountered at each stage, from drug administration to the cancerous lesion. Stabilizing circulation and functionalization of the targeting surface impart high tumor accumulation properties to nanocarriers. However, various cells can recognize and infiltrate the tumor microenvironment more efficiently than synthetic carriers via overexpression of adhesive ligands, particularly in inflamed stroma of tumors. Thus, a new field of nanomedicine, called biomimicry, has evolved to generate nanoparticles with the same biological characteristics as cells that naturally infiltrate tumors. Revolutionary synthetic processes have been developed to transfer the cell membrane of leukocytes and mesenchymal cells to synthetic carriers. In addition, cells can generate their own "nanocarriers," known as exosomes, to transport molecular messages to distant sites, while biomimicry of viral and bacterial agents allows high targeting efficiency towards inflammatory immune cells. Alterations in the protein expression in cancer cells caused by inflammation can also be exploited for drug delivery. Finally, new developments in biomimetic drug delivery focus on turning the infiltrating cells into microcarriers that can actively perfuse the tumor and eventually release their therapeutic payload. In this review, we summarize recent developments in biomimetic drug delivery with a particular focus on targeting the tumor inflammatory microenvironment.

CRISPR-Cas systems for diagnosing infectious diseases.

Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas3, Cas9, Cas12, Cas13, Cas14 etc.) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, lateral flow assay detection and other. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, CARMEN or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing COVID-19) and outbreaks of zoonotic viruses (African Swine Fever Virus etc.) urgently need the developing and distribution of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

Proteolytic Resistance Determines Albumin Nanoparticle Drug Delivery Properties and Increases Cathepsin B, D, and G Expression.

Proteolytic activity is pivotal in maintaining cell homeostasis and function. In pathological conditions such as cancer, it covers a key role in tumor cell viability, spreading to distant organs, and response to the treatment. Endosomes represent one of the major sites of cellular proteolytic activity and very often represent the final destination of internalized nanoformulations. However, little information about nanoparticle impact on the biology of these organelles is available even though they represent the major location of drug release. In this work, we generated albumin nanoparticles with a different resistance to proteolysis by finely tuning the amount of cross-linker used to stabilize the carriers. After careful characterization of the particles and measurement of their degradation in proteolytic conditions, we determined a relationship between their sensitivity to proteases and their drug delivery properties. These phenomena were characterized by an overall increase in the expression of cathepsin proteases regardless of the different sensitivity of the particles to proteolytic degradation.

Anticancer Nanotherapeutics in Clinical Trials: The Work behind Clinical Translation of Nanomedicine.

The ultimate goal of nanomedicine has always been the generation of translational technologies that can ameliorate current therapies. Cancer disease represented the primary target of nanotechnology applied to medicine, since its clinical management is characterized by very toxic therapeutics. In this effort, nanomedicine showed the potential to improve the targeting of different drugs by improving their pharmacokinetics properties and to provide the means to generate new concept of treatments based on physical treatments and biologics. In this review, we considered different platforms that reached the clinical trial investigation, providing an objective analysis about their physical and chemical properties and the working mechanism at the basis of their tumoritr opic properties. With this review, we aim to help other scientists in the field in conceiving their delivering platforms for clinical translation by providing solid examples of technologies that eventually were tested and sometimes approved for human therapy.

Nanomedicine for increasing the oral bioavailability of cancer treatments.

Oral administration is an appealing route of delivering cancer treatments. However, the gastrointestinal tract is characterized by specific and efficient physical, chemical, and biological barriers that decrease the bioavailability of medications, including chemotherapeutics. In recent decades, the fields of material science and nanomedicine have generated several delivery platforms with high potential for overcoming multiple barriers associated to oral administration. This review describes the properties of several nanodelivery systems that improve the bioavailability of orally administered therapeutics, highlighting their advantages and disadvantages in generating successful anticancer oral nanomedicines.

Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus.

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence and chronic hepatitis B. CRISPR/Cas9 nucleases can specifically target HBV cccDNA for decay, but off-target effects of nucleases in the human genome limit their clinical utility. CRISPR/Cas9 systems from four different species were co-expressed in cell lines with guide RNAs targeting conserved regions of the HBV genome. CRISPR/Cas9 systems from Streptococcus pyogenes (Sp) and Streptococcus thermophilus (St) targeting conserved regions of the HBV genome blocked HBV replication and, most importantly, resulted in degradation of over 90% of HBV cccDNA by 6 days post-transfection. Degradation of HBV cccDNA was impaired by inhibition of non-homologous end-joining pathway and resulted in an erroneous repair of HBV cccDNA. HBV cccDNA methylation also affected antiviral activity of CRISPR/Cas9. Single-nucleotide HBV genetic variants did not impact anti-HBV activity of St CRISPR/Cas9, suggesting its utility in targeting many HBV variants. However, two or more mismatches impaired or blocked CRISPR/Cas9 activity, indicating that host DNA will not likely be targeted. Deep sequencing revealed that Sp CRISPR/Cas9 induced off-target mutagenesis, whereas St CRISPR/Cas9 had no effect on the host genome. St CRISPR/Cas9 system represents the safest system with high anti-HBV activity.

Gene Editing by Extracellular Vesicles.

CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.

Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases.

Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6-50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed by CRISPRa-induced APOBEC3A/APOBEC3B, while the remaining DNA templates harbored frequent deaminated nucleotides. To conclude, the CRISPRa approach could be readily utilized for manipulating innate immunity and investigating the effects of the key effector molecules on foreign nucleic acids.

Age-Related Impaired Efficacy of Bone Marrow Cell Therapy for Myocardial Infarction Reflects a Decrease in B Lymphocytes.

Treatment of myocardial infarction (MI) with bone marrow cells (BMCs) improves post-MI cardiac function in rodents. However, clinical trials of BMC therapy have been less effective. While most rodent experiments use young healthy donors, patients undergoing autologous cell therapy are older and post-MI. We previously demonstrated that BMCs from aged and post-MI donor mice are therapeutically impaired, and that donor MI induces inflammatory changes in BMC composition including reduced levels of B lymphocytes. Here, we hypothesized that B cell alterations in bone marrow account for the reduced therapeutic potential of post-MI and aged donor BMCs. Injection of BMCs from increasingly aged donor mice resulted in progressively poorer cardiac function and larger infarct size. Flow cytometry revealed fewer B cells in aged donor bone marrow. Therapeutic efficacy of young healthy donor BMCs was reduced by depletion of B cells. Implantation of intact or lysed B cells improved cardiac function, whereas intact or lysed T cells provided only minor benefit. We conclude that B cells play an important paracrine role in effective BMC therapy for MI. Reduction of bone marrow B cells because of age or MI may partially explain why clinical autologous cell therapy has not matched the success of rodent experiments.

Dead Cas Systems: Types, Principles, and Applications.

The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particular, Cas proteins devoid of nucleolytic activity (dead Cas proteins; dCas) can be used to deliver functional cargo to programmed sites in the genome. In this review, we describe current CRISPR systems used for developing different dCas-based molecular approaches and summarize their most significant applications. We conclude with comments on the state-of-art in the CRISPR field and future directions.

m6A Methylation in Regulation of Antiviral Innate Immunity.

The epitranscriptomic modification m6A is a prevalent RNA modification that plays a crucial role in the regulation of various aspects of RNA metabolism. It has been found to be involved in a wide range of physiological processes and disease states. Of particular interest is the role of m6A machinery and modifications in viral infections, serving as an evolutionary marker for distinguishing between self and non-self entities. In this review article, we present a comprehensive overview of the epitranscriptomic modification m6A and its implications for the interplay between viruses and their host, focusing on immune responses and viral replication. We outline future research directions that highlight the role of m6A in viral nucleic acid recognition, initiation of antiviral immune responses, and modulation of antiviral signaling pathways. Additionally, we discuss the potential of m6A as a prognostic biomarker and a target for therapeutic interventions in viral infections.

Swelling, Rupture and Endosomal Escape of Biological Nanoparticles Per Se and Those Fused with Liposomes in Acidic Environment.

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment.

Common Marmoset Cell Lines and Their Applications in Biomedical Research.

Common marmosets (Callithrix jacchus; CMs) are small New World primates widely used in biomedical research. Early stages of such research often include in vitro experiments which require standardized and well-characterized CM cell cultures derived from different tissues. Despite the long history of laboratory work with CMs and high translational potential of such studies, the number of available standardized, well-defined, stable, and validated CM cell lines is still small. While primary cells and immortalized cell lines are mostly used for the studies of infectious diseases, biochemical research, and targeted gene therapy, the main current applications of CM embryonic stem cells and induced pluripotent stem cells are regenerative medicine, stem cell research, generation of transgenic CMs, transplantology, cell therapy, reproductive physiology, oncology, and neurodegenerative diseases. In this review we summarize the data on the main advantages, drawbacks and research applications of CM cell lines published to date including primary cells, immortalized cell lines, lymphoblastoid cell lines, embryonic stem cells, and induced pluripotent stem cells.

Hydroxychloroquine Enhances Cytotoxic Properties of Extracellular Vesicles and Extracellular Vesicle-Mimetic Nanovesicles Loaded with Chemotherapeutics.

Because of their high biocompatibility, biological barrier negotiation, and functionalization properties, biological nanoparticles have been actively investigated for many medical applications. Biological nanoparticles, including natural extracellular vesicles (EVs) and synthetic extracellular vesicle-mimetic nanovesicles (EMNVs), represent novel drug delivery vehicles that can accommodate different payloads. In this study, we investigated the physical, biological, and delivery properties of EVs and EMNVs and analyzed their ability to deliver the chemotherapeutic drug doxorubicin. EMNVs and EVs exhibit similar properties, but EMNVs are more effectively internalized, while EVs show higher intracellular doxorubicin release activity. In addition, these nanotherapeutics were investigated in combination with the FDA-approved drug hydroxychloroquine (HCQ). We demonstrate that HCQ-induced lysosome destabilization and could significantly increase nanoparticle internalization, doxorubicin release, and cytotoxicity. Altogether, these data demonstrate that, from the delivery standpoint in vitro, the internalization of EMNVs and EVs and their payload release were slightly different and both nanotherapeutics had comparable cytotoxic performance. However, the synthesis of EMNVs was significantly faster and cost-effective. In addition, we highlight the benefits of combining biological nanoparticles with the lysosome-destabilizing agent HCQ that increased both the internalization and the cytotoxic properties of the particles.

Technological aspects of manufacturing and analytical control of biological nanoparticles.

Extracellular vesicles (EVs) are cell-derived biological nanoparticles that gained great interest for drug delivery. EVs have numerous advantages compared to synthetic nanoparticles, such as ideal biocompatibility, safety, ability to cross biological barriers and surface modification via genetic or chemical methods. On the other hand, the translation and the study of these carriers resulted difficult, mostly because of significant issues in up-scaling, synthesis and impractical methods of quality control. However, current manufacturing advances enable EV packaging with any therapeutic cargo, including DNA, RNA (for RNA vaccines and RNA therapeutics), proteins, peptides, RNA-protein complexes (including gene-editing complexes) and small molecules drugs. To date, an array of new and upgraded technologies have been introduced, substantially improving EV production, isolation, characterization and standardization. The used-to-be "gold standards" of EV manufacturing are now outdated, and the state-of-art requires extensive revision. This review re-evaluates the pipeline for EV industrial production and provides a critical overview of the modern technologies required for their synthesis and characterization.

Transient and tunable CRISPRa regulation of APOBEC/AID genes for targeting hepatitis B virus.

APOBEC/AID cytidine deaminases play an important role in innate immunity and antiviral defenses and were shown to suppress hepatitis B virus (HBV) replication by deaminating and destroying the major form of HBV genome, covalently closed circular DNA (cccDNA), without toxicity to the infected cells. However, developing anti-HBV therapeutics based on APOBEC/AID is complicated by the lack of tools for activating and controlling their expression. Here, we developed a CRISPR-activation-based approach (CRISPRa) to induce APOBEC/AID transient overexpression (>4-800,000-fold increase in mRNA levels). Using this new strategy, we were able to control APOBEC/AID expression and monitor their effects on HBV replication, mutation, and cellular toxicity. CRISPRa prominently reduced HBV replication (∼90%-99% decline of viral intermediates), deaminated and destroyed cccDNA, but induced mutagenesis in cancer-related genes. By coupling CRISPRa with attenuated sgRNA technology, we demonstrate that APOBEC/AID activation can be precisely controlled, eliminating off-site mutagenesis in virus-containing cells while preserving prominent antiviral activity. This study untangles the differences in the effects of physiologically expressed APOBEC/AID on HBV replication and cellular genome, provides insights into the molecular mechanisms of HBV cccDNA mutagenesis, repair, and degradation, and, finally, presents a strategy for a tunable control of APOBEC/AID expression and for suppressing HBV replication without toxicity.

Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9.

CRISPR-Cas9 systems can directly target the hepatitis B virus (HBV) major genomic form, covalently closed circular DNA (cccDNA), for decay and demonstrate remarkable anti-HBV activity. Here, we demonstrate that CRISPR-Cas9-mediated inactivation of HBV cccDNA, frequently regarded as the "holy grail" of viral persistence, is not sufficient for curing infection. Instead, HBV replication rapidly rebounds because of de novo formation of HBV cccDNA from its precursor, HBV relaxed circular DNA (rcDNA). However, depleting HBV rcDNA before CRISPR-Cas9 ribonucleoprotein (RNP) delivery prevents viral rebound and promotes resolution of HBV infection. These findings provide the groundwork for developing approaches for a virological cure of HBV infection by a single dose of short-lived CRISPR-Cas9 RNPs. Blocking cccDNA replenishment and re-establishment from rcDNA conversion is critical for completely clearing the virus from infected cells by site-specific nucleases. The latter can be achieved by widely used reverse transcriptase inhibitors.

Hepatitis B virus markers in hepatitis B surface antigen negative patients with pancreatic cancer: Two case reports.

BACKGROUND: Hepatitis B virus (HBV) is a known carcinogen that may be involved in pancreatic cancer development. Detection of HBV biomarkers [especially expression of HBV regulatory X protein (HBx)] within the tumor tissue may provide direct support for this. However, there is still a lack of such reports, particularly in non-endemic regions for HBV infection. Here we present two cases of patients with pancreatic ductal adenocarcinoma, without a history of viral hepatitis, in whom the markers of HBV infection were detected in blood and in the resected pancreatic tissue. CASE SUMMARY: The results of examination of two patients with pancreatic cancer, who gave informed consent for participation and publication, were the source for this study. Besides standards of care, special examination to reveal occult HBV infection was performed. This included blood tests for HBsAg, anti-HBc, anti-HBs, HBV DNA, and pancreatic tissue examinations with polymerase chain reaction for HBV DNA, pregenomic HBV RNA (pgRNA HBV), and covalently closed circular DNA HBV (cccDNA) and immunohistochemistry staining for HBxAg and Ki-67. Both subjects were operated on due to pancreatic ductal adenocarcinoma and serum HBsAg was not detected. However, in both of them anti-HBc antibodies were detected in blood, although HBV DNA was not found. Examination of the resected pancreatic tissue gave positive results for HBV DNA, expression of HBx, and active cellular proliferation by Ki-67 index in both cases. However, HBV pgRNA and cccDNA were detected only in case 1. CONCLUSION: These cases may reflect potential involvement of HBV infection in the development of pancreatic cancer.

CRISPR/Cas and Hepatitis B Therapy: Technological Advances and Practical Barriers.

After almost a decade of using CRISPR/Cas9 systems to edit target genes, CRISPR/Cas9 and related technologies are rapidly moving to clinical trials. Hepatitis B virus (HBV), which causes severe liver disease, cannot be cleared by modern antivirals, but represents an ideal target for CRISPR/Cas9 systems. Early studies demonstrated very high antiviral potency of CRISPR/Cas9 and supported its use for developing a cure against chronic HBV infection. This review discusses the key issues that must be solved to make CRISPR/Cas9 an anti-HBV therapy.