Although treatment outcomes of glioblastoma, the most malignant central nervous system (CNS) tumor, has improved in the past decades, it is still incurable, and survival has only slightly improved. Advances in molecular biology and genetics have completely transformed our understanding of glioblastoma. Multiple classifications and different diagnostic methods were made according to novel molecular markers. Discovering tumor heterogeneity only partially explains the ineffectiveness of current anti-proliferative therapies. Dynamic heterogeneity secures resistance to combined oncotherapy. As tumor growth proceeds, new therapy-resistant sub clones emerge. Liquid biopsy is a new and promising diagnostic tool that can step up with the dynamic genetic change. Getting a ’real-time’ picture of a specific tumor, anti-invasion and multi-target treatment can be designed. During invasion to the peri-tumoral brain tissue, glioma cells interact with the extracellular matrix components. The expressional levels of these matrix molecules give a characteristic pattern, the invasion spectrum, which possess vast diagnostical, predictive and prognostic information. It is a huge leap forward combating tumor heterogeneity and searching for novel therapies. Using the invasion spectrum of a tumor sample is a novel tool to distinguish between histological subtypes, specifying the tumor grades or different prognostic groups. Moreover, new therapeutic methods and their combinations are under trial. These are crucial steps towards personalized oncotherapy. Glioblastoma multiforme (GBM) is the most malignant tumor in the CNS and one of the tumors with the poorest prognosis considering other locations as well. It is incurable and inevitably fatal; hence, therapy can only hinder its progression and prolong survival with a tolerable quality of life. GBM represents 14.7% of all primary CNS tumors and 47.7% of all primary malignant CNS neoplasms. Its incidence rate increases with age, with the highest rate in individuals between the ages of 75 and 84 years, along with a considerable prevalence in the middle-aged population. Most of the diagnosed patients is usually aged 45–65. Younger age is reported to associate with a better outcome, and male patients have a better prognosis compared to female ones. Its aggressiveness results in a poor 5-year survival rate of 5.6%. Despite intensive research in the last decades, the OS still remains under expectations. During the course of the disease, the majority of patients experience some degree of impaired self-care, and a considerable number requires frequent medical care and special assistance, placing a significant social and economic burden onto the healthcare system. GBM can be characterized with vast intra- and inter-tumoral genetic heterogeneity. Indeed, its name, ’multiforme’, refers to the histological diversity seen in specimens. A total of four histologic criteria are needed for a diagnosis: microvascular proliferation, presence of necrosis, nuclear atypia and high mitotic activity. In the past, glioblastoma was the sole diagnosis for all that fulfilled these criteria; thus, this entity comprised excessively heterogeneous types. Treatment opportunities were highly limited and unitary as well: early results with irradiation were disappointing, which was slightly improved by chemotherapy. Intensive research in the field of molecular genetics has led to the discovery of certain chromosomes, genes and molecules involved in GBM tumor biology, which widened our knowledge of glioma diagnostics, estimating survival and measuring the effectivity of a certain therapy. Later on, it became clear that creating a molecular profile can describe a tumor entity more precisely since the molecular markers seem to be superior to the histological markers. Applying these novel markers in routine patient management transformed the classification of GBM from time to time, creating new entities while eradicating others . The subHeterogeneity is a widely known feature of GBM, as implied by its name: ’multiforme’. Numerous subclones develop in the early stage due to rapid mitoses . As the The overall survival (OS) of GBM patients without postoperative treatment is 3–6 months. The introduction of radiotherapy (RT) increased the OS rates to 9–12 months. Temozolomide (TMZ) has been added to RT, resulting in yet another increase in survival. Despite this progress, the treatment protocol for GBM has remained essentially unchanged in the past years despite the enormous progress in diagnostics. As of 2006, patients receive standard post-surgical concurrent chemo-radiotherapy and subsequent temozolomide monotherapy regardless of the diagnostic subgroups . AccordiSince current histological analysis is not suitable for targeted therapy, a new concept using novel diagnostic tools is needed. Tissue samples represent only a fraction of the whole tumor at a certain of time. Diagnostic inaccuracy can be attributed to heterogeneity and dynamic changes. A key step in the management of glioma is the difficulty in obtaining tissue samples eligible for molecular analysis. These samples can only be obtained by surgical resection or stereotactic biopsy with a considerable risk of life-threatening complications. Besides the high risk these procedures pose, the size of the tumor samples is often limited and is not always representative of the whole tumor. Only a fraction of the whole molecular profile of the tumor can be determined, especially in cases of biopsies due to intratumoral heterogeneity. Chances of monitoring disease progression or therapeutic responsiveness are also restricted, since repeated invasive biopsies are not reasonable. Blood, cerebrospinal fluid and urine samples have been found to contain tumor components in different forms: cells, extracellular vesicles and circulating cell-free nucleic acids. A growing number of research studies supports the examination of these exosomes, DNA and RNA particles. These so called “liquid biopsy” samples can represent the entire tumor tissue and possess enormous diagnostic, prognostic and predictive potential. Their functions have been proved to contribute to the sophisticated background communication between various cell types, stroma, blood vessels, secreted factors and surrounding ECM. Liquid biopsy is a rapid and inexpensive way of obtaining the relevant information about tumors and can be performed several times during the clinical course of the disease . FurtherGlioma stem cells (GSCs), as a cellular origin of GBM, can be found in the necrotic and hypoxic center of the tumor and are believed to be the main source of heterogeneity and therapy resistance. They exist in two forms based on the cellular origin: proneural and mesenchymal GSCs. Proneural cells are less aggressive and can be found in secondary GBMs, while primary, highly invasive and fast-growing glioblastomas derive from the mesenchymal types . CulturiNucleic acids (both DNA and RNA) derived from tumor cells circulate in a cell-free form, are attached to lipid/protein structures or are found in circulating extracellular vesicles (EVs). These EVs can elicit different types of interactions with nearby cells . Circulating DNA types include cell-free DNA (cfDNA), circulating tumor DNA (ctDNA) and mitochondrial DNA (mtDNA). CfDNA has a 150–200 base-pair-long, double-stranded structure and a 10–15 ng/L concentration in healthy human plasma. In cancer patients, the plasma level is significantly increased, and the composition was also shown to be changing over time. CtDNA derived from tumor cells is the major cfDNA fraction . In caseCirculating RNA types include messenger RNA (mRNA), short and long non-coding RNA (snc and lncRNA), micro-RNA (miRNA) and circular RNA (circRNA). MiRNAs have the most prominent role; these 19–25 bp long nucleic acids are excellent biomarkers and are abundant in most bodily fluids. They are great examples of epigenetic mechanisms regulating the expression of multiple target genes by forming a “silencing complex” . MiRNAs Cells can release vesicular structures with a luminal center covered by the phospholipid bilayer of the plasma membrane. They are heterogeneous and three major subgroups could be distinguished: exosomes, microvesicles and apoptotic bodies. Exosomes are extracellular vesicles with a dimension of 40–150 nm in diameter. Nucleic acids, proteins, lipids and metabolites are enclosed in a double lipid membrane layer escaping degradation. Exosomes derived from tumor cells help cell communication by establishing its micro-environment. This communication can facilitate the development of resistance to irradiation or chemotherapy . Thus, elimination of tumor exosomes from serum has immense therapeutic potential . The rolTumor cells can migrate to neighboring tissue and invade the peri-tumoral brain. Extensive invasion of cancer cells to the adjacent parenchyma makes surgical removal impossible and leads to inevitable local recurrence. Previous studies have demonstrated that the extracellular matrix (ECM) is altered in composition in various cancer types. Invasion activity of glioma cells is based on interaction with well-defined ECM components. It is a complex process with transmembrane receptors, extracellular ligands and synthetizing and degrading enzymes . These dSearching for new therapeutic targets aims to inhibit multiple signaling pathways at the same time and to hinder the proliferation of therapy-resistant subclones. There are numerous studies targeting the elimination GBM via different mechanisms, but no breakthrough in treatment has been reached yet  33]. Th. Th33]. Besides irradiation, only two antitumor agents  and pulsating electric tumor-treating fields (TTF) have FDA approval for GBM at present . TTF areGlioblastoma is an immunologically ’quiet’ disease, which means that an immunosuppressed tumor microenvironment is provided with minimal antigene presentation, different antigene escape mechanisms and direct immunosuppression . ImmunotTumor cells are able to avoid cellular immune response and elimination by the following mechanism: certain antigens  that are expressed on the surface of T-cells bind to their corresponding ligands located on the tumor surface. This leads to reduced cytotoxic T-cell activation. Blocking these antigens selectively by MABs stimulates an immune response. Clinical trials with anti-CTLA4  or anti-PD1 (pembrolizumab) show encouraging preliminary results ,40. The Training the immune system with vaccination is a very hot topic nowadays, and it can be a useful tool against tumors as well. Tumor-associated and tumor-specific antigenes (TAA and TSA) generate the immune response: a protein vaccine against EGFRvIII (rindopepimut) has promising early results . AnotherUsing viral vectors with the ability to integrate into the host tumor genome is another type of immunotherapy. These vectors contain selected genes that code enzymes or other proteins with a potential lethal effect on tumor cells. Toca 511 and Toca FC is a combination of gene therapy and a prodrug. Retroviral vector Toca 511 (vocimagene amiretrorepvec) contains gene-encoding cytosine deaminase. Toca FC is a prodrug of 5-fluorouracil, a well-known chemotherapeutic agent that can cross the blood–brain barrier and transforms locally to 5-fluorouracil . Oncolytic viruses are genetically engineered viral particles that can infect and dismiss tumor cells selectively while leaving adjacent brain tissue intact. Neural stem cells (NSCs) have the potential to carry oncolytic viruses to the target, thus increasing effectivity. Three virus families have gained significance so far: adenoviruses , herpesviruses  and recombinant polio-rhinoviruses . Delta-24-RGD can elicit antitumor effects alone, but it also has promising results in combination with pembrolizumab . Trends Genetically modified autologous T cells are armed with specific receptors that can attach to tumor antigens and implement a lethal cytotoxic attack. These cells with chimeric antigen receptors are called CAR-T cells. Possible tumor antigens can be EGFRvIII, HER2 and IL13RA2 . There is vast potential in immunotherapy, but promising new attempts in other treatment modalities are also notable. Under physiological conditions, signal transduction cascades take part in everyday cell life, affecting proliferation, differentiation, mobility, cell–cell communication and survival. Tumors use these processes for excessive proliferation, abnormal angiogenesis or to evade apoptosis. In GBM, usually three major signaling pathways are dysregulated. Due to tumor heterogeneity, all the pathways can be affected in the same patient . MembersCell surface antigenes (like TK receptors) expressed on the surface of glioma cells are potential targets of antibody-drug conjugate (ADC) therapeutics. ADCs are molecules with intrinsic antitumor effects bound to a specific antibody (against a surface antigen) and delivered directly to the tumor cells. EGFR appears to be a perfect antigen and the antibodies could deliver cytokines, antimitotic agents, bacterial toxins or radioactive isotopes ,56,57. ISearching for novel ways of intervention at the genomic level has led to the development of epigenetic therapy. Epigenetic mechanisms are deregulated in GBM. Modifying gene expression, transcription and translation has the potential of the inhibition of oncogenes and supporting suppressor genes by impeding proliferation, provoking cell cycle arrest and promoting apoptosis. Besides miRNAs that also can have epigenetic effects, histone deacetylase inhibitors (HDACIs) are potential candidates to hinder glioma cell proliferation. There is ongoing research with vorinostat and panobinostat in glioblastoma, mostly in combination with another type of drug .Radioresistance to ionizing radiation is a well-known feature of glioblastoma. Different underlying mechanisms have been identified, including microRNAs, tumor heterogeneity, glioma stem cells, tumor microenvironment, hypoxia, metabolic alterations and DNA damage/repair. Radiosensitizers are used to increase the efficacy of irradiation without increasing the dose of radiotherapy, which elevates the likelihood of side effects and damage to the normal brain tissue. Among the different mechanisms, the most important seems to be DNA damage response (DDR). PARP enzymes repair DNA damage, reinforce genome stability and hinder apoptosis. Olaparib is the first PARP inhibitor that has already reached encouraging results in breast and ovarian cancers and is now under investigation in GBM with veliparib and fluzoparib ,61. DNA-Ionizing radiation is a well-known ‘ab ovo’ treatment modality of glioblastoma, which was used firstly as a sole option. Radioresistance and side effects that ruin QoL resulted in worse outcomes after radiation treatment in the past. Better instrumentation, the application of radiosensitizers and improvements in imaging technologies have led to the rebirth of radiation therapy. A constant upgrade in conformality and novel modalities deliver patient- and tumor-optimized radiation that spare normal tissues and minimize side effects. The evolution of increasing accuracy started with whole-brain radiotherapy (WBRT) in the beginning, which was later transformed to focal brain radiotherapy (FBRT). The development of three-dimensional techniques, such as intensity-modulated radiation therapy (IMRT) and volumetric arc radiation therapy (VMAT), allowed the delivery of increased doses sparing non-tumor tissue . RadiosuThe currently accepted standard therapy of GBM is maximal safe surgical resection followed by TMZ-based concurrent radio-chemotherapy. In case of recurrence, repeated surgery (if possible) and/or administration of bevacizumab are options. Our current molecular genetic knowledge highly surpasses the treatment options. In the past, glioblastoma was believed to be a single entity, but using our understanding of its genetic composition, GBM can now be divided into several distinct subtypes. These subtypes have excessively different diagnostic, predictive and prognostic potentials and can also be discriminated by their treatment targets and response. Liquid biopsy, as a new and emerging diagnostic tool, can perform molecular mapping of the heterogeneous tumor tissue and can follow the dynamic molecular changes during oncotherapy. It is clear that novel combined and personalized multi-target therapies are needed to further improve treatment outcomes. Most of the agents have already proved to be effective in other tumor types. In the future, the goal will be to match the combination of the available therapies with the subtype of the tumor. In general, there are a lot of ongoing studies, with promising early results, but the present therapy remains the gold standard. The most crucial criterion for prolonged survival is a good performance status of the patient after tumor resection so that nonsurgical oncotherapy can be started. The peritumoral invasion of glioblastoma makes complete tumor resection practically impossible; therefore, the previous surgical strategy of “maximal extension of tumor resection” is suggested to be replaced by “maximal safe tumor resection”. The effectiveness of antitumor therapy mainly depends on the radio- and chemo-sensitivity of the tumor cells, which shows great variation. To overcome this, a new treatment strategy could be the concept of combined therapy, with addition of anti-invasion agents to the currently used cyto-reductive, anti-proliferative agents. As a result, the demarcated tumor could be removed more radically by surgery or treated with stereotactic radiosurgery/radiotherapy. Since these treatment modalities can be repeated multiple times, recurrent tumors could be also treated effectively, which may prolong survival times.


Human cytomegalovirus (HCMV), a ubiquitous in humans, has a high prevalence rate. Young people are susceptible to HCMV infection in developing countries, while older individuals are more susceptible in developed countries. Most patients have no obvious symptoms from the primary infection. Studies have indicated that the virus has gradually adapted to the host immune system. Therefore, the control of HCMV infection requires strong immune modulation. With the recent advances in immunotherapy, its application to HCMV infections is receiving increasing attention. Here, we discuss the immune response to HCMV infection, the immune escape mechanism, and the different roles that HCMV plays in various types of immunotherapy, including vaccines, adoptive cell therapy, checkpoint blockade therapy, and targeted antibodies. Human cytomegalovirus (HCMV), also known as human herpesvirus 5 (HHV-5), is a linear double-stranded DNA β-herpesvirus that belongs to the family of herpesviruses A recent systematic review and meta-analysis estimated a global HCMV seroprevalence of 83% in the general population, 86% in women of reproductive age, and 86% in donors of organs or blood Currently used drugs, such as Ganciclovir (GCV), Cidofovir (CDV) and Foscavir (FOS), have problems such as poor antiviral effect, large side effects and drug resistance, which cannot meet the clinical needs. Therefore, it is urgent to develop new prevention and treatment methods The virion of HCMV is composed of double-stranded DNA (dsDNA), which is enclosed in a capsid comprising four integral protein  that are organized into 162 capsomeres (150 hexamers plus 12 pentamers) and 320 triplexes located between the capsomeres The life cycle of HCMV starts when it binds to receptors on the surface of host cells and enters into the cytoplasm. Current data support two models of HCMV entering the cytoplasm through receptor-mediated endocytosis and membrane fusion There are two types of cell infection status: latent infection and lysogenic infection. For a latent infection, latency-associated transcript (LAT) HCMV genes are transcribed and accumulated in host cells The reason why we have not found an effective way to combat HCMV is probably that the immune responses to HCMV infections are still poorly understood. Changes in the level of the immune response directly affect the severity of HCMV infection. However, how the immune system responds to HCMV infection is not yet clear.It is well known that cell-mediated immunity and antibody-mediated immunity are necessary to suppress HCMV infection Currently, CD4+ or CD8+ T cells and NK cells are considered to play significant roles in the host defense against HCMV, and they are the key participants in the cellular immune response. CD4+ or CD8+ T cells restrain viral replication and prevent disease, but do not eliminate the latently infected host cell. How do latently infected cells pose a health risk from a potential reactivation? HCMV may be reactivated by immunosuppression, inflammation, differentiation, or critical diseases HCMV can evade an immune response in three ways: evasion from immune recognition receptors, inhibition of immune cell activation, or suppression of effector function Figure .In terms of evading immune recognition receptors, TLRs comprise a significant signaling pathway required for antiviral defense. The HCMV-encoded glycoproteins US7 and US8 target the TLR3 and TLR4 signaling pathways by promoting the degradation of these TLRs; US7 targets TLR3 and TLR4 by a ubiquitin/proteasome system; and US8 promotes TLR3 and TLR4 destabilization. This results in the overall downregulation of TLR-mediated innate antiviral response In addition, in order to inhibit immune cell activation, previous studies have shown that there are many HCMV proteins that can inhibit immune cell activation, such as UL16 HCMV mainly breaks the link, or the interaction or the communication or the cross talk between cellular immunity and humoral immunity by inhibiting the engagement of HCMV-specific antibody Fc fragments to FcR, and then suppressing effector function. Antibody-dependent cellular phagocytosis (ADCP) occurs upon engagement of virus-specific antibody Fc fragments to FcR, resulting in the cytotoxic killing of infected cells and whole virion degradation. HCMV also encode their own viral FcRs, which recognize the Fc regions of host immunoglobulins. Mimicking host FcRs, vFcRs enable herpesviruses to reduce and evade antiviral immune responses In fact, there are multiple strategies for the “tricky” HCMV to achieve immune escape. At the same time, the elucidation of these immunosuppressive strategies of HCMV also provides ideas for controlling the progression of infection.Immunotherapy is the treatment of diseases by artificially enhancing or suppressing the immune function of the body. Immunotherapy has been widely used in the treatment of a large number of cancers, and has achieved good results in different cancers such as the brain Several types of HCMV vaccine candidates have been developed or are currently under development, including first-generation vaccines (live-attenuated Towne), second-generation vaccines , and third-generation vaccines (nucleic acid vaccines) The first-generation vaccine, Towne, attenuated by serial in vitro passaging, seems to have lost its ability for persistent immune induction, as indicated by gradually declining T lymphocyte responses The second-generation vaccine, based on early investigation, the chimeras Towne and Toledo of the magnitude and duration of the serologic responses were not notably more rapid or more robust The third-generation vaccine ASP0113 , a DNA vaccine containing two plasmids encoding CMV antigens (gB aimed at inducing the production of antibodies against CMV and PP65 inducing T-cell mediated responses), was found to be safe and well tolerated in Japanese recipients undergoing allogeneic hematopoietic stem-cell transplantation (HCT). VCL-CB01, a candidate CMV DNA vaccine that contains plasmids encoding CMV pp65 and gB to induce cellular and humoral immune responses, is formulated with poloxamer CRL1005 and benzalkonium chloride to enhance immune responses Some scholars propose that an mRNA-based gB vaccine may ultimately prove more efficacious than some second-generation vaccines and increase the durability and breadth of vaccine-elicited antibody responses that can prevent HCMV-associated disease Adoptive cell therapy (ACT) involves the extraction of immune cells from the body, in vitro genetic modification and amplification, and transfusion back into the patient ACT has become a therapeutic strategy for HCMV reactivation in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) CMV is a unique oncolytic virus that is produced by genetic modification of some virulent viruses existing in nature. It uses the inactivation or defect of tumor suppressor genes in target cells to specifically recognize and infect tumor cells, replicate in large quantities, and eventually destroy tumor cells. It also stimulates an immune response that attracts more immune cells to continue killing the remaining cancer cells, and this is a growing field aimed to identify new therapies for treating cancer. Recently, there has been interest in developing vaccines based on CMV, which can induce large numbers of CD8+ T cells that are specific for an epitope that the virus encodes, better known as memory inflation For a lot of “cold tumors,” tumors with low immunogenicity, the response to traditional methods such as radiotherapy, chemotherapy, and immunotherapy is limited Whether the CMV can be used as an oncolytic virus is a question that needs to be considered and solved. We have to consider whether CMV has oncogenic properties, infects other normal cells, and produces severe viral infections that exacerbate the disease.HCMV viremia in renal transplant recipients also appears to upregulate PD-1 expression on CD4 T cells Targeted antibodies are antibodies designed to target antigens on cancer cells, primarily to disrupt the unrestricted growth and proliferation of infected cells. One approach uses antibody peptide epitope conjugates (APECs) to deliver suitable antigens to the tumor surface, which directs pre-existing CMV immunity against tumor cells and activates specifically CMV-reactive effector T cells, whereas a bispecific T-cell engager activates both effector and regulatory T cells In general, it is obvious that the development of vaccines designed to target HCMV proteins as antigens and stimulate the body to produce effective antibodies and immunocytes has still a long way to go. In adoptive immunotherapy, HCMV is often used as a target antigen to activate anti-CMV-specific T lymphocytes, thereby killing tumor cells infected with CMV. In terms of immune checkpoints, HCMV infection upregulates the expression of genes encoding immune checkpoint genes as well as other immunocyte markers. Although antibody therapy is very limited for CMV because of its intracellular presence in a latent form, interrupting the FcR-associated immune escape pathway and targeting immune checkpoints are promising strategies. In oncolytic virus therapy, CMV is often used as a vector to express oncogenes and induce the anti-tumor ability of T lymphocytes. Of course, due to safety concerns, research in the area of oncolytic viruses is cautious, and the existing research is based on cell and animal models, instead of patients.HCMV continues to be one of the most significant pathogens affecting the short-term and long-term outcomes of immunocompetent and immunocompromised patients. Firstly, HCMV asymptomatic latent infection and its severity once reactivated are the main obstacles of fundamental research and clinical therapeutics. Considering standardization and personalized immunotherapy, it is difficult to design a relatively perfect immunotherapy regimen to account for the particular complexity of commonly infected patients, different organ transplants, the heterogeneity of tumors (hot and cold tumors), and co-infection with other pathogens simultaneously. Secondly, identification of targets for HCMV immunotherapy, clinically meaningful inclusion criteria, and markers of immune responses, such as peripheral blood cell changes and inflammatory cytokines  is crucially important. Thirdly, the efficacy of single-target immunotherapy or single immunotherapy is limited. However, multi-target or multi-combination immunotherapy may cause unpredictable inflammatory cell infiltration, even cytokine storm, which are worthy of consideration. Finally, advances in immunotherapy technology coupled with the recent fundamental advances in the understanding of HCMV infection have created opportunities for the development of effective immunotherapy for HCMV infection. It is likely that combinatorial regimens with complementary mechanisms of action are required to achieve a broad and durable anti-HCMV benefit.


Orthotospovirus in the family Tospoviridae and order Bunyavirales. TSWV, transmitted by several species of thrips, causes significant disease losses to agronomic and horticultural crops worldwide, impacting both the yield and quality of the produce. Management strategies include growing virus-resistant cultivars, cultural practices, and managing thrips vectors through pesticide application. However, numerous studies have reported that TSWV isolates can overcome host-plant resistance, while thrips are developing resistance to pesticides that were once effective. RNA interference (RNAi) offers a means of host defence by using double-stranded (ds) RNA to initiate gene silencing against invading viruses. However, adoption of this approach requires production and use of transgenic plants and thus limits the practical application of RNAi against TSWV and other viruses. To fully utilize the potential of RNAi for virus management at the field level, new and novel approaches are needed. In this review, we summarize RNAi and highlight the potential of topical or exogenous application of RNAi triggers for managing TSWV and thrips vectors.Tomato spotted wilt virus (TSWV) is the type member of the genus  Among the most economically important plant viruses, tomato spotted wilt virus (TSWV) is considered the second most significant virus species in global agricultural industries . TSWV haFrankliniella occidentalis [The reason for the prevalence and significance of TSWV as a disease in horticultural crops is its insect vector, thrips. Thrips feed on plant tissue utilising a “punch-and-suck” method . Brieflydentalis , while identalis ,9. Interdentalis . Adults dentalis ,11,12.F. occidentalis (western flower thrips), the most prolific vector of TSWV [Considering that TSWV and its thrips vectors have wide and overlapping host ranges, an integrated disease management approach has to be used to manage TSWV and other thrips-transmitted viruses ,12. Beca of TSWV . However of TSWV , especia of TSWV . This haCultural practices for TSWV management largely consist of methods to remove virus reservoir plants outside the crop field to prevent the movement of vector thrips from reservoirs into crops. These strategies can include outright disposal of green bridges for TSWV , plantinLycopersicon hirsutum, L. hirsutum f glabratum, and L. pennellii being poor hosts to thrips nymphs, preventing feeding and subsequent infection with TSWV [There has also been significant efforts to produce varieties of tomato plants that are resistant to thrips, with ith TSWV ,28. Howeith TSWV ,30,31 anith TSWV ,33. As wOrius genus (commonly known as minute pirate bugs), such as O. niger, O. armatus, and O. laevigatus, can significantly reduce the thrips populations when introduced under glasshouse conditions and in seedling nurseries [Biological control strategies aim to reduce the populations of insect pests through the use of natural predators . Studiesurseries . Howeverurseries .RNA interference (RNAi) is a mechanism of genetic regulation observed across plants, animals, and fungi that utilises small RNA (sRNA) as trigger molecules for the manipulation of gene expression. sRNAs are derived from double-stranded RNA (dsRNA) and come in a variety of forms, each differing in structure, function, and biogenesis. For example, micro RNA  are transcribed from plant genomes to regulate expression of endogenous genes , while sThe mechanisms of RNAi have been well-studied and several reviews have been published on the topic ,40. The Viral genomes often encode a silencing suppressor to repress or subvert plant RNAi functions to prevent their own inhibition. Silencing suppressors are a common theme across plant viruses, and utilise many different mechanisms to counter host antiviral RNAi responses  as well The NSs protein of tospoviruses was shown to be the silencing suppressor , and funThe full extent of the NSs’ function to suppress silencing in a host plant is presently unclear. A study that utilised the NSs protein alongside viruses deficient in their own silencing suppressors concluded that while NSs did not appear to prevent the initial biogenesis of vsiRNAs or their systemic spread through the plant, the reduced accumulation of vsiRNAs compared to controls suggested that the suppressor rather interferes with the amplification of those trigger molecules . This maTsw resistance gene [Tsw and suppression activity at once, although those functions were supposedly not inextricably linked [Systemic expression of the TSWV’s NSs protein in capsicum plants in comparison to the virus’s other non-structural proteins demonstrated that this silencing suppressor is responsible for attracting thrips to TSWV-infected plants through suppression of terpene synthesis, which would otherwise assist in repelling these insects . NSs alsnce gene ,60, withnce gene ,61. Invey linked . Finallyy linked .While plant viruses are typically able to overcome RNAi defences in their hosts using silencing suppressors, transgenic RNAi defences have nonetheless demonstrated significant value in creating plants with resistance to specific viruses. The early years of RNAi research are full of examples where researchers introduced viral transgenes into plants, often expressing coat protein sequences, and observed delayed symptom development or outright resistance ,65,66. TThe first published study that used transgenic RNAi to create TSWV resistance did so inadvertently; Gielen et al.  transforN. benthamiana infected with TSWV resulted in limited viral protection in comparison to results targeting the N gene [Thereafter, the use of RNAi to induce resistance against TSWV has been thoroughly investigated. A study that produced transgenic plants expressing RNA targeting other genes within the TSWV genome for silencing found that significant resistance was only achieved when RNA matching the non-structural movement protein (NSm) or N genes were expressed, suggesting that the RNAi response is sequence specific, and that not all genes are equally as effective as RNAi targets . Similare N gene . A simile N gene ; in shorGroundnut ringspot virus, Tomato chlorotic spot virus, and Watermelon silver mottle virus) resulted in resistance to all of these viruses, as plants were able to produce siRNAs matching each virus from a single dsRNA substrate [Begomovirus genus, provided cross-protective resistance to both viruses [Watermelon silver mottle virus genome, providing resistance to several other tospoviruses due to sufficient conservation exhibited across tospoviruses in the targeted sequences [Transgenic RNAi strategies can be used to create plants resistant to multiple viruses. Developing chimeric constructs containing an N gene sequence from TSWV and three other tospoviruses species  were infested onto those leaf discs, still in contact with the dsRNA solution, and lethality was recorded through direct observation, as well as quantitating gene knock-down through qPCR. Their results demonstrated a significant decrease in the transcription levels of the associated genes, indicating that an RNAi response had been initiated by dsRNA uptake, and the authors were able to shortlist a number of genes that were successful in inducing mortality in the thrips through dsRNA feeding [There has been a limited amount of research on thrips and RNAi. Badillo-Vargas et al.  reportedgulation . Whittengulation  also achn et al.  develope feeding .F. occidentalis through direct feeding on dsRNA with a vATPase-B sequence; they found that attempting to feed the thrips on dsRNA in an artificial diet solution was ineffective due to dsRNA degradation induced by the feeding process. However, feeding the insects on plants that had taken up that dsRNA by a petiole-dip method did result in significant thrips mortality, suggesting that RNAi in thrips is possible through indirect feeding methods, albeit with some caveats that may impose restrictions on the delivery method. This is a critical point, as more direct methods like microinjection and dsRNA-producing bacteria are unlikely to translate to practical outcomes as a pest management strategy for thrips.Andongma et al.  examinedWhile RNAi holds great potential to develop TSWV-resistant crop plants, there are issues with using transgenic plants to bolster RNAi-based virus resistance. Viruses are capable of overcoming RNAi sequences targeted against them, as the positive selection pressure induced by resistant plants can result in spontaneous mutations in the viral genome, reducing the homology between the siRNAs and target transcripts. This has been demonstrated in transgenic plants utilising RNAi against TSWV to silence the viral N gene, with positive pressure being exerted on specific amino acids of the sequence ,103. FurN. tabacum using in vitro synthesized dsRNA [N. benthamiana [Topical application of RNAi is an emerging space for crop protection, ranging from viruses to insect pests to fungi ,107. In ed dsRNA , and hased dsRNA , sugarcaed dsRNA , and cuced dsRNA . Recentlthamiana . While mthamiana .While promising, there are limitations to the emerging strategy of topical RNAi. DsRNA on the surface of a plant is vulnerable to degradation from UV radiation and environmental nucleases, resulting in topical RNAi having a limited window of efficacy, typically just 5–7 days ,112. AddC. annuum). Under glasshouse conditions, TSWV-susceptible capsicum cv. Yolo Wonder plants were sprayed with solutions of water, a non-specific GFP dsRNA, or N gene dsRNA produced by bacterial expression. One day after spray application, the plants were challenged with TSWV by mechanical inoculation. Fourteen days after viral challenge, the TSWV infection incidence among the groups was determined by qualitative ELISA. Changepoint analysis [As part of ongoing research for the development of novel delivery approaches to trigger RNAi in plants for crop protection, the effect of dsRNA targeting the N gene of TSWV for silencing was evaluated as a topical spray on capsicum plants (analysis  demonstraquaporin homolog gene in F. occidentalis, previously shown to be effective in inducing lethality through RNAi [F. occidentalis adults. Over six days, mortality was determined by direct observation before extracting total RNA to determine the expression change of the targeted aquaporin gene. A trend towards increased mortality was observed in thrips feeding on the aquaporin dsRNA-treated leaves compared to the controls, accompanied by a reduction in relative gene expression (In addition, we have also conducted studies targeting thrips using exogenous application of dsRNA to further manage TSWV by directly reducing the populations of thrips vectors. As previously discussed, studies have successfully demonstrated the potential to utilise dsRNA in inducing mortality in thrips through feeding on plant tissue soaked in dsRNA solutions ,100. Thepression .Further consideration in the deployment of RNAi is the risk of off-target effects. RNAi mechanisms are present across plant species, as well as insects and animals. While it is easy enough to generate a dsRNA sequence for homology against a targeted sequence, it is a significantly greater hurdle to ensure the dsRNA sequence does not possess sufficient homology against unintended gene targets, and thus be toxic to unintended organisms ,122. To TSWV continues to be a serious threat to agriculture, given its ability to break host resistance and the capability of its thrips vectors to develop resistance to traditional pesticides. For this reason, there is a need to explore novel and effective strategies to introduce resistance to TSWV and its thrips vectors in crop plants. Researchers from around the world are developing new pest management options that are totally different formulations to what we currently think of as pesticides. In laboratory and glasshouse trials, topical or exogenous application of RNAi is showing some very attractive properties compared to traditional pesticides, including zero reduced crop residues, minimal off-target impacts, and low risk for resistance. Furthermore, a body of evidence is being generated addressing the environmental stability of RNAi and regulatory paradigms for its wider application. Presently, the cost of producing dsRNA, as the RNAi-triggering, biologically active agent, is being researched to ascertain its economic feasibility. Another area that is gaining momentum is novel carriers of RNAi triggers to make it suitable for field applications. The progress made so far in developing novel and effective delivery methods for exogenous introduction of dsRNA could be a new addition to the existing toolbox for managing TSWV and thrips vectors.


Genome editing, involving precise manipulation of cellular DNA sequences to alter cell fates and organism traits, offers the potential to both understand human genetics and cure genetic disease as never before. Scientific, technical and ethical aspects of employing CRISPR technology for therapeutic applications in humans are discussed, focusing on specific examples that highlight both opportunities and challenges. Genome editing is or will soon be in the clinic for several diseases, with more applications in the pipeline. The rapid pace of the field demands active efforts to ensure responsible use of this breakthrough technology to treat, cure and prevent genetic disease. In the nearly seventy years since the discovery of the DNA double helix, technologies have advanced for determining, analyzing and altering genome sequences and gene expression patterns in cells and organisms. These molecular tools are the foundation of molecular biology, fueling the therapeutic industry by enabling profound advances in understanding the genetics of normal and disease traits. The ability to diagnose genetic diseases has developed rapidly with reductions in genome sequencing costs, extensive comparative analyses of human genome sequences and applications of high-throughput genomic screening. However, the dearth of therapies, much less cures, for genetic diseases has created a growing disconnect between diagnostics and treatments, underscoring the urgent need to develop therapeutic options. Mitigation or correction of disease-causing mutations is a tantalizing goal with tremendous potential to save and improve lives, representing a convergence of technical and medical advances that could eventually eradicate many genetic diseases.5. In particular, fundamental research showing how clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbes with adaptive immunity has propelled transformative technological opportunities afforded by RNA-guided proteins. CRISPR-Cas9 and related enzymes have been used to manipulate the genomes of cultured cells, animals and plants, vastly accelerating the pace of fundamental research and enabling breakthroughs in agriculture and synthetic biology  and disease severity. These data in turn reveal DNA sequence alterations or corrections that could provide a genetic cure by either disrupting function of a toxic or inhibitory gene or restoring function of an essential gene.Sickle cell disease and muscular dystrophy, two common human genetic disorders, provide instructive examples of diseases that could be treated or cured by genome editing in the foreseeable future. Sickle cell disease results from a single base pair change in DNA that in turn generates a defective protein with destructive consequences in red blood cells. Duchenne muscular dystrophy belongs to a set of muscle-wasting diseases resulting from DNA sequence changes that disrupt normal production of a protein required for muscle strength and stability. A closer look at each of these diseases illustrates the ways that genome editing could offer therapeutic benefit to patients.11 and mapped to a genetic locus in the 1950s12, a single A to T mutation results in a glutamate-to-valine substitution in β-globin  to correct a genetic defect associated with disease. Muscular dystrophy exemplifies this type of disorder because it involves weakening and disruption of skeletal muscles over time , to create a molecular entity capable of binding and cutting DNA1. Importantly, DNA binding occurs at a 20-base pair DNA sequence that is complementary to a 20-nucleotide sequence in the guide RNA and can be readily altered by the experimenter20  and transcription activator-like effector nucleases (TALENs) demonstrated the promise of therapeutic genome editing. These early technologies enabled inactivation of the HIV co-receptor gene CCR5 in somatic cellsmenter20 . The DNAS. pyogenes (SpCas9) is the CRISPR-Cas enzyme most commonly used for genome editing and genetic manipulation, a growing collection of natural and engineered Cas9 homologs and other CRISPR-Cas RNA-guided enzymes is expanding the genome manipulation toolbox24. It is the intrinsic programmability present in this diversity of enzymes that underscores the utility of CRISPR-Cas technology for genome editing and other applications including gene regulation and diagnostics 28, and for transcriptional control 30 ISPRa)30 . In addiISPRa)30 , 32. Whi35. The second approach is to activate expression of ɣ-globin, the fetal form of hemoglobin typically silenced in adult cells, by disrupting ɣ-globin repressors41 or their binding sites in the ɣ-globin gene promoter43. These genome-editing strategies require harvesting a patient’s hematopoietic progenitor/stem cells (HPSCs), either to correct the β-globin mutation or to restart expression of ɣ-globin, and then re-introducing the edited cells into the bone marrow. Major progress in delivering to44 and handling HPSCs has resulted in formidable efficiencies of mutation correction or mitigation47 that are expected to be curative.Two strategies to mitigate or cure sickle cell disease take advantage of demonstrated strategies for site-specific genome editing , 2. The in vivo delivery technology may one day enable treatment without requiring bone marrow transplantation, which would reduce both expense and patient hardship.Such an approach, while requiring bone marrow transplantation, would remove the need for a compatible bone-marrow donor and thus provide a path for treating and potentially curing many more people than can be treated at present. As discussed below, improvements in in vivo editing may resolve some of the issues with ex vivo sickle cell therapies, studies in muscular dystrophy illustrate that other challenges arise when attempting in situ gene correction. Three reports highlight both the tremendous potential and the significant remaining challenges to using genome editing to treat or cure muscular dystrophy in humans. In the first study, a DMD mouse model was created using CRISPR-Cas9 to generate a common deletion (ΔEx50) in the dystrophin gene that occurs in DMD patients48. The severe muscle dysfunction in the ΔEx50 mice was corrected by systemic delivery of adeno-associated virus (AAV) encoding CRISPR-Cas9 genome editing components, restoring up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. The second study used CRISPR-Cas9-mediated genome editing to remove a mutation in exon 23 in the mdx mouse model of DMD, providing partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle49. In the third study, dogs harboring the ΔEx50 mutation corresponding to a mutational “hotspot” in the human DMD gene were treated using CRISPR-Cas950. After virus-mediated systemic delivery in skeletal muscle, dystrophin levels were restored to 3–90% of normal, and the muscle tissue appearance in treated dogs was improved. Although promising, these reports, as well as early-stage data from patients treated with in vivo gene editing using ZFNs, highlight the gap between animal studies and applications in humans53 and underscore the need for improved methods for in situ delivery, as discussed in the next section. An early stage clinical trial using in vivo CRISPR-Cas9 delivery to the eye to treat congenital blindness54 and a close-to-the-clinic program for liver gene editing55 will shortly provide key first-in-human data to inform the direction of that effort.While ex vivo delivery to primary cells is electroporation of Cas9 as a preformed protein-RNA  complex56. In vivo delivery, which is much more challenging, is currently conducted using viral vectors  or lipid nanoparticles bearing Cas9 mRNA and an sgRNA. The difficulty of ensuring efficient, targeted delivery into desired cells in the body currently limits the clinical opportunities of in vivo genome editing, although this is an area of increasing research and development.For any of these genome editing methods to be useful clinically, the CRISPR-Cas enzymes, associated guide RNAs and any DNA repair templates must make their way into the cells in need of genetic repair. To produce a functional genome editing complex, Cas9 and sgRNA can be introduced to cells in target organs in formats including DNA/DNA, mRNA/sgRNA, or protein/sgRNA, respectively. All three formats are currently, or shortly to be, used in the clinic, using viral vectors, nanoparticles and electroporation of protein-RNA complexes, and each has distinct benefits and limitations . The cur57. Notably, the FDA has approved AAV for gene replacement therapy in spinal muscular atrophy and congenital blindness, and clinical trials are in progress58.Viral delivery vehicles, including lentivirus, adenovirus and adeno-associated virus (AAV), offer advantages of efficiency and tissue selectivity . AAV is S. pyogenes Cas9. As a result, in applications calling for corrective gene insertion, a second AAV vector encoding the sgRNA and/or a template sequence for homology-directed DNA repair must be used, reducing efficiency due to the need for cells to acquire both AAV vectors at once60. Smaller genome editing proteins, such as the S. aureus Cas9, C. jejuni Cas9 and newly identified CRISPR-Cas enzymes, may circumvent this issue65. Second, long-term expression of genome editing molecules may expose patients to undesired off-target editing or immune reactions67. Third, the production of AAV at scale and the employment of good manufacturing process (GMP) methods at affordable cost for clinical use remains a formidable challenge70.There are significant challenges to using AAV for therapeutic delivery of CRISPR-Cas components, however. First, the AAV genome can only encode ~4.7 kb of genetic cargo, less than other viral vectors and not much larger than the 4.2 kb length of the gene encoding 74. When combined with the highly anionic sgRNA, the cationic Cas9 protein forms a stable RNP complex that has anionic properties suitable for encapsulation by cationic lipid nanoparticles, potentially enabling delivery into cells through endocytosis and macropinocytosis. Cationic lipid-based delivery is a relatively easy, low-cost process to deliver CRISPR components into cells75. This approach has been used for one-shot delivery of Cas9 RNPs into mice to achieve therapeutically useful levels of genome editing in the liver55. Disadvantages of this approach include significant toxicity of the LNPs76 and the sometimes undesired selectivity of cell-type specific uptake of the particles.Nanoparticles offer an alternative to viral-based delivery of Cas9 and sgRNAs and are suitable for delivering genome editing components in the form of DNA, mRNA, or ribonucleoprotein (RNP) . For exa79. Such assemblies, complexed with polymers capable of disrupting endosomes and including DNA templates for homology-directed repair, were found to promote correction of dystrophin gene mutations in mice80. Ongoing research continues to advance nanoparticle delivery technology, such as for endothelial cells that could enable access to lung and other organs81.Inorganic nanoparticles are another type of delivery vehicle with advantages including tunable size and surface properties. Gold nanoparticles, in particular, are attractive materials for molecular delivery because of the intrinsic affinity of gold for sulfur, enabling functionalized molecules to be coupled to the gold particle surface. Gold nanoparticles were used originally for nucleic acid delivery by conjugating to thiol-linked DNA or RNA  manages a scientific consortium aimed at measuring and standardizing such outcomes as genome editing technology advances100.The clinical utility of genome editing depends fundamentally on accuracy and precision. Accuracy refers to the ratio of on-versus off-target genetic changes, whereas precision relates to the fraction of on-target edits that produce the desired genetic outcome. Inaccurate (off-target) genome editing occurs when CRISPR-induced DNA cleavage and repair happens at genomic locations not intended for modification, typically sites that are close in sequence to the intended editing site  and CRISPR activation (CRISPRa) employ catalytically deactivated forms of Cas9 (dCas9) that are fused to transcriptional repressors or activators104. Notably, genome editing therapies that involve ex vivo editing, such as for sickle cell disease, are not as affected by either immunogenicity or pre-existing CRISPR-Cas antibodies, with residual Cas9 protein in the ex vivo edited cells being a manageable issue. The potential for inadvertent selection of genome-edited cells with undesired genetic changes came to light with the observation that selection for inactivation of the p53 pathway, which is associated with rapid cell growth and cancer, can occur during laboratory experiments on cells that are not used clinically106. Subsequent experiments showed that p53 inactivation can be controlled or avoided through protocol optimization107. As for long-term safety and efficacy of genome-edited cells in vivo, much remains to be determined. However, the recent report of a single HIV-positive patient who received CRISPR-Cas9-edited hematopoietic progenitor cells showed that although the number of edited cells was too low to mitigate HIV infection, no adverse outcome was detected over 19 months after transplantation of the edited cells108. Together, these findings suggest that there are, at present, no known insurmountable hurdles to the eventual development of safe and effective clinical applications of genome editing in humans.Other factors affecting clinical applications of genome editing include the immunogenicity of bacterially-derived editing proteins, the potential for pre-existing antibodies against CRISPR components to cause inflammation and the unknown long-term safety and stability of genome editing outcomes. Immunogenicity of CRISPR-Cas proteins could be managed by high-efficiency one-time editing treatments and by using different editing enzymes. Pre-existing Cas9 antibodies and reactive T-cells have been detected in humans exposed to pathogenic bacteria harboring CRISPR systems, although it is unknown whether these are present at concentrations sufficient to trigger an immune response to the genome editing enzymesThe clinical potential of genome editing exemplified by applications in sickle cell disease, muscular dystrophy and other monogenetic disorders could be stymied by extreme pricing of such next-generation therapeutics. Although CRISPR technology itself is a democratizing tool for scientists, extension of its broad utility in biomedicine requires addressing the costs of development, personalization for individual patients and the intrinsic difference between a chronic disease treatment versus a one-and-done cure  T-cell effectiveness, treat sickle cell disease and other inherited blood disorders, and stop or reverse eye disease103. With its potential to address unmet medical needs, clinical use of genome editing will ideally spur changes to regulatory guidelines and cost reimbursement structures that will benefit the field more broadly as these therapies continue to advance.For muscular dystrophy, the genetic diversity among patients lends itself to personalization that is an inherent strength of the CRISPR genome editing platform, yet also complicates clinical testing strategies. In addition, progressive diseases like muscular dystrophy require early treatment to be most effective, raising questions about coupling diagnosis and treatment. Beyond these examples, many rare genetic disorders will be treatable in principle if a streamlined strategy for CRISPR therapeutic development can be implementedNotably, all of the genome editing therapeutics under development aim to treat patients through somatic cell modification. These treatments are designed to affect only the individual who receives the treatment, reflecting the traditional approach to disease mitigation. However, genome editing offers the potential to correct disease-causing mutations in the germline, which would introduce genetic changes that would be passed on to future generations. The scientific and societal challenges associated with human germline editing are distinct from somatic cell editing and are discussed in the next section.Human germline genome editing can introduce heritable genetic changes in eggs, sperm or embryos. Germline genome editing is already in widespread use in animals and plants and has been employed in human embryos for research purposes. A report of alleged use of human embryo editing resulting in the birth of twin baby girls with edited genomes has focused global attention on an application of genome editing that must be rigorously regulated, as underscored by international scientific organizations.113. Although none of the published work involves implantation of the edited embryos to initiate a pregnancy, such work was reported at a conference on human genome editing in November 2018, leading to international condemnation in light of clear violations of ethical and scientific guidelines.Human germline editing differs from somatic cell editing because it results in genetic changes that are heritable if the edited cells are used to initiate a pregnancy . Germlin110 was not substantiated by later publications, but the mechanism by which double-stranded DNA breaks are repaired in early human embryos remains under debate. Some results were interpreted to indicate repair of a CRISPR-Cas9targeted gene allele by homology-directed repair with the cell’s other allele as the donor template114. Other scientists argued that such repair would be impossible given the apparent physical separation of sister chromatids early in embryogenesis, and suggested the data could also be consistent with large deletions in the embryo genomes115. Resolving this fundamental question will require further experiments. Human embryo editing has also begun to reveal differences in the genetics of early development in mice versus humans111, underscoring the potential value of research that will be enabled by precision genome modification.This work and the accompanying discussion around human germline editing have raised important questions that affect the future direction of the science as well as the societal and ethical issues that accompany any such applications. First, research using CRISPR-Cas9 in human embryos has challenged current understanding of DNA repair mechanisms and developmental pathways that occur in these cells. A report of inaccurate CRISPR-Cas9-based genome editing in non-viable human embryos116, UK Association of Genetic Nurses and Counsellors, Canadian Association of Genetic Counsellors, International Genetic Epidemiology Society, US National Society of Genetic Counselors, American Society for Reproductive Medicine, Asia Pacific Society of Human Genetics, British Society for Genetic Medicine, Human Genetics Society of Australasia, Professional Society of Genetic Counselors in Asia, and Southern African Society for Human Genetics. Key points on which these groups agree: (1) At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline genome editing that culminates in human pregnancy; (2) in vitro germline genome editing on human embryos and gametes should be allowed, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing, and there should be no prohibition on public funding of this research; (3) future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input.A second question raised by applications of genome editing in human embryos concerns the appropriate professional and societal response. Organizations including the National Academy of Sciences, the National Academy of Medicine, the Royal Society and their equivalents in other countries have sponsored meetings and reports, as have professional societies including the American Society of Human Genetics (ASHG)The third question raised by applications of CRISPR-Cas9 in human embryos is how to move the technology forward while ensuring responsible use. At the time of this writing, international commissions convened by the World Health Organization (WHO) and by the US National Academy of Sciences and National Academy of Medicine, together with the Royal Society, are drafting detailed requirements for any potential future clinical use. Medical needs must be defined so that risks versus possible benefits can be evaluated. Most importantly, procedures by which patients could be informed about the technology, its risks and a process for monitoring health outcomes must be determined.Therapeutic genome editing will be realized, at least for some diseases, over the coming 5–10 years. This profound opportunity to change healthcare for many people requires scientists, clinicians and bioethicists to work with healthcare economists and regulators to ensure safe, effective and affordable outcomes. The potential impact on patients is too important to wait.


Characterizing the diverse cell types that make up the nervous system is essential for understanding how the nervous system is structured and ultimately how it functions. The astonishing range of cellular diversity found in the nervous system emerges from a small pool of neural progenitor cells. These progenitors and their neuronal progeny proceed through sequential gene expression programs to produce different cell lineages and acquire distinct cell fates. These gene expression programs must be tightly regulated in order for the cells to achieve and maintain the proper differentiated state, remain functional throughout life, and avoid cell death. Disruption of developmental programs is associated with a wide range of abnormalities in brain structure and function, further indicating that elucidating their contribution to cellular diversity will be key to understanding brain health. A growing body of evidence suggests that tight regulation of developmental genes requires post-transcriptional regulation of the transcriptome by microRNAs (miRNAs). miRNAs are small non-coding RNAs that function by binding to mRNA targets containing complementary sequences and repressing their translation into protein, thereby providing a layer of precise spatial and temporal control over gene expression. Moreover, the expression profiles and targets of miRNAs show great specificity for distinct cell types, brain regions and developmental stages, suggesting that they are an important parameter of cell type identity. Here, we provide an overview of miRNAs that are critically involved in establishing neural cell identities, focusing on how miRNA-mediated regulation of gene expression modulates neural progenitor expansion, cell fate determination, cell migration, neuronal and glial subtype specification, and finally cell maintenance and survival. Understanding the biological basis of the vast cellular diversity found in the nervous system remains a high priority for neuroscience research. Recent advances in single-cell transcriptomics have enabled the exploration of neural cell diversity with increasing spatial and temporal resolution, generating unprecedented quantitative and comprehensive datasets characterizing the transcriptomes, morphology and electrophysiology of neuronal subtypes . MoreoveThere is increasing evidence that microRNAs (miRNAs) can act as key regulators of cellular identity. miRNAs are non-coding RNAs that function as post-transcriptional repressors of mRNA expression. A distinctive feature of miRNAs is that evolution has favored a continual expansion in the miRNA repertoire with increasing number of distinct cell types in an organism  across metazoans. This is in clear contrast to protein-coding genes, whose number show no correlation to biological complexity. Once miRNAs are added to metazoan genomes and integrated into gene regulatory networks, they are strongly conserved in primary sequence and rarely secondarily lost . These fThe expression patterns of miRNAs in the brain show an impressive specificity for distinct developmental stages, brain regions and cell types. A single miRNA is capable of regulating hundreds of different targets and these targets can also vary according to specific cell types and developmental stages . This suThe dynamic expression patterns of miRNAs, their ability to facilitate developmental transitions and fine-tune protein levels, their conservation as well as their evolutionary expansion with increasing biological complexity all make miRNAs well-suited to instruct and maintain the astonishing cellular diversity found in the nervous system. In this review, we discuss current evidence supporting critical roles for miRNAs in determining cell identity across their developmental trajectory. We describe miRNAs and their targets that are critical throughout neural development from neurulation to neural progenitor expansion, fate determination, neuronal and glial subtype specification, and finally maintenance and survival of these cell types. Furthermore, we also discuss how miRNAs regulate migration, lamination, morphology, and functional connectivity of neurons and glia, all aspects that are integral to cellular identity. A summary of the miRNAs and targets involved in all of these processes are listed in Developmental programs generate distinct cell types as opposed to continuous diversity by making distinct lineage decisions, which sequentially narrow the range of possible forms and functions. Early lineage decisions, including those that affect progenitor cells, have enormous power over the developmental trajectories a newly born cell can follow. Progenitor state, which is known to change over developmental time and in response to signaling, sets daughter cells along the path to a neuronal fate or glial fate, biases them to specify into early-born or late-born cell types, and determines their responsiveness to extrinsic maturation cues . In thisIt is common to interrogate the overall function of miRNAs by examining phenotypes induced by removal of all miRNAs. Mature, functional miRNAs are processed from precursor miRNAs by the endonuclease activity of the enzyme DICER. Thus, DICER knockout prevents miRNA biogenesis and eventually results in functional ablation of the vast majority of miRNAs . While gA study examining miRNA ablation via DICER knockout in Emx1-expressing cortical cells and progenitors found no defect in proliferation or neurogenesis, only defects in later differentiation and survival . HoweverWhile studying the roles of these miRNAs, it is important to remember that miRNAs function in complex networks with their targets, other miRNAs, and other non-coding RNAs. For example, one or multiple miRNAs may target multiple aspects of a cellular pathway or biological process in order to cooperatively regulate neuronal progenitors. This allows miRNAs to substantially boost the magnitude of repression and fine tune the timing of regulation in response to several intersecting cues. In addition, miRNAs and their targets may form regulatory loops for controlling the timing of critical transitions and strongly reinforcing fate decisions. The lin-28 gene supports pluripotency in proliferating stem cells, where it is highly expressed, and simultaneously suppresses the production of mature let-7 and miR-125 . When liMicroRNA regulation of the progenitor pool is the first step required for proper development of the brain architecture and thus foundational to maintaining proper developmental trajectories. Below, we discuss the following stages that are necessary to setting up the cell diversity in the brain.Neuronal fate appears as progenitors switch from expanding the progenitor pool to producing terminally differentiated neurons or neurogenic progenitors with restricted potency. miRNAs are crucial regulators of these processes. For example, cyclin D1, a key component of cell cycle regulation that affects the balance between progenitor proliferation and neuronal differentiation, both regulates and is regulated by miR-20a, miR-20b, and miR-23a, forming a regulatory loop controlling the transition to neurogenesis and differentiation . Other mAdult neurogenesis requires miRNAs to do the opposite: stably maintain progenitor fate to allow proliferation throughout life. Adult stem cells undergo asymmetric divisions to repopulate the stem cell pool while producing more differentiated progenitors, fated to produce differentiated neurons. miR-184 and miR-34a upregulate Notch signaling in the daughter cell fated for neuronal differentiation by targeting the Notch repressor Numbl . Meanwhiin vitro experiments suggest that this is mediated by co-expression of Foxg1 and Elavl2, an RNA binding protein that may block miR-9 from targeting Foxg1’s 3′UTR. At these ages, miR-9 seems to decrease cortical progenitor proliferation by repressing other transcription factors such as Pax6, Meis2, and Nr2e1  and failure to produce late-born cell types. These progenitors did not express late progenitor markers, reflecting a defect in progenitor developmental trajectory that ultimately disrupted neurogenesis . This efAfter neurogenesis creates populations of immature neurons, more mechanisms involving miRNAs activate to reinforce neuronal fate and set up the molecular machinery underlying neuronal function.in vitro showed that manipulating these miRNAs changes the proportions of cells differentiating into neurons versus glia  of layer V and callosal projection neurons (CPNs) of layers II/III and V form the corticospinal tract and corpus callosum, two main white matter tracts that are unique to placental mammals and that contribute to the functional and cell type complexity of these brains. miR-409-3p suppresses CPN fate in favor of CSMN development by downregulating LMO4, a transcription factor known to promote CPN areal identity. Layer V CSMN and CPN are born from the same progenitors and initially all express LMO4, so miR-409-3p is thought to mediate divergence of these two cell types . Meanwhiin vitro and in vivo  migrate radially from the ventricular progenitor zones. The combinatorial expression levels of miR-128, miR-9, and let-7b in radial glia modulate the lamination of the PNs they produce, including the proportions of cells that migrate into each layer. While miR-128 and let-7b seem to encode deep- and upper-layer identity in a dose-dependent manner, miR-9 specifically promotes development of layer V identity . Since tIn some cases, we know that miRNAs directly regulate the molecular mechanisms of migration. Neurons follow fibers extended by radial glia across the cortical plate to climb into the cortical layers; they interact with these fibers through N-cadherin on the cell membrane. The miR-379/410 cluster regulates the expression of N-cadherin in both cortical progenitors and neurons. Simultaneously manipulating the expression of three miRNAs in this cluster—miR-396-3p, miR-496, and miR-543—reversibly controls the rate of migration out of the ventricular zone. Interestingly, overexpression of these miRNAs individually did not affect N-cadherin expression or induce a migration phenotype, but overexpressing pairs of miRNAs can bias neurons to migrate into the deep or upper layers . ElucidaMicroRNA control of migration, whether through affecting large programs coordinating layer identity or by directly affecting the molecular migration machinery, is an important contributor to the developmental trajectory of a cell. Laminar positioning then contributes to how a neuron integrates into circuits and takes on mature functions. Next, we continue along this developmental trajectory by examining miRNA functions in morphological and functional development.Neuronal morphology was historically the main way of categorizing cells and remains a key indicator of cell type and function. For example, parvalbumin (PV)-expressing interneurons share fast-spiking electrophysiological characteristics and of course expression of PV, but can be categorized further into basket cells, chandelier cells, and translaminar cells by their morphologies, which reflect diverging connectivity and circuit functions. By regulating the growth and elaboration of neuronal compartments, miRNAs control the development of cell type-specific characteristics.MicroRNA function can have drastic effects on neurite outgrowth, and hence affect the function of individual cell types. This is evidenced by experiments where DICER was knocked out in postmitotic cortical pyramidal neurons. Loss of miRNAs caused dramatic reductions in soma size and neurite growth, which compounded into decreased brain size . miRNA ain vitro and in adult-born hippocampal neurons  neurons reduced axon growth in axons . Axon exin axons . miR-409in axons .Fos expression in 1-week-old interneurons following exposure to various odor stimuli. Thus miR-125 seems to control the timing of functional development in adult-born interneurons  low-threshold mechanoreceptors (LTMRs) terminate peripherally in the skin and participate in touch sensation. There are three types of myelinated LTMRs: Aδ LTMRs, Aβ rapidly adapting LTMRs and Aβ slowly adapting (Aβ SA) LTMRs. Conditional loss of the miR-183/96/182 cluster in mice leads to a failure to extinguish expression of the transcription factor SHOX2 during development and an increase in the proportion of Aδ LTMRs at the expense of Aβ SA-LTMRs. Conversely, overexpression of the miR-183/96/182 cluster that represses SHOX2 expression, or loss of SHOX2, both increase the Aβ SA-LTMR population at the expense of Aδ LTMRs . The miRIn recent years, numerous studies have demonstrated the importance of miRNAs in the development of various glial cell lineages such as Schwann cells (SCs) in the peripheral nervous system (PNS) and astrocytes, oligodendrocytes and microglia in the CNS. Below we summarize the critical miRNAs and their targets that regulate the differentiation, morphology, and function of these four glial subtypes.Microglia are the resident macrophages of the CNS that represent the first line of immune defense in the brain and spinal cord. They originate from early yolk sac myeloid progenitors and infiltrate the brain during early development. In their steady state, microglia mainly function to maintain brain homeostasis and upon injury or infection microglia transform to an activated state. Emerging evidence continue to show that microglia are in fact a multifunctional housekeeping cell type that contributes to many aspects of brain development such as neural circuit wiring, neuronal survival, synaptogenesis, synaptic transmission and myelination . MicroglOligodendrocytes are a unique glial cell type in the CNS that synthesizes multilamellar myelin membranes which ensheath axons. Myelination electrically insulates axons to promote rapid, energy-efficient action potential propagation and is thus crucial for the development and function of the CNS. Two independent studies demonstrated that DICER ablation in oligodendrocyte precursor cells (OPCs) disrupts proper CNS myelination and delays oligodendrocyte differentiation . These sSox2, Jun, and Ccnd1, providing some basis for the arrested differentiation of SCs. Further experiments will be required to determine the specific miRNAs and their targets that regulate SC differentiation and function (Schwann cells are the principal glial cells in the PNS, where they play essential roles in the development, maintenance, function and regeneration of peripheral nerves. SCs can be categorized into two major classes - myelinating and nonmyelinating SCs. Myelinating SCs provide the myelin ensheathment of all large-diameter peripheral axons, while nonmyelinating SCs typically associate with smaller axons. Several studies have shown that DICER ablation in SCs fully arrests their differentiation and ability to myelinate. Gene expression analyses from these studies revealed drastic downregulation of promyelinating transcription factors KROX20  and EGR2function . Followifunction . A miRNAin vitro (in vitro study showed that miR-125b positively regulates astrogliogenesis and promotes astrocyte proliferation (in vivo was shown to upregulate a brain-enriched miRNA, miR-128-3p, which in turn suppressed developmental expression of astroglial mGluR5. mGluR5 expression was not altered in FMRP-deficient neurons, highlighting an astroglia-specific mechanism (Astrocytes are the most abundant cell type in the CNS and are crucial regulators of neuronal development, function and connectivity. Thus, it is not surprising that astrocyte-specific DICER ablation results in drastic phenotypes across multiple regions in the brain. These phenotypes range from deficits in dendritic spine development in cortical and hippocampal neurons  to majorin vitro . Anotherechanism . These sSeeing as miRNAs are necessary for developing the characteristics of cell type—including laminar position, morphology, connectivity, and molecular markers—it is not surprising that they also play key roles in maintaining these cell types. Differentiated neurons must tightly regulate gene expression to remain functional throughout life and avoid cell death. Continual miRNA regulation buffers potentially harmful changes in gene expression and stabilizes a cell’s identity over its lifetime. Decreased miRNA function, by allowing harmful changes in identity and gene expression networks, may contribute to neurodegeneration. Recent studies have also suggested that many miRNAs repress pro-apoptotic genes, such that miRNA misexpression or gain-of-function mutations may provide cancerous cells with a mechanism of escaping apoptosis, making miRNA dysfunction a potential “second hit” during oncogenesis . The folReduced miRNA function caused by hemizygous DICER knockout in adult DA neurons reduced levels of DA and other molecules in DA metabolic pathways, while complete DICER knockout led to severe DA neuron death, especially in the substantia nigra . DysfuncDuring development, oligodendrocytes are greatly overproduced and only by competing for limiting amounts of target-derived growth factors is their number adjusted according to the number and length of the axons requiring myelination. The miR-17/92 cluster was shown to regulate this culling process of oligodendrocytes by promoting their proliferation through activation of the Akt signaling pathway . PostnatWithout miR-218-1 and miR-218-2, spinal MNs are born in normal numbers but fail to innervate muscles and die off in large numbers before birth . DICER kIn addition to developmental establishment of cellular diversity, miRNA function throughout life is necessary to maintenance of cell identity and health. Dysfunction of miRNA networks can induce degenerative phenotypes and may contribute to the mechanisms or the cell type-specificity of some neurodegenerative diseases.Numerous reports over the last two decades have shown that miRNA-mediated posttranscriptional regulation is indispensable at basically all stages of brain assembly. Although the majority of these studies are focused on developmental transitions, here we argue that the same mechanisms also demonstrate that miRNAs are key determinant of cell identity. By reinforcing the transcriptional programs underlying lineage decisions, miRNAs instruct the emergence and maintenance of many cell types of the brain. Further, miRNAs are necessary building blocks in the molecular pathways controlling lamination, differentiation and proper connectivity, processes through which neurons take on the characteristics essential to their cell type. Despite this wealth of data, we believe that the field is still in its infancy. In fact, miRNA roles have only been tested in a small percentage of brain cell types. Even for the ones tested, detailed miRNA mechanisms are for the most part still missing. Finally, owing to advances in sequencing technologies, the field is rapidly progressing from studying a single miRNA and a few of its targets to identifying miRNA-target networks associated with distinct cell types and developmental stages . This efNZ and JD wrote the review with help from FZ and GL. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anderson-Fabrydisease is an X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-galactosidase A. This results in pathological accumulation of glycosphingolipids in several tissues and multi-organ progressive dysfunction. The typical clinical phenotype of Anderson-Fabry cardiomyopathy is progressive hypertrophic cardiomyopathy associated with rhythm and conduction disturbances. Cardiac imaging plays a key role in the evaluation and management of Anderson-Fabry disease patients. The present review highlights the value and perspectives of standard and advanced cardiovascular imaging in Anderson-Fabry disease. Anderson-Fabry disease (AFD) is classified as an X-linked storage disorder caused by the abnormal activity of a lysosomal enzyme called α-galactosidase A. AFD results in a pathological accumulation of glycosphingolipids in several tissues that generates different disease phenotypes according to the extent and severity of the involved organ ,2,3 RenaThe prevalence of AFD varies depending on the screening method used. Neonatal screening programs reported an unexpectedly high incidence ranging from 1:1250 to 1:7800 . BecauseTherefore, prompt diagnosis and treatment of patients with Fabry disease seem warranted.LVH should always be evaluated together with family history and extracardiac symptoms. LVH is a more common finding in AFD male patients than female (53% versus 33% in untreated patients) . In maleStandard echocardiography  is the fAFD cardiomyopathy is characterized by concentric remodeling usually progressing to concentric hypertrophy . In AFD In the past, the echocardiographic binary appearance of the LV endocardial border, namely the binary sign, was considered the pathognomonic sign to discriminate patients suffering from AFD from those with familial HCM . This siLV diastolic dysfunction occurs early in the AFD disease stages  and reprDiastolic dysfunction in AFD cardiomyopathy is related to progressive myocardial wall thickening. Its severity increases in the advanced stages of LVH ,34,35. HDiastolic function indices, such as mitral flow Doppler parameters and the Tei index, a marker for combined diastolic and systolic function, are often altered in AFD patients with LVH . The TeiDiastolic dysfunction leads to the enlargement of the left atrium. In AFD, left atrial dilation occurs in the early stages of Fabry disease, even before the development of LVH ,40,41. AThis suggests that the evaluation of atrial damage may be useful in the early diagnosis of AFD.In most cases, atrial dilatation occurs as mild or moderate, becoming severe only in the presence of significant mitral valve disease, myocardial fibrosis and major LVH ,44.Right ventricular hypertrophy (RVH)  is frequIn AFD, a remodeling process may affect the aorta. Barbey et al.  found aoThe process of accumulation of glycosphingolipids may involve the valvular apparatus . A thickSpeckle tracking echocardiography (STE)  has an iThe reduction in the inferolateral region strain has shown to be the most accurate parameter to identify the presence of LGE on CMR . The posSTE advancements now make available the analysis of layer-specific myocardial deformation. In newly diagnosed, untreated AFD patients, our group demonstrated that layer-specific strain imaging highlights an impairment of LV longitudinal deformation, mainly involving subepicardial strain and causing an increase in LS myocardial gradient. These findings could be useful for identifying the mechanisms underlying early LV dysfunction in AFD patients .Furthermore, there is evidence that STE-derived left atrial peak longitudinal deformation and RVLS are impaired in AFD patients compared to healthy controls, even when standard echocardiographic parameters are in the normal range ,58.Mechanical dispersion assessed with STE was observed by Cianciulli et al. in 76% of patients with AFD and LVH, while it was not evident when LVH was absent . The useThree-dimensional echocardiography has some advantages over standard two-dimensional echocardiography. It provides more accurate information on left ventricular volumes, mass and ejection fraction, which have a better correlation with those measured using CMR . HoweverCMR provides relevant information that contribute to diagnosing, monitoring and treating patients with AFD . One of Tissue characterization is crucial for a better evaluation of AFD. CMR is the primary imaging modality for myocardial tissue characterization. Parametric mapping techniques with CMR allow the quantification of changes in myocardial composition  based on changes in T1, T2 and T2* relaxation times and extracellular volume . Unlike The presence of fibrosis can be assessed by the amount and distribution of the LGE after gadolinium-based contrast agents administration. Replacement fibrosis is considered a sign of disease progression  and may The typical LGE pattern is at the mid-wall in the basal inferolateral area of the left ventricle . TherefoHanneman et al. demonstrated that the amount of LGE was strongly correlated with the incidence of cardiovascular events , stressing the prognostic value of CMR in risk stratification of AFD patients .The use of speckle tracking analysis is less common in CMR than echocardiography. It can be reliably estimated, using image feature tracking methods applied to SSFP CMR images .Few studies have analyzed CMR-derived GLS and GCS in AFD patients. No significant differences in GLS between AFD patients and controls have been found . RegardiAs shown by Knott et al. , AFD patIn the last few years, the emerging role of nuclear imaging techniques in AFD has been studied. Glycosphingolipids’ storage also affects endothelial and smooth muscle cells causing microvascular dysfunction at different levels, including the myocardium. This process results in a global reduction in coronary flow detected by single-photon emission computed tomography (SPECT) imaging with 99mTc sestamibi .Myocardial hypoperfusion can also be estimated using positron emission tomography (PET) imaging. Tomberli et al. demonstrated that a global reduction in coronary flow reserve was an early sign of cardiac involvement, regardless of sex and LVH . ERT theFew studies show that PET imaging, and in particular hybrid cardiac PET-MR imaging, is also useful to detect myocardial inflammation, present in an early phase of the disease, and to identify different stages of disease progression. The presence of 18F-FDG uptake correlates with impaired LV longitudinal function at echo  and LGE Using hybrid positron emission tomographymagnetic resonance imaging, Imbriaco et al. demonstrated that focal 18F-FDG uptake was associated with a trend towards a pseudo-normalization of abnormal T1 mapping values in female AFD patients that may represent an intermediate stage before the development of myocardial fibrosis, suggesting a potential relationship between progressive myocyte sphingolipid accumulation and inflammation . Hybrid Nuclear imaging is useful to study the cardiac effect of autonomic nervous system dysfunction in AFD . The cheCurrently, SPECT and PET imaging are used only for investigation reasons, but they look promising in detecting early cardiac involvement in AFD patients.“Fabry disease-often seen, rarely diagnosed” is how Hoffmann and Mayatepek titled their AFD review . CurrentAn integrated multi-modality imaging approach including both echocardiography and CMR might be optimal for the management of AFD patients. In the future, echocardiography, by its large availability and low cost, will remain the initial imaging modality of choice in patients with proven or suspected AFD, but the role of CMR will be likely to increase so much as toalso become an essential diagnostic test in the initial evaluation.