Sodium glucose cotransporter-2 (SGLT2) inhibitors inhibit the development of diabetic nephropathy (DN). We determined whether changes in perirenal fat (PRAT) by a SGLT2 inhibitor ipragliflozin (Ipra) contribute to the suppression of DN development. High-fat diet (HFD)-fed mice were used as a DN model and were treated with or without Ipra for 6 weeks. Ipra treatment reduced urinary albumin excretion (UAE) and glomerular hypertrophy in HFD-fed mice. In the PRAT of Ipra-treated mice, adipocyte size was increased, and inflammation, fibrosis, and adipocyte death were suppressed. In conditioned medium made from PRAT (PRAT-CM) of Ipra-treated mice, the concentration of leptin was significantly lower than PRAT-CM of mice without Ipra treatment. Serum leptin concentration in renal vein positively correlated with UAE. PRAT-CM from HFD-fed mice showed greater cell proliferation signaling in mouse glomerular endothelial cells (GECs) than PRAT-CM from standard diet-fed mice via p38MAPK and leptin-dependent pathways, whose effects were significantly attenuated in PRAT-CM from Ipra-treated mice. These findings suggest that Ipra-induced PRAT expansion may play an important role in the improvement of DN in HFD-fed mice. In vitro experiments suggest that reduced PRAT-derived leptin by Ipra could inhibit GECs proliferation, possibly contributing to the suppression of DN development. Chronic kidney disease (CKD) is a common disease and is a risk factor for cardiovascular, cerebrovascular, and end-stage renal diseases (ESRD). Metabolic diseases, such as diabetes mellitus, hypertension, obesity, and dyslipidemia, have been strongly associated with CKD development, with diabetic nephropathy (DN) being one of the most prevalent primary diseases in ESRD .Sodium glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents that promote urinary glucose excretion by inhibiting glucose absorption at the proximal tubule. They have rapidly gained popularity because of their insulin-independent glycemic control activities and the ability to induce caloric loss and increase insulin sensitivity. Large-scale clinical trials among patients with type 2 diabetes revealed that SGLT2 inhibitors prevented macrovascular events such as myocardial infarction, stroke, and heart failure  and alsoThe amount of adipose tissue around the heart, blood vessels, and kidneys is correlated with the risk of developing cardiovascular diseases . FurtherThe aim of this study was to investigate whether SGLT2 inhibitors induce “healthy adipose expansion” in PRAT and whether it affects the pathogenesis of DN by altering PRAT-derived secretory factors.Atgl, Hsl, and Pck1 (HFD-fed wild-type (WT) mice were divided into two groups, one treated with Ipra for 6 weeks and the other not. Similar to our previous findings ,18, Ipraand Pck1 k.As previously reported , IL-15 gEmr1, Tnf, Ccl2, and Ccr2) and fibrosis  in PRAT compared to SD-fed mice, with Ipra treatment significantly weakening or tending to weaken the upregulation  to M2-type ATMs (CD11c-CD206+ cells) in PRAT  showed that Ipra significantly suppressed the M1/M2 ratio, the ratio of M1-type ATMs , which was significantly or modestly decreased by Ipra treatment (Fabp1) and fatty acid synthesis-related genes (Fas) in the kidney, which were suppressed by Ipra treatment, and suppressed lipolysis-related genes (Cpt1a), which were increased by Ipra treatment  through modulating leptin secretion from PRAT, by ex vivo experiments. Leptin concentrations in the conditioned media (CM) prepared from PRAT (PRAT-CM) were higher in HFD-fed mice than in SD-fed mice, and these increases were suppressed by Ipra treatment a. We perP38 phosphorylation and PCNA expression were significantly greater in GECs treated with PRAT-CM from HFD-fed mice than from SD-fed mice, and this upregulation was suppressed by Ipra treatment –e. FurthIn the present study, we found that Ipra treatment ameliorated DN in HFD-fed wild-type mice, accompanied by an expansion of PRAT. The expanded PRAT displayed improved insulin signaling and suppressed inflammation and fibrosis, consistent with healthy adipose expansion. PRAT-CM of HFD-fed mice treated with Ipra promoted lower cell proliferation signals in GECs than PRAT-CM from HFD-fed mice without Ipra treatment. These results provide new insights into the mechanism by which SGLT2 inhibitors suppress DN and the establishment of therapies focused on PRAT.Several reports have shown that PRAT affects the progression of DN. One report found that the improvement in PRAT inflammation through genetic and exogenous PAI-1 inhibition suppressed the development of DN in obese mice . The othSGLT2 inhibitors as in the present study, thiazolidinediones , DPP4 inGiven that leptin concentrations in the renal vein showed solid and positive correlations with UAE, leptin circulating in the kidney may play a crucial role in developing DN in the present study. Because the correlation was weaker in systemic leptin concentration than in the renal vein, reduced production of PRAT-derived leptin by Ipra was more likely to contribute to attenuation of DN in HFD-fed mice than leptin in the systemic circulation. In vitro studies using PRAT-CM and GECs are consistent with this proposed mechanism: PRAT-CM from HFD-fed mice enhanced cell proliferation signaling in GECs via p38MAPK- and leptin-dependent pathways.As well as leptin, PRAT synthesizes and secretes various adipokines and pro-inflammatory cytokines, including adiponectin, visfatin, resistin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and -1β, some of whose gene expression in PRAT were altered by Ipra . These cSlc5a2) was hardly expressed in adipose tissue of mice [The Ipra-induced functional alternation of PRAT was consistent with “healthy adipose expansion”, an increase in the accumulation without inflammation or fibrosis as we previously reported in Epi and PVAT ,16,17,18Studies using genetically modified mice suggested the presence of three possible pathologies for this healthy adipose expansion: (i) activation of insulin signaling in adipocytes (observed in adipocytes in adipocyte-specific phosphatase and tensin homolog (PTEN) knockout mice ); (ii) rAs with Epi of PTEN knockout mice, Ipra attenuated insulin resistance of PRAT in HFD-fed mice. Moreover, as with mincle knockout mice, Ipra-treated mice exhibited reduced ATMs in PRAT, indicating amelioration of PRAT inflammation. Although the current study did not examine oxidative stress in PRAT, reports have suggested that induction of 3-HBA suppresses oxidative stress by inhibiting histone deacetylases . The incPck1 [Pck1, suggesting that this pathway may have been involved in the pathogenesis of the healthy adipose expansion. Taken together, these various factors, including increased insulin signaling, decreased macrophage infiltration, and increased ketone bodies could contribute to induce the healthy adipose expansion in PRAT of Ipra-treated mice.Moreover, we recently reported a novel mechanism by which elevated 3-HBA in Ipra-treated mice contributes to the healthy adipose expansion of Epi by suppressing IL-15 expression in ATMs and increasing the expression of lipogenesis-related genes, such as Because Ipra treatment caused an elevation in fasting serum NEFA levels in HFD-fed mice via attenuation of hyperinsulinemia, Ipra appeared to enhance lipolysis in adipose tissue. In fact, increase of perilipin 1 in Ipra-treated mice, which allows a low level of basal lipolysis by reducing the access of cytosolic lipases to stored triacylglycerol in lipid droplets in adipocytes ,37, was We also observed a decrease in TUNEL-positive cells apart from adipose expansion in the PRAT of Ipra-treated mice. These results are consistent with those in our previous reports on Epi  and PVATEach observation in the present study was consistent with a conclusion that Ipra reduced urinary albumin excretion as a result of an inter-organ network, which was primally triggered by increased urinary glucose excretion. In particular, we assumed that PRAT mediated the inter-organ communication through secretory factors including leptin and inflammatory cytokines. However, strictly speaking, we could not precisely demonstrate the inter-organ network because interventional in vivo experiments were lacking in the present study. Further experiments are awaited to demonstrate the SGLT2 inhibitor-triggered inter-organ network.In conclusion, we propose a novel mechanism by which the SGLT2 inhibitor Ipra induces adipocyte hypertrophy without increasing inflammation, fibrosis, or adipocyte death in PRAT of HFD-fed mice. Our findings also imply a novel inter-organ network between kidney and PRAT, possibly mediated by leptin, for the development of DN, and thus suggests new clinical implications for the prevention and treatment of DN through pharmacological intervention.Additional details of the Materials and Methods are included in the v/v) and added to the drinking water. During HFD feeding experiments, 8-week-old WT mice were fed an HFD  for 14 weeks, followed by an HFD with the vehicle or Ipra for 6 weeks. Ipra concentrations in the drinking water were changed every week based on daily water consumption and body weight, adjusting to 10 mg/kg/day. Age-matched control male WT mice were fed an SD throughout the experiment period. Body weight and blood glucose were measured every 2 weeks. At the end of the experiment, animals were sacrificed using intraperitoneal pentobarbital anesthesia (30 mg/kg) after 16 h of fasting. Both kidneys, PRAT, Epi, and liver were carefully dissected and removed. After careful removal of the vessels and connective tissue surrounding adipose tissue, the remaining PRAT, Epi, and liver were weighed on an electronic balance. The kidney and PRAT were divided for histological and mRNA/protein analyses, respectively.Male C57BL/6J WT mice were purchased from CLEA Japan, Inc.  and Charles River Laboratories Japan, Inc. . The animals were allowed free access to water and a standard diet . Ipra, provided by Astellas Pharma Inc. , was dissolved in dimethyl sulfoxide  at 0.04% . The leptin antagonist triple mutant (leptA), a mutant leptin analog that functions as a competitive inhibitor, was purchased from PROSPEC . Recombinant mouse leptin was purchased from PeproTech .p < 0.05 indicating statistical significance.Normally distributed values were expressed as mean ± standard error of the mean and compared using Student’s t-test or analysis of variance with post hoc testing. Non-Gaussian distributed values were expressed as box-and-whisker plots with median values and 10th, 25th, 75th, and 90th percentiles. Nonparametric statistical analysis was performed using the Mann–Whitney or Kruskal–Wallis tests with Dunn post hoc tests. All statistical analyses were performed using Prism 7 , with


HBG alleles complements defective HBB alleles underlying β-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed.Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact  Hemoglobinopathies are the world's most common group of monogenic disorders with an estimated 7% of the global population carrying these diseases Piel, . ParticuBy applying X-ray crystallography to hemoglobin, Max Perutz pioneered the use of structural information to uncover how mutations lead to disease at the molecular and atomic levels Perutz, . These aThe ongoing development of various candidate genetic therapies raises the possibility that autologous hematopoietic stem cell transplantation (auto-HSCT) will complement, or perhaps even replace, allo-HSCT as a preferable treatment modality. Importantly, the efficient and safe collection of long-term repopulating hematopoietic stem cells (HSCs) from patients with severe hemoglobinopathies is crucial for the success of genetic therapies. The development of plerixafor-based mobilization regimens has resulted in an efficient and safe manner of collecting long-term engrafting HSCs from both SCD and β-thalassemia patients -mediated gene addition, or gene editing based on programmable nucleases, e.g., zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases (RGNs) based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) proteins (CRISPR-Cas), is required to permanently rescue pathological phenotypes through the transplantation of genetically modified HSCs.As mentioned before, knowledge on the molecular, cellular and developmental processes underlying hemoglobinopathies is extensive. As such, it provides a rich foundation on which novel genetic therapy concepts can be built upon and tested. Currently, there are two main categories of genetic therapies being developed for treating hemoglobinopathies, i.e., gene therapy and gene editing involving exogenous gene addition and direct modification of endogenous DNA, respectively. Backed by decades of fundamental and pre-clinical research, gene therapy is the first modality of genetic therapy entering clinical trials targeting diseases of the hematopoietic system , then as fetal hemoglobin (α2ɤ2) and, finally, shortly after birth, acquires the composition of adult hemoglobin (α2δ2 and α2β2). These hemoglobin variants are expressed in the embryonic yolk sac, fetal liver, spleen  and bone marrow, respectively , the duplicated α-globin genes , two transcriptionally active genes with unknown function (μ and θ) and 3 pseudogenes . This cluster is located near the telomeric end of the short arm of chromosome 16  deletion upstream of the α-globin locus  and Hb Bart's (ɤ4), respectively. These insoluble tetramers precipitate intracellularly and cause the disruption of the RBC membrane leading to hemolytic anemia. In the case of Bart's hydrops fetalis syndrome, the fetus lacks hemoglobin tetramers containing α-globin altogether making it dependent on the expression of Hb Portland (ζ2ɤ2) , namely, silent carrier, carrier with symptoms, Hemoglobin H disease and α-thalassemia major. These forms range in severity from no symptomology to a lethal condition named Bart's hydrops fetalis , of which the most common deletions are -α3.7 and -α4.2 , internal heme-pocket sites and α-b-globin helix-structures . Characterized by severe mental retardation and dysmorphic features this syndrome shows striking similarities among patients. The molecular cause of ATR-X are point mutations in the ATRX gene (Xq13.3) encoding a chromatin-associated protein belonging to the SNF2 family of helicase/adenosine triphosphatases , which is inactive   or reduced α-globin expression, can have a major influence on the clinical presentation of β-thalassemia  is an autosomal recessive disorder caused by a large spectrum of mutations (>300 known) that reduce or abolish the production of the β-globin chain expressed from the e Thein, . When a a Thein, . Additioa Thein, . Interesa Thein, . These na Thein, . The rarh Thein, , 2018.By virtue of the in-depth knowledge about the complex genetics of β-thalassemia and advanced DNA sequencing technologies, it is currently possible to guide clinical management on the basis of the patient's genotype, i.e., causative mutations and genetic modifiers   do not favor pathologic HbS polymerization  leading to ɤ-globin persistence into adulthood can ameliorate the clinical severity of SCD  is an autosomal recessive disease affecting normal β-globin function. However, in contrast to β-thalassemia, SCD is caused by a single T → A substitution leading to the translation of valine instead of glutamic acid at position 6 of the β-globin chain Ingram, . Due to 0-thalassemia that the LCR regulates β-globin expression over a long distance  analysis revealed the involvement of a hitherto unsuspected trans-acting factor (Spt5) encoded by SUPT5H, which, when haplo-insufficient, reduced β-globin gene expression  techniques such as whole exome sequencing (WES) and WGS.ex vivo and reinfused back to the patient. In order to appreciate the full potential of genetic therapies for the treatment of hemoglobinopathies, it is important to understand the wide range of genetic toolsets that are under development. Therefore, in the following sections, we briefly cover the currently available genetic techniques and discuss their testing for the treatment of β-thalassemia and SCD.Although the outcomes of allo-HSCT have significantly improved in recent decades, the treatment remains suboptimal due to limited donor availability and associated risks, such as, graft-versus-host disease. Therefore, numerous efforts are being directed to treatments based on auto-HSCT in which the patient's own stem cells are harvested, genetically modified Initially, realistic prospects for gene therapy of hematological disorders arose with the introduction of ɤ-retroviral vectors (ɤ-RVs), such as those based on the Moloney murine leukemia virus, owing to their ability to stably integrate exogenous DNA into target-cell chromosomes. After the emergence of serious adverse events caused by insertional oncogenesis in a few clinical trials using ɤ-RVs, self-inactivating (SIN) lentiviral vectors (LVs) based on the human immunodeficiency virus type 1 (HIV-1), were introduced  found in the ɤ-globin chain. Of note, this mutation prevents the polymerization of this recombinant β-globin chain with residual HbS hemoglobin molecules in SCD patients, making the treatment applicable to treat both β-thalassemia and SCD  located in the vector LTRs. The enhancer-blocking properties of the cHS4 insulator aims at reducing the chance for insertional oncogenesis due to spurious LCR-driven activation of nearby proto-oncogenes  to increase vector RNA synthesis during its production yielding, as a result, higher titers of functional vector particles  transgene addition , (ii) shnockdown  and (iiinockdown  (Breda enockdown . The firnockdown . For insnockdown . An initT87Q-globin or hybrid βɤ-globin), (iii) absence or presence of insulators  within this intron. This redesigned vector can be produced at 6-fold higher titers and transduces HSCs at 4- to 10-fold higher rates than their “reverse-oriented” counterparts , constructed for inducing fetal ɤ-globin synthesis, has also progressed to a clinical trial stage  resulting in a 3- to 5-fold increase in HbF amounts when compared to mock-transduced cells  conditions, has permitted the initiation of a phase 1/2 clinical trial  end-to-end ligation of chromosomal termini, involving non-homologous end joining (NHEJ) pathways, e.g., classical NHEJ and microhomology-mediated end joining (MMEJ), and (ii) exogenous (donor) DNA-templated repair, involving homology-directed repair (HDR) , each designed to recognize a DNA triplet, fused to a catalytic domain derived from the FokI restriction enzyme. Each ZFN monomer recognizes a specific sequence of 12 to 18 nucleotides with the induction of a DSB requiring the dimerization of two FokI catalytic domains brought together by target DNA binding of a working pair of ZFN monomers , are capable of recognizing specific DNA sequences through their DNA-binding units called TALE repeats , (ii) codon-optimization of the Cas9 open reading frame; and (iii) addition of nuclear localization signals to the Cas9 protein  of Cas9  assembling single-guide RNAs (gRNAs) by fusing sequence-tailored CRISPR RNAs (crRNAs) to a common scaffolding Similarly to ZFNs and TALENs, RGNs can induce DSBs at off-target sequences  covalently linked to either a cytidine or adenine deaminase capable of inducing C → T or A → G transitions, respectively  covalently linked to an engineered Molony murine leukemia virus reverse transcriptase (RT) optimized owing to five point mutations enhancing its stability and processivity , that simultaneously provides a primer and a template for the RT. Specifically, the pegRNA consists of a conventional sequence-tailored gRNA linked to a primer binding site (PBS) and a RT template encoding the edit of interest. Via the PAM-interacting domain of the Cas9 nickase, the prime editor recognizes the PAM and, after hybridization between the crRNA portion of the pegRNA and the target sequence, the PAM-containing DNA strand is nicked. The resulting single-stranded genomic DNA anneals to the PBS of the pegRNA providing a primer for RT-mediated cDNA synthesis. The resulting DNA copy containing the edit eventually hybridizes to the complementary chromosomal target sequence with non-hybridizing DNA flap removal leading to edit installation at the target site presumably following DNA replication or mismatch repair  recognizes a T-rich PAM , (ii) requires a crRNA and no tracrRNA and (iii) seems to have a single RuvC-like domain mediating the staggered cleavage of double-stranded DNA strands with 4-5 nt overhangs distal from the PAM  HBB mutation-specific.The tailoring of genome editing strategies for the genetic correction of hemoglobinopathies is progressing rapidly. These strategies can be divided in two main categories: (i) HBB gene itself via the introduction of programmable nucleases and donor DNA templates into HSCs , so far resulted in 6 patients treated of which 3 patients remained vaso-occlusive crisis free (VOC-free) with fetal hemoglobin levels up to 48%   . Other c ongoing .de novo-created mutations yielding a HPFH-like phenotype have recently been presented in yet to be peer reviewed data . Exposing HSCs ex vivo to small-molecule drugs and growth factors favoring survival and limited cell cycle entry can improve gene editing frequencies  donors  or shRNA molecules into murine β-thalassemic primary erythrocytes  are particularly common , life-long monitoring for the emergence of potentially hazardous monoclonal expansions of HSC progenies, is warranted. Importantly, LV-based gene therapies are showing promising results in terms of achieving clinically relevant β-like globin expression levels in initial and ongoing clinical trials  viral vectors, such as adenoviral (AdV) vectors and adeno-associated viral (AAV) vectors, are promising agents for introducing gene editing components into HSCs, in particular, certain capsid pseudo-typed variants  closely monitoring the impact and precision of gene repair procedures in target cells regardless of their type and replication status (Maggio and Gonçalves, Equally regarding the ultimate performance of gene editing interventions, it is worth mentioning that various types of stem and progenitor cells, including HSPCs, are particularly susceptible to p53-dependent cell cycle arrest and apoptosis, even when subjected to a limited number of targeted DSBs (Haapaniemi et al., Hence, looking ahead, besides seeking to enhance absolute gene editing efficiencies, an equally important priority will be continuing to improve the safety profile of gene editing procedures as a whole. To this end, macromolecular enzymatic complexes that bring about targeted and precise genomic modifications without catalytic induction of DSBs might become particularly valuable and include, nicking RGNs and their derivative base and prime editor proteins as well as engineered or molecularly evolved site-specific recombinases and CRISPR-based transposases and recombinases (Komor et al., ex vivo transduction and autologous transplantation of HSCs into afflicted patients. This being said, in addition to regulatory requirements, robust and affordable GMP-grade platforms for up-scaling and downstream processing of AMTPs will be crucial before genetic therapies for hemoglobinopathies become broadly available to those in need (Staal et al., In conclusion, knowledge from hemoglobin biology and clinical genetics studies, together with the herein covered rapid expansion of gene editing techniques, are accelerating the development of genetic therapies for treating hemoglobinopathies. These advances are in turn expected to capitalize and build upon gene transfer and stem cell technologies underlying HZ wrote the manuscript with the contributions from the other authors. All authors have reviewed and edited the work.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.


The prognosis for metastatic prostate cancer patients remains poor. Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer and is a promising target for both imaging and therapy. In this review paper, we provide an overview of the evidence for PSMA-targeted imaging in prostate cancer, focusing on different imaging modalities and their theranostic applications. We will also review PSMA-targeted radioligand therapy, focusing on lutetium-177 radioligand therapy and alpha-emitting radioligand therapy with actinium-225. Combination regimens with lutetium-177 and other systemic therapy agents will be reviewed. Antibody-based radioimmunotherapy will also be discussed along with other noteworthy radionuclide agents.Introduction: Prostate-specific membrane antigen (PSMA) is a promising novel molecular target for imaging diagnostics and therapeutics (theranostics). There has been a growing body of evidence supporting PSMA theranostics approaches in optimizing the management of prostate cancer and potentially altering its natural history. Methods: We utilized PubMed and Google Scholar for published studies, and clinicaltrials.gov for planned, ongoing, and completed clinical trials in PSMA theranostics as of June 2021. We presented evolving evidence for various PSMA-targeted radiopharmaceutical agents in the treatment paradigm for prostate cancer, as well as combination treatment strategies with other targeted therapy and immunotherapy. We highlighted the emerging evidence of PSMA and fluorodeoxyglucose (FDG) PET/CT as a predictive biomarker for PSMA radioligand therapy. We identified seven ongoing clinical trials in oligometastatic-directed therapy using PSMA PET imaging. We also presented a schematic overview of 17 key PSMA theranostic clinical trials throughout the various stages of prostate cancer. Conclusions: In this review, we presented the contemporary and future landscape of theranostic applications in prostate cancer with a focus on PSMA ligands. As PSMA theranostics will soon become the standard of care for the management of prostate cancer, we underscore the importance of integrating nuclear medicine physicians into the multidisciplinary team. Prostate cancer represents a significant public health problem in the world. It is the second most common cancer and fifth leading cause of cancer death among men in 2020 . While mProstate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein receptor that is overexpressed  in most prostate cancers (>90%). PSMA is normally expressed in the renal tubules and duodenum, in which it plays an essential role in the processing and uptake of dietary folates, and in the brain, where it plays a major role in modulating the output of glutamate signaling. The biological function of PSMA in prostate cancer remains elusive, although evidence suggests its role in activation of PI3K-Akt signaling in prostate cancer through release of glutamate as messenger molecule . Even th68Ga-PSMA-11), gallium-68-labeled PSMA-I&T (68Ga-PSMA-I&T), and fluorine-18 (18F) labeled PSMA ligands, including 18F-DCFPyL and 18F-PSMA-1007. There is no consensus as to the optimal PSMA radioligand. Currently, 68Ga-PSMA-11 and 18F-DCFPyL are the only FDA-approved radioligands for PSMA-targeted PET imaging in men with prostate cancer [Many PSMA-targeting small molecule and antibody agents have been developed and tested for imaging by single-photon emission computerized tomography (SPECT) and positron emission tomography (PET). Compared to anti-PSMA antibodies, small-molecule PSMA inhibitors are preferable as PET imaging agents due to faster tumor uptake and more rapid excretion, which enhance contrast and reduce radiation exposure ,7. Curree cancer .18F-PSMA-1007 has been found to have diagnostic accuracy comparable to 68Ga-PSMA-11 for detection of biochemically recurrent prostate cancer, with the advantage of minimal excretion in the urinary tract, which allows for better detection of prostate bed recurrences [18F-PSMA-1007 PET scans, as there have been reports of focal unspecific bone uptake, defined as focal mild-to-moderate uptake (SUVmax < 10.0) not obviously related to a benign or malignant cause [The fluorinated agents have the advantage of a longer half-life which allows for central production and distribution over longer distances as well as lower positron energy that can lead to improved imaging quality. nt cause .PSMA-targeted imaging has been extensively explored in primary staging and restaging of prostate cancer. Conventional imaging with CT, MRI and bone scan have limited sensitivity and specificity for detecting occult metastatic disease, particularly in the setting of low PSA values . Prospec18F-DCFPyL PET/CT prior to MDT. The treating radiation oncologists were blinded to the result of PSMA scans and treatment plans were based on conventional imaging. The PSMA scans were compared to the SBRT treatment plans, and patients were categorized as having total or subtotal consolidation of PSMA-avid lesions. In the SBRT arm, disease progression at six months was 10% vs. 61% in the observation arm (p = 0.005). For those who received total consolidation, rate of new metastases at six months was 16% vs. 63% in those with subtotal consolidation (p = 0.006). Remarkably, median distant metastasis-free survival was 29 months in those with total consolidation vs. six months in those with any untreated lesions [Recent evidence has suggested that treatment of the primary tumor or metastasis-directed therapy (MDT) in carefully selected patients with low-volume metastatic prostate cancer may confer a survival advantage . Improve lesions . This stWith the advent PSMA-targeted imaging, there are many ongoing clinical trials in oligometastatic-directed therapy  in both PSMA-targeted imaging has been explored as a biomarker for assessment of treatment response. However, this can be challenging as PSMA avidity can be affected by a multitude of factors such as castration-sensitive versus castration-resistant disease, timing and type of therapy received, and location of the lesions. PSMA expression appears to be upregulated with ADT and androgen-axis inhibitors such as enzalutamide in the first three months of treatment initiation ,23. PSMAAccording to consensus statements on PSMA PET/CT response assessment criteria, from a panel of international experts recruited by European Association of Nuclear Medicine (EANM) and EAU (European Association of Urology), PSMA PET/CT should not be performed to assess response within three months after initiation of systemic therapy in hormone sensitive prostate cancer. The panel also recommended that PSMA PET/CT response assessment should be implemented and evaluated only in the context of clinical trials . PSMA im223Ra) has been established for metastatic prostate cancer localized to the bone [Prostate cancer is sensitive to radiotherapy (RT), in which various forms of RT, such as external beam RT (EBRT) and brachytherapy, have become standard treatment options for localized prostate cancer, and SBRT is currently explored in patients with oligometastatic prostate cancer. Systemic radiation with alpha particle-emitting calcium mimetic Radium-223 (the bone . ConcurrThe intensity of uptake (avidity) in PSMA PET/CT represents the PSMA expression in prostate cancer cells. The level of avidity generally increases with tumor dedifferentiation and castration resistance. However, dedifferentiated neuroendocrine prostate cancer maybe an exception in which PSMA-avidity can be low owing to suppression of PSMA gene (FOLH1) . Pretrea18F-fluorodeoxyglucose (FDG) is a PET tracer that is preferentially taken up by cancer cells due to increased glucose metabolism known as “Warburg effect” [18F-FDG and 18F-fluorodihydrotestosterone (FDHT), which is an androgen receptor probe. Patients with more than 12 FDG-avid lesions had a significantly worse OS than their counterparts. The study also demonstrated an increased risk of death in patients with discordant lesions (positive FDG-avid lesion but negative androgen receptor expression with 18F-FDHT) [ effect” . FDG-PET effect” . In pros effect” ,35. This effect” ,37. FDG-8F-FDHT) .177Lu-DOTATATE in patients with neuroendocrine tumors, in which FDG-PET is recommended to be used in conjunction with somatostatin-receptor (SSR) imaging to exclude patients with discordant lesions (FDG-positive/SSR-negative) from receiving PRRT treatment [Tumor sites with low PSMA-avidity and high FDG-avidity represent sites of aggressive disease which cannot be effectively targeted by radioligand therapy. Discordance between PSMA-PET and FDG-PET has been observed in very advanced mCRPC, with dismal OS of 2.5 months based on the LuPSMA trial . Thus, Freatment .177Lu) is a beta-emitting radioisotope that has a mean energy of 133.6 keV with maximum penetration depth of <2 mm and 6.7-days half-life [177Lu labelled PSMA has been studied extensively to date and it has become a promising new therapeutic approach for treatment of mCRPC. The most studied of the 177Lu labelled PSMA radioligands include 177Lu-PSMA-I&T (imaging and therapy) and 177Lu-PSMA-617, with the latter being preferred owing to reduced kidney uptake [177Lu-PSMA radioligand therapy [177Lu-PSMA radioligand therapy in the 3rd line setting, 44% of treated patients had PSA decline ≥50% or more, with median overall survival of 14 months [Lutetium-177  and grade 3–4 thrombocytopenia in 27% of patients [In the first prospective single-arm phase 2 study (LuPSMA), patients .177Lu-PSMA-617 (6.0–8.5 GBq intravenously every six weeks for up to six cycles) vs. cabazitaxel (20 mg/m2 intravenously every three weeks for up to 10 cycles) for mCRPC patients who have progressed on docetaxel. The majority (91%) of patients on the trial had prior ARAT. In this study, the 177Lu-PSMA-617 arm had an improved PSA response rate (PSA reduction ≥ 50%) of 66% vs. 37% for cabazitaxel, as well as improved one-year PFS rate of 19% vs. 3%  with a median follow-up of 18.4 months. Objective response rate (ORR) was significantly greater in 177Lu-PSMA-617 arm . Pain response was also numerically higher with 177Lu-PSMA-617  versus 54% for cabazitaxel [177Lu-PSMA-617 is a promising alternative to cabazitaxel in men who have progressed following docetaxel for mCRPC.Cabazitaxel is a chemotherapy agent for patients with mCRPC who have progressed after previous treatment with docetaxel. Based on the CARD study, cabazitaxel is the current standard of care for treatment for patients who have progressed on one of androgen-receptor-axis-targeted therapies (ARATs)  and docetaxel . TheraP azitaxel . This st177Lu-PSMA-617 (7.4 GBq every six weeks × six cycles) plus best standard/standard-of-care (SOC) versus best SOC care alone in heavily-pretreated mCRPC patients who have received at least one ARAT and were previously treated with 1–2 taxane chemotherapy regimens. The study enrolled 831 patients with 551 patients allocated to 177Lu-PSMA-617 + SOC. Over a median follow-up of 20.9 months, treatment with 177Lu-PSMA-617 significantly improved OS by a median of 4.0 months ; p < 0.001, one-sided), compared to SOC alone. The radiological PFS (rPFS) was also improved by treatment with 177Lu-PSMA-617 + SOC compared to SOC alone ; p < 0.001, one-sided). Overall 177Lu-PSMA-617 therapy was well tolerated. This is the largest and latest prospective study to date investigating 177Lu-PSMA-617 in treatment of mCRPC. The positive results of this study support the adoption of 177Lu-PSMA-617 as a standard armamentarium against advanced PSMA-positive mCRPC [The VISION study (NCT03511664) is an international phase 3 randomized trial of ve mCRPC .177Lu-PSMA-I&T has similar PSMA-affinity, dosimetry and pharmacokinetics profile compared to 177Lu-PSMA-617 [177Lu-PSMA- I&T consisted of 100 mCRPC patients, who underwent 319 cycles with a mean dose of 7.4 GBq. PSA decline over 50% was seen in 38% of patients with PSA PFS and OS of 4.1 and 12.9 months, respectively. High grade hematological toxicities included 9% of anemia, 6% of neutropenia, and 4% of thrombocytopenia. High grade non-hematological toxicities were not observed [PSMA-I&T is a small molecule PSMA inhibitor initially developed in Germany in 2015. PSMA-617 . In a rePSMA-617 . The larobserved .177Lu-PSMA-I&T (AKA. 177Lu-PNT2002) versus abiraterone or enzalutamide in delaying radiographic PFS in the second line setting after progression on 1st line ARAT  in mCRPC patients. This study recently started recruitment in March 2021 with estimated enrollment of 415 participants (NCT04647526).SPLASH is a phase 3, open-label, randomized study evaluating the efficacy of 177Lu-PSMA radioligand therapy with ARAT may lead to improved tumor control in CRPC.Preclinical observational studies have shown upregulation of PSMA messenger RNA production and PSMA receptor expression during AR inhibition . In pati177Lu-PSMA-617 vs enzalutamide monotherapy as first-line therapy in patients with mCRPC who have sufficient PSMA expression on PSMA PET/CT. This study aims to recruit 160 men with mCRPC who are deemed at high-risk for early failure on enzalutamide monotherapy  [177Lu-PSMA-617 in combination with 160 mg enzalutamide daily. Primary endpoint is PSA-PFS. Secondary endpoints include rPFS, OS, PSA-response rate, quality of life and adverse events. This study is expected to be completed in 2023.ENZA-P (NCT04419402) is a phase 2 randomized multicenter study evaluating enzalutamide in combination with  trials) ,51. Pati177Lu-PSMA-617 in combination with standard of care vs. standard of care in patients with metastatic hormone-sensitive prostate cancer (mHSPC). In this study, the standard of care is defined as a combination of ARAT and ADT. Approximately 1126 patients will be randomized in this study. This study is anticipated to start recruiting in May 2021.PSMAddition (NCT04720157) is a phase 3 randomized study comparing 177Lu-PSMA-617, it will be important to determine long term safety and the impact of early use of 177Lu-PSMA-617 on subsequent lines of therapy, such as chemotherapy. Other important questions that need to be answered will include the consideration of optimal dose, schedule, and patient selection.Although there is a biological basis to support combining ARAT with Radiation induces single-stranded and double-stranded DNA breaks through formation of oxidative free radicals. The enzyme poly ADP ribose polymerase (PARP) is crucial in repairing radiation-induced single-stranded DNA breaks, which allows for radio-resistance of cancer cells . Thus, P177Lu-PSMA-617 with olaparib in men with mCRPC who have progressed on prior ARAT, and have PSMA-avid disease on imaging. 177Lu-PSMA-617 will be administered up to 6 cycles with the dose of olaparib escalated from 50 mg to 300 mg, in six increments. The primary endpoints are to establish dose limiting toxicities, maximum tolerated dose and recommended phase 2 dose. Secondary endpoints include safety and efficacy. Tissue and liquid biopsies (circulating tumor cells and DNA) will be collected and analyzed to identify predictive biomarkers of response and resistance.Preclinical studies have shown synergistic anti-tumor effect of combination PARP inhibitor and radiation, including radioligand therapy. PARP inhibitors appear to be especially effective in enhancing radiation-induced tumor cell death at low doses ,60. LuPAOther DDR inhibitors that may have synergy with radioligand therapy include DNA-PK inhibitor AZD7648, ATM inhibitor AZD0156, and RNA polymerase I inhibitor CX-5461. Preclinical studies for these agents have shown synergy with radiation therapy ,63.Anti-PD-1 monoclonal antibodies such as nivolumab and pembrolizumab, and anti-CTLA-4 monoclonal antibodies, such as ipilimumab, together referred to as immune-checkpoint inhibitors (ICI) are an important class of cancer therapies that have demonstrated significant activity, including some durable responses, in a number of cancers, including melanoma, non-small cell lung cancer, bladder cancer, and kidney cancer. These agents are now used as single agents or in combination with chemotherapies as first or second lines of treatment for approximately 50 cancer types with more than 3000 active clinical trials as of 2020 . In pros177Lu-PSMA-617 in mCRPC patients who have progressed on ARAT. Patients with PSMA-avid disease are enrolled to receive pembrolizumab up to two years (35 cycles every three weeks) in combination with up to six cycles of 177Lu-PSMA-617 (six weeks apart). The primary endpoints include PSA response rate and safety. Secondary endpoints include rPFS, PSA-PFS, and OS. The study aims to enroll 37 patients with completion expected in October 2021.PRINCE (NCT03658447) is an Australian phase Ib/II study examining the combination of pembrolizumab with 177Lutetium PSMA radioligand therapy earlier in the prostate cancer natural history. The LuTectomy trial (NCT04430192) is an Australian open label, phase I/II non-randomized study evaluating neoadjuvant 177Lu-PSMA-617 (one or two cycles) followed by radical prostatectomy + pelvic lymph node dissection in patients with high risk localized or locoregional advanced prostate cancer and PSMA-avid disease. Accrual for this study has begun with estimated primary completion date in August of 2022.Clinical trials are underway to examine the role of 177Lu-PSMA-I&T in oligometastatic HSPC. Patients with PSMA-avid disease with five or less metastatic lesions are randomized to either two cycles of 177Lu-PSMA-I&T given six weeks apart vs. standard of care. The estimated enrollment size is 58 patients with estimated primary completion in January 2023. The important clinical rationale of this study is that many patients with oligometastatic prostate cancer do not qualify for local MDT treatment such as surgery or EBRT due to tumor location or prior therapy, in which targeted radioligand therapy for these lesions maybe beneficial.The Bullseye trial (NCT04443062) is a Dutch randomized, open label, multi-center, phase 2 study testing 177Lu-PSMA-617 given every six weeks followed by docetaxel chemotherapy every three weeks for six cycles, versus docetaxel chemotherapy on its own in patients with newly-diagnosed high volume mHSPC, defined as PSMA-avid visceral metastases, or ≥4 PSMA-avid bone lesions with one or more outside the vertebral column and pelvis as seen on 68Ga-PSMA PET/CT. The study aims to enroll 140 patients with estimated primary completion in April of 2024.UpFrontPSMA (NCT04343885) is an Australian phase 2 randomized clinical trial comparing the efficacy of two cycles 131I)-MIP-1095 is a PSMA-targeted small-molecule inhibitor radionuclide therapy developed by Molecular Insight Pharmaceuticals, Inc. It was one of the first agents used in radiopharmaceutical therapy against prostate cancer. 131I is a beta-emitter having a similar particle range and half-life as 177Lu , but with much more abundant gamma emission, which makes it less ideal from toxicity and radiation safety perspectives [Iodine-131, thrombocytopenia lasted multiple months [131I-MIP-1095 in combination with enzalutamide compared to enzalutamide alone in patients with PSMA-avid mCRPC who have progressed on abiraterone.A study testing a single cycle of e months . In a sue months . ARROW (177Lu-PSMA-R2 is being developed by Advanced Accelerator Applications (a subsidiary of Novartis). PSMA-R2 is a urea-based PSMA-targeting small molecule inhibitor. In preclinical studies, 177Lu-PSMA-R2 has rapid and specific uptake in mice bearing prostate cancer tumors, with rapid elimination through urinary system [177Lu-PSMA-R2 in patients with PSMA-avid mCRPC is currently being conducted, with planned completion in June 2022 (NCT03490838).y system . A phase223Ra, which is a first-in-class alpha particle-emitting agent approved for mCRPC with symptomatic bone metastases [223Ra treatment in mCRPC is limited by the fact that it is a bone-seeking agent that does not target soft-tissue metastases, and even within bone metastases the tumor cells are only reached via cross-fire radiation. The discovery of PSMA allowed for the delivery of alpha-emitting radionuclides more selectively to the tumor.Alpha particle radiation is characterized by a significantly higher linear energy transfer (LET) compared to beta particle radiation (50–230 keV/μm vs. 0.2 keV/μm), which allows for a greater chance of inducing unrepairable DNA double-strand breaks in cancer cell with increased cytotoxic potential. Furthermore, alpha particles have a shorter path in tissue (50–100 μm) compared with beta articles  ,74. The tastases . However225Ac)-PSMA-617 has been the most studied PSMA-targeting alpha particle therapy. Based on a preliminary report from a single-center study, 225Ac-PSMA-617 (100 kBq/kg every two weeks) resulted in complete PSA and imaging response in two patients who received greater than eight prior systemic therapies [225Ac-PSMA-617 is also active in mCRPC patients who have progressed after 177Lu-PSMA-617. In a group of 26 heavily pre-treated (median of six prior lines of systemic therapy) mCRPC patients who have progressed on 177Lu-PSMA-617, all of them received 225Ac-PSMA-617 under a compassionate use program in Germany. This study reported substantial PSA decline of >50% in 65% of patients with PFS and OS of 4.1 and 7.7 months, respectively. High grade hematological toxicities were anemia (35%), leucopenia (27%), and thrombocytopenia (19%). All patients experienced xerostomia, which led to treatment discontinuation in about a quarter of patients [Actinium-225  is a prospective phase 1, open-label, international, dose escalation study to evaluate the safety of 213Bi) is a mixed alpha and beta emitter with a short half-life of 45.6 min. It demonstrated pre-clinical activity when tagged to PSMA-targeting antibody J591 [213Bi-PSMA-617 in which one patient with mCRPC was treated with two cycles. This patient achieved PSMA-imaging response and biochemical response with decrease in PSA level from 237 μg/L to 43 μg/L [225Ac-PSMA-617 based on a dosimetry study [Bismuth-213  [The extracellular domain of PSMA also makes an accessible target for antibodies. Anti-PSMA radioimmunotherapy actually predates the development of small molecule-based radioligand therapy. A notable example is J591 antibody, which was initially characterized in 1997, and it has been the most explored antibody for PSMA-targeted radioimmunotherapy to date . 177Lu-J 60 days . In a phctively) .177Lu-J591 appears to be a promising strategy to increase cumulative radiation in order to improve anti-tumor activity while mitigating hematological toxicity. In a phase 1/2 study, 49 mCRPC patients received fractionated doses of 177Lu-J591 ranging from 20 to 45 mCi/m2 × 2 two weeks apart. The recommended phase 2 doses were 40 mCi/m2 and 45 mCi/m2 × 2. At the higher dose, median OS was 42.3 months vs. 19.6 months in patients who received lower dose. The higher dose led to higher grade 4 thrombocytopenia and neutropenia than lower dose , with no high-grade hemorrhagic episodes in any of the patients [Dose-fractionation of patients .177Lu-J591 is likely attributed to monoclonal antibodies being large molecules with slower clearance from the circulation compared to small molecules such as PSMA-617 [225Ac-J591 could mitigate the xerostomia and nephrotoxicity seen in 225Ac-PSMA-617. In a phase 1 trial, 22 mCRPC heavily-pretreated patients were treated with seven dose levels of 225Ac-J591. The treatment was very well tolerated and the MTD was not reached. Only one patient treated with 80 KBq/kg had grade 4 anemia and thrombocytopenia. There were no DLT reported in 6 patients that were treated at the highest planned dose of 93.3 KBq/kg. Xerostomia was only seen in patients with prior 177Lu-PSMA-617 therapy. PSA decline was seen in 64% of patients with 41% of patients experienced ≥ 50% PSA decline. Enrollment in the expansion cohort is currently being completed [177Lu-J591, a phase I/II dose-escalation study of fractionated and multiple dose 225Ac-J591 is currently ongoing for mCRPC patients (NCT04506567).The cause of higher hematological toxicity for PSMA-617 . Howeverompleted . Given t227Th) is an alpha-particle emitter with a long half-life of 18.7 days, which allows for ease of transportation and preparation of the radiopharmaceutical. 227Th can be efficiently complexed with octadenate 3, 2-hydroxypryridinone  chelator convalently linked to antibodies at ambient room temperature. 227Th-PSMA-TTC is a novel, fully human antibody attached to 227Th, which has recently been developed by Bayer and has demonstrated very strong antitumor efficacy in animal models with prostate cancer [Thorium-227 (e cancer . CurrentBiTE (bispecific T-cell engager) therapy is a novel targeted immunotherapy approach that engages the patient’s own T cells to kill cancer cells. The BiTE molecule binds to a tumour-associated antigen on targeted cells and CD3 on T cells which leads to T-cell proliferation, cytokine production, and T-cell-mediated killing of cancer cells. The advantage of this treatment approach over conventional ICI is such that it can bypass the conventional pathway of T-cell receptor activation and may be active, independent of the tumour’s genetic background . BiTE thPasotuxizumab (AMG 212) is a 55kDa BiTE molecule that binds to CD3 on T cells and also PSMA on prostate cancer cells, thereby activating the patient’s own T cells to eliminate PSMA-expressing prostate cancer cells. Preclinical studies have demonstrated delayed tumour growth and shrinkage in human cancer xenograft models . A phaseBased on extensive research conducted thus far, PSMA theranostics represents a rapidly emerging strategy in the management of prostate cancer. PSMA-targeted imaging has demonstrated excellent sensitivity and specificity for the detection of prostate cancer compared to conventional imaging, with preliminary evidence demonstrating that the result impacts clinical practice. There are many potential clinical applications of PSMA-targeted imaging, with the most encouraging being the earlier detection of disease, leading to earlier therapeutic interventions. However, the impact on survival of an earlier initiation of therapy based on PSMA-targeted imaging is not yet apparent. Furthermore, PSMA-targeted radioligand therapy is a highly promising way to treat advanced prostate cancer, with recent evidence of rPFS and OS benefits. More research is underway to validate efficacy as well as improve safety and outcomes at earlier stages of the disease. Future research directions include the development of standardized methods for patient selection, response assessment, cost–benefit analysis, and the optimization of production and supply of radiopharmaceuticals. Considering that PSMA theranostics will soon become a standard of care in the management of prostate cancer, it will be crucial to integrate nuclear medicine physicians into the multidisciplinary team alongside medical oncologists, radiation oncologists, urologists, and radiologists in order to optimize the care of patients with this complex malignancy.


Since the beginning of the COVID-19 pandemic, divergent variants of concern (VoCs) of SARS-CoV-2 have evolved and become the most prevalent SARS-CoV-2 variants in distinct locations at different times. Currently, the Delta variant (B.1.617.2) dominates infection events in large parts of the world. Immunization campaigns, however, still use SARS-CoV-2 vaccines based on the spike (S) protein of the original Wuhan virus.The S protein of the Delta variant of SARS-CoV-2 harbors mutations that support replication and transmission but also weaken the binding of neutralizing antibodies. The Delta variant has been reported to evade control by antibodies induced upon infection and, arguably more relevant, after BNT162b2 (BNT) vaccination .In addition to homologous prime-boost protocols, millions of ChAd-primed vaccinees received heterologous boost immunization with mRNA-based SARS-CoV-2 vaccines, as vaccination with ChAd was halted due to an increased risk of thrombotic events.The results from randomized , 5 and on = 31, 20 females) or heterologous BNT  prime-boost protocols [We assessed plasma from 85 individuals at a mean of 68 days (range 45–91 days) after ChAd priming and a mean of 17 days (range 13–23 days) after either homologous ChAd (While homologous ChAd boosting only slightly increased neutralization of the Delta variant, heterologous ChAd/BNT vaccination led to a ninefold increase in neutralizing titers Fig. , resultiThe overall robust inhibition of the Delta variant further supports heterologous boosting with BNT of vaccinees initially primed with ChAd. However, in contrast to Alpha, Beta, and Gamma variants, homologous BNT prime-boost vaccination might be even more efficient in neutralizing the Delta variant.Our data emphasize a high level of complexity in antibody responses that is affected not only by the vaccine or the vaccinee’s immune system but also by the VoC to be combatted. Moreover, our data indicate the urgent need for detailed studies to evaluate which vaccine schedules are best suited to combat COVID-19, once humoral immunity starts diminishing and the risk for infection and disease rises again in the vaccinated population.Supplementary Material and Methods


Dear Editor,3. Because mitochondria membrane4 insulated mtDNAs’ access from large ribonucleoprotein complex of Cas9 and sgRNA, mtDNAs are intractable for genetic modifications with RNA-guided CRISPR/Cas9 system commonly used for effectively editing nuclear DNA of different species. Although ZFN and TALEN have been previously engineered to successfully cut and eliminate mtDNA in a programmable way9, correction of disease-causing point mutations in mtDNA remained challenging. Recently, DddA-derived cytosine base editors (DdCBEs) have been developed to specifically induce C-to-T conversion in mtDNA by fusion of sequence-programmable TALE and split deaminase derived from interbacterial toxins10. DdCBE has been demonstrated to generate mutant zebrafish and mice carrying base-modified mtDNA by injecting DdCBE mRNA into zygotes12. However, the efficacy of DdCBE for specific installation of C-to-T conversion in mtDNA of human embryos remained to be investigated.Mitochondrial diseases could be caused by heritable mutations in both mtDNA and nuclear DNAND4 gene on mitochondrial genome. Split deaminase pair DddAhalf G1397-C and G1397-N, along with SOD2/COX8A mitochondrial targeting signal and uracil glycosylase inhibitor, were further fused with the TALE pair respectively to generate ND4-DdCBE that targets ND4 locus. After in vitro transcription, we injected the DdCBE mRNA into clinically discarded human embryos with three pronuclei (3PN) to check protein expression and cellular localization. Immunostaining results using anti-HA and anti-FLAG antibodies revealed high expression of the DddA-TALE fusion deaminase pairs and their proper co-localization with mitotracker signal in the blastomere cell of human embryos  injected with both ND4-DdCBE and GFP mRNA showed around 25% C•G-to-T•A conversions while dead DdCBE-injected, uninjected (GFP−), or GFP only ones remained intact on target loci  potentially resulted from ND4-DdCBE off-target effects on mitochondrial genome. Most of SNVs identified in 2PN GFP+ embryos were centered around on-target loci  and interrogate off-target effects of DdCBE, we performed mitochondrial genome-wide sequencing analysis after injecting yos Fig. . The othoci Fig. . We thenoci Fig. , probablces Fig. , potentiits Fig.  and S4d,Taken together, our results demonstrate that DdCBE is an effective base editor for inducing point mutations in mtDNA of human embryos, and the efficiency is much higher in 8-cell embryos. Our 8-cell injection method could help generate mitochondrial disease models as well as derived embryonic stem cells for functional investigation of disease-associated mutations in mtDNA. The current study is performed on clinically discard 3PN and 2PN embryos from healthy people. Therefore, it warrants further study to test the capability of DdCBE to correct disease-causing mutations in mtDNA of the embryos from donor patients, which may provide the alternative way for prevention of heritable mtDNA mutations leading to the untreatable mitochondrial diseases after birth. Given the bystander and off-target editing profile, DdCBE remains to be further optimized for both basic and therapeutic research in the future.Supplementary information


Immunotherapy has moved to the forefront of modern oncologic treatment in the past few decades. Various forms of immunotherapy currently are emerging, including oncolytic viruses. In this therapy, viruses are engineered to selectively propagate in tumor cells and reduce toxicity for non-neoplastic tissues. Adenovirus is one of the most frequently employed oncolytic viruses because of its capacity in tumor cell lysis and immune response stimulation. Upregulation of immunostimulatory signals induced by oncolytic adenoviruses (OAds) might significantly remove local immune suppression and amplify antitumor immune responses. Existing genetic engineering technology allows us to design OAds with increasingly better tumor tropism, selectivity, and antitumor efficacy. Several promising strategies to modify the genome of OAds have been applied: capsid modifications, small deletions in the pivotal viral genes, insertion of tumor-specific promoters, and addition of immunostimulatory transgenes. OAds armed with tumor-associated antigen (TAA) transgenes as cancer vaccines provide additional therapeutic strategies to trigger tumor-specific immunity. Furthermore, the combination of OAds and immune checkpoint inhibitors (ICIs) increases clinical benefit as evidence shown in completed and ongoing clinical trials, especially in the combination of OAds with antiprogrammed death 1/programed death ligand 1 (PD-1/PD-L1) therapy. Despite remarkable antitumor potency, oncolytic adenovirus immunotherapy is confronted with tough challenges such as antiviral immune response and obstruction of tumor microenvironment (TME). In this review, we focus on genomic modification strategies of oncolytic adenoviruses and applications of OAds in cancer immunotherapy. With the global population aging, cancer has become a leading cause of death and a major barrier to improving quality of life in most countries . As the + T cells, which increases efficacy of ICIs (Oncolytic viruses (OVs) are naturally occurring or engineered viruses that selectively propagate in tumor cells while minimizing disruptions to normal ones . OVs can of ICIs .Currently, there are numerous OVs under preclinical or clinical investigations, including but not limited to adenoviruses , herpes Adenovirus (Ad) is a non-enveloped, double-stranded linear DNA virus with icosahedral capsid that mainly consists of hexon, penton, and fiber proteins . The penOAd is a therapeutic agent that infects tumor cells via the interaction of adenoviral fiber knob to receptors on the surface of the cells . DiffereAlthough wild-type adenoviruses preferentially infect tumor cells due to the defective viral sensing mechanism of most tumor cells , OAds caAd5 is the most commonly used backbone for conventional OAds design, whereas CXADR, the natural receptor of Ad5, is absent or low expressed in many cancer cells . UnfortuThe first modification strategy was chimeric fibers. Replacement of the Ad5 fiber knob with Ad3 fiber knob (5/3 chimerism) has been proven to increase infectivity for cancer cells, while retaining the safety and replication competent of the parental Ad5 . Notablyvβ3 or αvβ5 integrins (The second strategy is an incorporation of arginine-glycine-aspartic acid (RGD) peptides in the fiber knobs of Ad5, which allows the virus to enter cells using αntegrins . A phasentegrins .As it is difficult for Ad5 to access tumor cells, researchers have constructed fully serotype 3 OAds instead. In mice, Ad3 was more immunogenic and induced higher amount of cytokines but less liver damage than Ad5 and Ad5/3 . In 15 pA major restriction of natural Ads is the lack of the ability to selectively target tumor cells . To enhaHowever, relying solely on viral genes deletion is insufficient for resolution of unwanted tissue damage . AnotherThe growing recognition that immune activation induced by OAds is an essential component of cancer treatment has led researchers to construct potent vectors for immune factors through adding immunostimulatory transgenes into the adenoviral genome. This allows these immunostimulatory factors to accumulate at tumor sites, thereby increasing antitumor efficacy and decreasing systemic adverse reaction. Several common immune factors carried by OAds are described below.+ T cells. Two patients with the best overall survival showed the most prominent infiltration of CD8+ T cells to tumors as well as systemic induction of tumor-specific CD8+ T cells. Importantly, the upregulated expression of PD-L1 in the post-treatment of mesothelioma patients further highlighted that ONCOS-102 can act as an immunostimulatory agent to facilitate immune checkpoint blockade (Granulocyte-macrophage colony-stimulating factor (GM-CSF) is best known for its critical role in immune modulation and hematopoiesis . Some stblockade . More deInterleukin-12 (IL-12) is a proinflammatory cytokine that initiates antitumor immune responses by promoting the generation of tumor-specific cytotoxic T lymphocytes (CTLs) and activating natural killer (NK) cells and T cells . AlthougAs multiple cells in the tumor stroma, such as the infiltrating immune cells, express CD40 and 4-1BB, immunotherapies targeting CD40 and 4-1BB have gained growing interest . CD40 shex vivo tumor model derived from ovarian cancer (OVCA) patient samples -mediated antigen-specific signal transduction, optimal activation of T cells requires antigen engagement with positive secondary signals provided by costimulatory molecules such as OX40 (CD134), a tumor necrosis factor receptor super family member . OX40 cain vivo and simultaneously reflecting the host’s immune response. Immunodeficient mice bearing human tumor-derived xenografts are used in virtually all in vivo experiments of OAds since human Ads cannot be allowed to replicate in mouse cells , an oncofetal glycoprotein antigen involved in cell adhesion, is present in the intestine, liver, and pancreas of the fetus. Measurement of CEA has been recommended as a prognostic indicator in colorectal cancer as well as a surveillance tool for monitoring the tumor recurrence .+ and CD8+ T cells in a postimmunization sample with high CMI activity. Unfortunately, comparisons for significance in survival time in this study cannot be made since there was no active control group , which belongs to tissue kallikrein-related peptidase family . PSA is In a murine prostate cancer model, Ad5 with the insertion of a full-length PSA gene (Ad5-PSA) activated a strong anti-PSA immune response that suppressed growth rate of PSA-generating tumor . LubarofThe potential advantages of adenoviruses as basis for cancer vaccines have been proven in several preclinical and clinical trials . Althoug3) established prostate tumors in mice were efficiently eliminated by injection of Ad5-PSA in combination with recombinant canarypox viruses (ALVAC) encoding IL-12, IL-2, or TNF-α 7 days later. Melanoma-associated antigen 3 (MAGE-A3) is specifically expressed in the placenta and germline cells of the testis but frequently overexpressed in sarcoma and other tumor tissues  1.1. There were no DLTs, and the most common AEs were chills and fever associated with Ad replication. Disappointingly, one patient was diagnosed with infectious colitis related to ONCOS-102.As noted above, ONCOS-102 is an OAd-encoding GM-CSF transgene, and it was found to correlate with upregulated PD-L1 expression in mesothelioma patients by Furthermore, an ongoing phase I/II study combines ONCOS-102 with durvalumab, an anti-PD-L1 antibody, for the treatment of advanced peritoneal malignancies (NCT02963831). The combination therapy seems to be a promising strategy to activate immune responses in anti-PD-1/PD-L1 refractory cancers.Delta-24-RGD is an engineered adenovirus with a 24-base pair deletion and an insertion of the RGD-4C peptide motif into the adenoviral fiber knob. These modifications allowed Delta-24-RGD to replicate more efficiently in low-CXADR-expressing glioma cell lines . Delta-2Delta-24-RGDOX is a variant based on the virus backbone of Delta-24-RGD, which expresses immune costimulator OX40L to induce superior tumor-specific immunity. Since Delta-24-RGDOX injection induced upregulation of PD-L1 expression on the glioma cells, Anti-CTLA4 antibodies can prevent the dysfunction of T cells and potentiate tumor-specific immune responses by blocking the activity of CTLA-4 . PromisiSubsequently, an OAd expressing a complete human anti-CTLA4 monoclonal antibody (Ad5/3-D24aCTLA4) was designed . T-cell Anti-PD-L1 antibodies have been extensively investigated to improve durable response rate and overall survival of advanced cancer . Tanoue In spite of superior antitumor effectiveness and tolerable safety profiles, current oncolytic adenovirus immunotherapy still faces challenges that limit the clinical application of it. To overcome the limitations that include antiviral immune response and obstruction of TME, some potential solutions are provided.Most individuals have preexisting neutralizing antibodies (NAbs) against different adenovirus types, because these viruses are the cause of common respiratory infections . AntivirAdditionally, the TME represents as an obstruction against virotherapy. In solid tumors, TME is composed of a complex mixture of malignant cells and non-malignant tissues that contain myeloid-derived suppressor cells, fibroblasts, endothelial cells, and extracellular matrix (ECM) . The disWith increasing numbers of genomic modification strategies, impressive preclinical and clinical outcomes for modified oncolytic adenoviruses are continuously emerging, and multiple clinical trials are ongoing. OAds induce direct oncolysis and upregulate the immunostimulatory signals intratumorally, which can dramatically reduce local immunosuppression and enhance tumor-specific immunity. Good safety and tolerability of OAds have been confirmed in most extant clinical trials. Nevertheless, virtually no monotherapy will likely defeat all immune evasion mechanisms. OAds in combination with immune checkpoint inhibitors provides a promising approach to achieve long-term tumor control in patients who are unresponsive to systemic immune checkpoint blockade. Further research of OAd requires more attention for confronting the challenges, thus breaking its limitations and maximizing efficacy of treatment with minimal systemic toxicity.YZ, ZL, LL, JW, and HuZ: original draft preparation. ZL, LL, TL, and HaZ: manuscript reviewing and editing. BX: project administration and final manuscript supervision. 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.


Although biological males and females are equally likely to become infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), evidence has mounted that males experience higher severity and fatality compared to females.The objective of this review is to examine the existing literature on biological mechanisms underlying sex-based differences that could contribute to SARS-CoV-2 infection clinical outcomes. Sex-based differences in immunologic response and hormonal expression help explain the differences in coronavirus disease 2019 (COVID-19) outcomes observed in biological males and females. X inactivation facilitates a robust immune response to COVID-19 in females, who demonstrate a more profound antibody response and faster recovery when compared to males. Low testosterone levels also help explain the dysregulated inflammatory response and poor outcomes observed in some males with COVID-19. Gender differences in health expression and behaviors further compound these observed differences.Understanding the biology of sex-based differences in COVID-19 severity and mortality could help inform preventative measures, treatment decisions, and development of personalized, sex-specific therapies. Although most COVID-19 patients develop only mild disease, some mount a pronounced inflammatory response that results in severe pneumonia, respiratory failure, and death.Male sex is a known risk factor for severe outcomes in COVID-19.Biological sex-based differences in COVID-19 severity and mortality are influenced by genetic and hormonal differences at the cellular and immunologic level.Biological sex-based differences are further complicated by environmental factors, namely social and cultural norms, that define gender roles which impact risk of infection and severe outcomes.Understanding sex-based differences in COVID-19 outcomes will help inform clinical decision algorithms and treatment strategies to optimize the care of COVID-19 patients. Coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in December 2019 and quickly overwhelmed healthcare systems across the globe. The World Health Organization (WHO) declared COVID-19 as a public health emergency on January 30, 2020 and a pandemic on March 11, 2020. Although most COVID-19 patients develop only mild symptoms, some develop severe pneumonia, acute respiratory distress syndrome (ARDS), and multi-organ failure; as of February 2022, there were nearly 400 million reported cases and 6 million deaths worldwide [p < 0.001) across all age groups, race/ethnicity, payers, and preexisting comorbidities [Emerging epidemiologic studies have identified compelling and consistent sex differences in COVID-19 outcomes. The COVID-19 Sex-Disaggregated Data Tracker, developed by the Global Health 50/50 research initiative, showed similar numbers of COVID-19 cases in people identifying as men and women. However, men were more likely to develop severe illness and have higher case fatality rates, especially when older than 60 years . In a mebidities –10, 11. bidities , 13.Sex-based differences in COVID-19 outcomes are mediated by immune response. Females mount a stronger immune responses to a variety of infections, likely due to the influence of genetic and hormonal differences on the immune system . AlthougMultiple elements of the immune response to viral infection vary by sex. TLR7, an endosomal receptor expressed on dendritic and B cells, recognizes viral infections and triggers a type I interferon (IFN) response . TLR7 exSex hormones play an important role in immune function and response to viral infections. Differences in sex hormone levels help explain the immune response and COVID-19 severity in males compared to females. In a single-center cohort study of patients with COVID-19, lower testosterone levels and increased estradiol to testosterone ratio during hospitalization were associated with higher inflammatory cytokine concentrations, increased disease severity, ventilator use, ICU admission, and mortality . This diTestosterone has suppressive effects on immune function, and estrogen may have either positive or negative effects, depending on the level . For malLow testosterone levels correlate with COVID-19 severity in males , 36. Tes48 These clinical outcomes potentially reflect social and cultural norms rooted in unmeasured gender differences.In addition to biological sex-based differences, gender, as defined by social and cultural norms, plays a role in risk for infection and progression to severe outcomes. Gender differences are commonly described for groups identifying as women and men, leaving unmeasured variation in transgender or individuals of other gender identities, who may not be identifiable in existing studies, thereby confounding all gender-specific measures. In the United States, approximately 0.4% of the population identify as transgender . In termAdditional studies are needed to understand the impact that genetics, hormones, and innate and adaptive immune functional differences play in COVID-19 outcomes. Identifying hormone levels and biomarkers associated with sex-based differences in clinical outcomes may allow for earlier prediction of anticipated clinical course and inform therapeutics. This approach will likely have applications beyond COVID-19, and facilitate understanding of response to other viral infections in preparation for the next pandemic.Sex-based differences in COVID-19 outcomes are multifactorial. Understanding the mechanisms underlying these differences could help predict patient outcomes and inform clinical decision-making to facilitate early treatment and disposition decisions. Recognition of sex-based differences can also help inform future research efforts to develop targeted, sex-specific therapies in COVID-19.


The multifaceted self-renewal process is regulated by factors, including transcription factors, miRNAs, and the bone marrow niche. A developed classical hierarchical model that outlines the hematopoiesis in a lineage-specific manner through in vivo fate mapping, barcoding, and determination of self-renewal regulatory factors are still to be explored in more detail. Thus, an in-depth study of the self-renewal property of HSCs is essentially required to be utilized for ex vivo expansion. This review primarily focuses on the Hematopoietic stem cell self-renewal pathway and evaluates the regulatory molecular factors involved in considering a targeted clinical approach in numerous malignancies and outlining gaps in the current knowledge.Hematopoietic stem cells (HSCs) possess two important properties such as self-renewal and differentiation. These properties of HSCs are maintained through hematopoiesis. This process gives rise to two subpopulations, long-term and short-term HSCs, which have become a popular convention for treating various hematological disorders. The clinical application of HSCs is bone marrow transplant in patients with aplastic anemia, congenital neutropenia, sickle cell anemia, thalassemia, or replacement of damaged bone marrow in case of chemotherapy. The self-renewal attribute of HSCs ensures long-term hematopoiesis post-transplantation. However, HSCs need to be infused in large numbers to reach their target site and meet the demands since they lose their self-renewal capacity after a few passages. Therefore, a more in-depth understanding of  The bone marrow resident hematopoietic stem cells (HSCs) direct the process of hematopoiesis . The earFetal BM HSCs were isolated for the first time in 1992, disclosing their property of asymmetric cell division upon functional characterization . HSCs di− Cre model is a common in vivo fate mapping system specific to HSCs wherein the Tie2 gene was manipulated using the Cre/loxP system. The Tie2+ labeled LT-HSCs amplify the ST-HSCs and multipotent progenitors (MPPs) populations validating the spot of LT-HSCs at the top of the lineage map , B-cell acute lymphoblastic leukemia (B-ALL), T-cell ALL (T-ALL), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), chronic lymphocytic leukemia (CLL), and follicular lymphoma (FL) .Leukemic growth and HSCs maintenance is also regulated by the metabolic pathways. Among the 22 amino acids, leukemic niche is heavily dependent on amino acid metabolism for sustenance of leukemic stem cells (LSCs), unlike HSCs which are only affected negatively by valine deficiency . The nicex vivo expansion strategies to enhance the self-renewal and proliferation of HSCs need to be devised. For instance, it has been shown that stem cell factor (SCF) and Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) inhibitor, 8-Hydroxy-7-diazenyl-quinoline-5-sulfonic acid, Disodium Salt (NSC87877) synergism activates c-Kit and inhibits c-Kit negative regulators (SHP-1/SHP-2) that can be used clinically to enhance cellular proliferation of both erythroid and megakaryoblast cells  are 30 to 10,000 nm sized cargo encapsulating lipid bilayers, and can be further categorized into exosomes and microvesicles based on their size. HSCs secreted exosomes typically fall within the range 30–120 nm . The EVsA better understanding of the transcriptional process regulating the self-renewal of HSCs is essential to enhance the shelf-life and potency of HSCs so that the frequency of HSCs isolation from donors and the invasiveness of the process can be minimized. This review summarizes the intrinsic factors that affect HSCs homeostasis and lineage commitment .In vivo evidence and colony-forming assays revealed two primary characteristics of HSCs-self-renewability and multipotency  family markers, particularly CD150 and CD229, segregate mouse HSCs based on their potency. CD229- LT-HSCs commit to myeloid lineage, while CD229+ HSCs commit to lymphoid lineage  and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) revealed heterogeneity within the HSCs population . These cwability . The subThe transition within these three cell types is modulated by TFs such as Runx1, Tal1/SCL, Gfi1, Sall4, Etv6, Sox17, GATA-2, and FoxO3a. NFIL3 is expressed in CLPs and is required in innate lymphoid cell development within the BM . SimilarHSCs constitute <0.01% cells of the total BM population and maintain the hematopoietic system’s stability, owing to their self-renewal and differentiation potency . The selExtrinsic and intrinsic factors control the dynamic cellular organization within the hematopoietic system. Extrinsic factors include growth factors, hypoxia, and morphogens that activate signaling pathways, whereas intrinsic factors encompassing TFs, cell cycle regulators, epigenetic proteins, and miRNAs, as summarized in + and CXCL12hi cells  . The sinhi cells . The hemal cells . This thal cells .via osteopontin surface expression.The BM is the primary site of hematopoiesis and this niche includes the endosteal and vascular niches . The endThe second type of niche is within the blood vessels called the vascular niche . The vas0 phase. The Mef TF facilitates the quiescent HSCs transition from G0 to G1 phase  is an oncogenic TF of the SET/PR domain protein family . It has ficiency .The TFs belonging to Forkhead O (FoxO) family play a crucial role in diverse physiological processes, including apoptosis, regulating cell-cycle dynamics, and stress resistance . Four meThe GATA family proteins, inclusive of the three isoforms GATA-1, GATA-2, and GATA-3, are essentially identified by two highly conserved zinc fingers of the C2H2 type that recognize the motif “WGATAR” . GATA-1 Growth factor independence 1 (Gfi1), a transcriptional repressor composed of Snail/Gfi1 (SNAG) domain and six zinc-finger motifs, is a part of the oncogenic complementation system as Bmi-1 . The mosThe homeobox (HOX) genes are a family of highly conserved homeodomain-containing TFs that specify cell type identity in the early stages of development. These can be categorized into four separate clusters- HOXA, HOXB, HOXC, and HOXD, located on different chromosomes. The expression of HOX proteins follows a specific pattern, with the HOXA gene expressed in myeloid cells, HOXB in erythroid cells, and HOXC in lymphoid cells . HOX 1-6Mef or ELF4 is a member of the E26 transformation-specific (ETS) family of winged helix-turn-helix TFs and primarily acts on LT-HSCs . Its expvia TGF-β and MAPK signaling  is an essential TF functioning at multiple hematopoiesis stages, including definitive HSCs formation and differentiation into granulocytes, B-cell, and megakaryocytes . StructuMutations in the RUNX1 gene correlate with the occurrence of AML and myelodysplasia, suggesting that this TF has a tumor-suppressive function. The conserved RUNX family has three isoforms, RUNX1, RUNX2, and RUNX3. Molecules regulating RUNX1 activity include ERK, HIPK2, cyclin-dependent kinases, or methylators like PRMT1 . RUNX1 w0 phase compared to more actively proliferating HSCs in the G1/S/G2 phase , Sonic hedgehog (Shh), and Smad, control the LT-HSCs population. Moreover, the IL-6 and IL-11 receptor, gp130 protein, and c-Kit play an essential role in HSCs self-renewal . Intracevia a better understanding of the epigenetic regulators. A crucial epigenetic regulator is MLLT3 or AF9, which in conjugation with DOT1L, acts as histone reader to methylate H3K79 and maintains the self-renewability of HSCs. MLLT3 levels decrease in HSCs cultured in vitro; thus, stabilizing MLLT3 levels in culture and causing a 12-fold increase in expansion of transplantable HSCs , and stress resistance in cancer stem cells (CSCs) , MSCs, intestinal stem cells (in s (CSCs) .+  for their ex vivo expansion  under normoxic conditions activates the Notch pathway in ST-HSCs by triggering the transcriptional activity of HES and HEY genes responsible for self-renewal  activates protease-activated receptor-1 signaling and displays a transcriptome similar to that of freshly isolated HSCs  play a significant role within the hematopoietic niche in maintaining homeostasis through autocrine and paracrine signaling . Identifvia miR-155 and miR-146 shuttling that inhibits T, B, and NK cells activity since these miRNAs regulate innate immune response by regulating vesicle trafficking (+ and CD34+ EVs have been shown to reverse graft-versus-host disease (GvHD) and hindlimb ischemia by suppressing Th1-mediated inflammation . Early-onset side-effects include mucositis, hemorrhagic cystitis, GvHD and graft failure, pulmonary complications, thrombotic microangiopathy, and prolonged pancytopenia . On the ex vivo expansion using extracellular vesicles can create HSCs without immunogenicity, making HSCs more suitable for therapeutic purposes as their infusion may be performed across histocompatibility locus antigen barriers without risk of rejection.Hematopoiesis is a complex developmental process regulated by a closely integrated network of TFs, miRNAs, epigenetic modifications, cytokines, and growth factors. These components maintain a fine balance between the self-renewal and differentiation properties of HSCs to generate a functionally stable hematopoietic pool. The BM niche components modulate the heterochromatic expression of multiple genes to maintain HSCs progression into a specific lineage, preventing leukemogenesis occurrence. Dysregulation in these networks leads to a diverse range of hematological malignancies such as ALL, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid (CML), myeloma, aplastic and sickle-cell anemia, GvHD and lymphoma. In addition to intrinsic factors, various extrinsic factors such as ionization, obesity, and lack of exercise also impact the hematopoietic niche, causes oxidative stress and ablation of long-term renewability of HSCs. Therefore, a more lucid image of the hematopoietic development system is required to strategize natural therapies. Advanced technologies such as single-cell RNA sequencing, multiphoton intravital microscopy, the building of more pragmatic animal models like zebrafish, and high-throughput genetic screening, have deepened our insight into this system. However, a better comprehension of inflammation-upregulated HSCs and EVs trafficking might offer opportunities to discerning novel therapies for HSCs homing in an organ-specific manner to be used in clinical settings. The underlying mechanisms of action of TFs and miRNA of bone marrow niche in interconnecting therapeutic strategies are still poorly understood. It is conceivable that over the years, their role in influencing self-renewal and differentiation will become more apparent as the biological interactions of these microenvironmental factors are unraveled through a continuous moving from bench to bedside and vice versa. Engineered HSCs and controlled