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

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

Comparison of minimal residual disease measurement by multicolour flow cytometry and PCR for fusion gene transcripts in infants with acute lymphoblastic leukaemia with KMT2A gene rearrangements.

This study aimed to evaluate the concordance between minimal residual disease (MRD) results obtained by multicolour flow cytometry (MFC) and polymerase chain reaction for fusion gene transcripts (FGTs) in infants with acute lymphoblastic leukaemia (ALL) associated with rearrangement of the KMT2A gene (KMT2A-r). A total of 942 bone marrow (BM) samples from 123 infants were studied for MFC-MRD and FGT-MRD. In total, 383 samples (40.7%) were concordantly MRD-negative. MRD was detected by the two methods in 441 cases (46.8%); 99 samples (10.5%) were only FGT-MRD-positive and 19 (2.0%) were only MFC-MRD-positive. A final concordance rate of 87.4% was established. Most discordance occurred if residual leukaemia was present at levels close to the sensitivity limits. Neither the type of KMT2A fusion nor a new type of treatment hampering MFC methodology had an influence on the concordance rate. The prognostic value of MFC-MRD and FGT-MRD differed. MFC-MRD was able to identify a rapid response at early time-points, whereas FGT-MRD was a reliable relapse predictor at later treatment stages. Additionally, the most precise risk definition was obtained when combining the two methods. Because of the high comparability in results, these two rather simple and inexpensive approaches could be good options of high clinical value.

Genetic characteristics and treatment outcome in infants with KMT2A germline B-cell precursor acute lymphoblastic leukemia: Results of MLL-Baby protocol.

The aim of this study was to present the diagnostic and outcome characteristics of infants with germline status of KMT2A gene (KMT2A-g) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated consistently according to the MLL-Baby protocol, a moderate-intensity protocol. Of the 139 patients enrolled in the MLL-Baby study, 100 (71.9%) carried different types of rearranged KMT2A (KMT2A-r), while the remaining 39 infants (28.1%) had KMT2A-g. KMT2A-g patients were generally older (77% older than 6 months), less likely to have a very high white blood cell count (greater than 100 × 109 /L), less likely to be central nervous system (CNS)-positive, and more likely to be CD10-positive. The 6-year event-free survival and overall survival rates for all 39 patients were 0.74 (standard error [SE] 0.07) and 0.80 (SE 0.07), respectively. Relapse was the most common adverse event (n = 5), with a cumulative incidence of relapse (CIR) of 0.13 (SE 0.06), while the incidence of a second malignancy (n = 1) and death in remission (n = 3) was 0.03 (SE 0.04) and 0.08 (SE 0.04), respectively. None of the initial parameters, including genetics and the presence of recently described fusions of NUTM1 and PAX5 genes, was able to distinguish patients with different outcomes. Only rapidity of response, measured as minimal residual disease (MRD) by flow cytometry, showed a statistically significant impact. Moderate-intensity therapy, as used in the MLL-Baby protocol in infants with KMT2A-g BCP-ALL, yields results comparable to other infant studies. Patients with a slow multicolor flow cytometry (MFC)-MRD response should be subjected to advanced therapies, such as targeted or immunotherapies.

A simple procedure to identify children with B-lineage acute lymphoblastic leukemia who can be successfully treated with low or moderate intensity: Sequential versus single-point minimal residual disease measurement.

Sequential monitoring of minimal residual disease (MRD) by molecular techniques or multicolor flow cytometry (MFC) has emerged over the past two decades as the primary tool to optimize treatment in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The aim of our study was to compare the prognostic power of repeated MFC-MRD measurement with single-point MRD assessment in children with BCP-ALL treated with the reduced-intensity protocol ALL-MB 2008. Data from consecutive MFC-MRD at day 15 and day 36 (end of induction, EOI) were available for 507 children with Philadelphia-negative BCP-ALL. They were stratified into standard risk (SR, n = 265), intermediate risk (ImR, n = 211), and high risk (HR, n = 31) according to the initial clinical characteristics defined in the ALL-MB 2008 protocol. Quantitative (relative to quantitative thresholds) and kinetic (logarithmic reduction) assessments of MFC-MRD at both time points effectively separated patients into three groups with different risk of recurrence. On the other hand, starting with low (for the SR group) and moderate (for the ImR group) induction therapy, a single MFC-MRD measurement at EOI proved sufficient to unequivocally identify patients in whom this therapy is highly effective and distinguish them from those who cannot be successfully treated with such therapy. Therefore, initiating treatment with low or moderate treatment from the start, together with careful consideration of initial clinical risk factors and just one EOI-MFC-MRD measurement is simple, inexpensive, and entirely sufficient for treatment optimization. Furthermore, for a large proportion of patients, this approach allows better adjustment, in particular also reduction of therapy intensity than sequential MRD measurements.

Characterization of the complete mitochondrial genome of Plagiorchis multiglandularis (Digenea, Plagiorchiidae): Comparison with the members of Xiphidiatan species and phylogenetic implications.

Plagiorchis multiglandularis Semenov, 1927 is a common fluke of birds and mammals, with significant impacts on animals and also human health. However, the systematics of Plagiorchiidae remain ambiguous. In the present study, the complete mitochondrial (mt) genome of P. multiglandularis cercariae was sequenced and compared with other digeneans in the order Xiphidiata. The complete circular mt genome of P. multiglandularis was 14,228 bp in length. The mitogenome contains 12 protein-coding genes and 22 transfer RNA genes. The 3' end of nad4L overlaps the 5' end of nad4 by 40 bp, while the atp8 gene is absent. Twenty-one transfer RNA genes transcribe products with conventional cloverleaf structures, while one transfer RNA gene has unpaired D-arms. Comparative analysis with related digenean trematodes revealed that A + T content of mt genome of P. multiglandularis was significantly higher among all the xiphidiatan trematodes. Phylogenetic analyses demonstrated that Plagiorchiidae formed a monophyletic branch, in which Plagiorchiidae are more closely related to Paragonimidae than Prosthogonimidae. Our data enhanced the Plagiorchis mt genome database and provides molecular resources for further studies of Plagiorchiidae taxonomy, population genetics and systematics.

Incidence and prognostic value of central nervous system involvement in infants with B-cell precursor acute lymphoblastic leukemia treated according to the MLL-Baby protocol.

AIM: The aim of the study was to evaluate the incidence and prognostic impact of central nervous system (CNS) involvement in infants with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), as well as its relation with minimal residual disease (MRD) data. METHODS: A total of 139 consecutive infants with BCP-ALL from the MLL-Baby trial were studied. Cerebrospinal fluid (CSF) samples were investigated by microscopy of cytospin slides. MRD was evaluated according to the protocol schedule by flow cytometry and PCR for fusion gene transcripts (FGT). RESULTS: Involvement of the CNS at any level was found in 50 infants (36.0%). The incidence of CNS involvement was higher in patients with KMT2A gene rearrangements (44.0% for KMT2A-r vs. 15.4% for KMT2A-g, p = .003). The outcome of CNS-positive infants was significantly worse than that of CNS-negative infants, although this prognostic impact was limited to the KMT2A-r group (event-free survival 0.21 for CNS-positive vs. 0.48 for CNS-negative infants, p = .044). CNS-positive infants could not be treated successfully by conventional chemotherapy alone, irrespective of the rapidity of MRD response. In contrast, the combination of initial CNS negativity and FGT-MRD negativity identified a group comprising up to one-third of infants with KMT2A-r ALL who can be treated with chemotherapy and achieve very good outcomes (disease-free survival above 95%), and remaining patients should be allocated to receive other types of treatment. CONCLUSION: We can conclude that this combination of initial CNS involvement and MRD data can significantly improve risk-group allocation in future clinical trials enrolling infants with ALL.

Lineage Conversion in Pediatric B-Cell Precursor Acute Leukemia under Blinatumomab Therapy.

We report incidence and deep molecular characteristics of lineage switch in 182 pediatric patients affected by B-cell precursor acute lymphoblastic leukemia (BCP-ALL), who were treated with blinatumomab. We documented six cases of lineage switch that occurred after or during blinatumomab exposure. Therefore, lineage conversion was found in 17.4% of all resistance cases (4/27) and 3.2% of relapses (2/63). Half of patients switched completely from BCP-ALL to CD19-negative acute myeloid leukemia, others retained CD19-positive B-blasts and acquired an additional CD19-negative blast population: myeloid or unclassifiable. Five patients had KMT2A gene rearrangements; one had TCF3::ZNF384 translocation. The presented cases showed consistency of gene rearrangements and fusion transcripts across initially diagnosed leukemia and lineage switch. In two of six patients, the clonal architecture assessed by IG/TR gene rearrangements was stable, while in others, loss of clones or gain of new clones was noted. KMT2A-r patients demonstrated very few additional mutations, while in the TCF3::ZNF384 case, lineage switch was accompanied by a large set of additional mutations. The immunophenotype of an existing leukemia sometimes changes via different mechanisms and with different additional molecular changes. Careful investigation of all BM compartments together with all molecular -minimal residual disease studies can lead to reliable identification of lineage switch.

Prognostic value of minimal residual disease measured by fusion-gene transcript in infants with KMT2A-rearranged acute lymphoblastic leukaemia treated according to the MLL-Baby protocol.

The prognostic value of minimal residual disease (MRD) measured by fusion-gene transcript (FGT) detection was investigated in 76 infants (aged ≤1 year) with acute lymphoblastic leukaemia (ALL) with lysine methyltransferase 2A (KMT2A) rearrangements. Either at the end of induction or at later time-points, FGT-MRD-positivity was associated with poor outcome. FGT-MRD-positivity after first consolidation or first high-risk block detected 46·5% of infants with extremely poor outcome [disease-free survival (SE) 0·06 (0·06), cumulative incidence of relapse (SE) 0·91 (0·05)], which was also confirmed in multivariable analysis. Thus, FGT-MRD measurement at a single time-point clearly identifies infants with ALL who are curable with conventional chemotherapy and those who would benefit only from other treatment approaches.

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

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

Rituximab and reduced-intensity chemotherapy in children and adolescents with mature B-cell lymphoma: interim results for 231 patients enrolled in the second Russian-Belorussian multicentre study B-NHL-2010M.

The value of adding rituximab to chemotherapy in children with aggressive B-cell non-Hodgkin lymphoma (B-NHL) is still insufficiently studied. We enrolled 231 patients [mean age 9 years old (range 2-17); male:female ratio 3·4:1] with Burkitt (BL, 179 patients, 76·7%), diffuse large B-cell (32 patients, 14%), primary mediastinal B-cell (14 patients, 6%), and other (6 patients, 2·6%) B-cell lymphomas in a prospective study of immuno-chemotherapy. Stages were I-II in 32% and III-IV in 68% of the patients. Four doses of 375 mg/m2 rituximab were added to the Berlin-Frankfurt-Munster-NHL-90-like chemotherapy, with methotrexate being reduced or omitted in the first 2 induction blocks. The complete remission rate was 100% in limited-stage and 91·4% in advanced-stage patients. Five advanced-stage patients (2·2%) died in induction and 1 patient with stage 2 B-NHL died in remission; 11 patients in the high-risk group progressed on therapy (3 non-BL are alive after salvage) and 5 relapsed. Sixteen patients (9·7%) with advanced stage disease proceeded to transplant. With a median follow-up of 46 months, 98·5 ± 1% of patients with limited disease and 88·1 ± 2% (88·1% in Risk Group 3; 82·6% in Risk Group 4) in advanced stages are alive. This study confirmed that combined immunochemotherapy for B-lymphomas is highly effective in children, despite reducing the intensity of the induction blocks.

The effect of trematode infection on the markers of oxidative stress in the offspring of the freshwater snail Lymnaea stagnalis.

Most invertebrate species exhibit immunological responses that can inactivate and eliminate penetrating parasites. Such immune responses in particular involve the formation of potentially toxic reactive oxygen species (ROS). We explored the immune capabilities of the first-generation (F1) offspring of naturally infected freshwater snails, Lymnaea stagnalis, in response to infection by trematode cercariae under laboratory conditions. The rates of ROS formation and peroxidase activity in the hemolymph of the F1 offspring of L. stagnalis parents infected by an asexual stage of trematodes were significantly higher than in F1 offspring of uninfected parents. Compared to offspring from uninfected parents, the growth rate of F1 snails from infected parents was higher, but survival was lower. After infection of F1 snails by trematode cercariae of Echinoparyphium aconiatum under laboratory conditions, the rate of ROS formation and peroxidase activity in the hemolymph of F1 offspring of uninfected parents increased compared to control snails. This pattern persisted throughout the entire 3-week observation period. In contrast, the rate of ROS formation in the hemolymph of F1 snails from infected parents after experimental infection by E. aconiatum cercariae did not differ from controls, and peroxidase activity even decreased. Thus, trematode parthenitae infection of parents could alter the immune response of their offspring.

Prevalence of opisthorchiid and other endoparasitic infections among cats and dogs in Novosibirsk oblast (Western Siberia, Russia).

To determine the current reservoir status and prevalence of Opisthorchis felineus infection in opisthorchiosis-endemic areas, feces samples were collected from 103 cats and 101 dogs admitted to shelters or discount sterilization centers from various districts of Novosibirsk city and from villages and towns along the Ob River (Novosibirsk Oblast). The feces samples were evaluated using two methods simultaneously: the formalin-ether sedimentation assay and a modified flotation assay. The prevalence of opisthorchiid infection was higher among cats (12.6%; 95% confidence interval 6.9-20.6%) than among dogs (4.0%; 95% confidence interval 1.1-9.8%). Overall prevalence of endoparasites among cats was 42.7% (five helminthic and four protozoan species) and among dogs 51.5% (eight helminthic and five protozoan species). Among the parasites found in the dogs, the prevalence of Sarcocystis sp. was the highest (16.8%), and in cats, the highest prevalence was shown by Giardia sp. and Toxocara cati (14.6% for each species). These results confirm the considerable contribution of stray cats and dogs to the maintenance of opisthorchiid eggs in the environment; these data should be taken into account for the development of effective control and education programs.

Temperature dependence of Opisthorchis felineus infection in the first intermediate host snail, Bithynia troschelii.

Opisthorchiasis is one of the most serious trematodiases in Russia, where the world's largest focus is located in the Ob basin. Temperature is an important factor affecting the metabolism of cold-blooded animals. It determines the development of the causative agent of opisthorchiasis, Opisthorchis felineus, and the success of infection of an intermediate host, the snail Bithynia troschelii. In the present study, the effect of water temperature on the development of the liver fluke O. felineus in the host snail was assessed, as was the temperature threshold at which B. troschelii hibernation initiates. Adult uninfected B. troschelii individuals collected from natural bodies of water were infected with O. felineus and maintained at different temperatures of water (18-30 °C, intervals of 3 °C) in the laboratory. Each snail was fed with embryonated uterine eggs of O. felineus at 24 °C. O. felineus infection in snails was detected using polymerase chain reaction (PCR) using specific primers. The prevalence of O. felineus infection in B. troschelii depends on the water temperature in which the snails are maintained. The highest infection rate of 45.2 % ± 12.1 % was observed at 27 °C (p ≥ 0.1). The longest lifespan of infected and uninfected B. troschelii was recorded at water temperatures of 24 and 27 °C. The snails were more successfully infected at the beginning of the warm season. Among the infected individuals, the majority (up to 85 %) were large snails. Cercarial shedding was not detected in experimentally infected snails. Apparently, this is due to the natural physiological state of Bithynia snails during the autumn-winter diapause, when opisthorchiids development in snails stops. At 10 °C, complete hibernation of all B. troschelii snails was observed, and infection by the trematodes became impossible. The highest prevalence of infection was recorded at 27 °C, suggesting that during climate warming, an increase in opisthorchiid infection of snails may occur, which must be considered when epidemiological measures are planned.

m6A Methylation in Regulation of Antiviral Innate Immunity.

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

Blinatumomab as postremission therapy replaces consolidation and substantial parts of maintenance chemotherapy and results in stable MRD negativity in children with newly diagnosed B-lineage ALL.

The bispecific T cell-binding antibody blinatumomab (CD19/CD3) is widely and successfully used for the treatment of children with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we report the efficacy of a single course of blinatumomab instead of consolidation chemotherapy to eliminate minimal residual disease (MRD) and maintain stable MRD-negativity in children with primary BCP-ALL.Between February 2020 and November 2022, 177 children with non-high-risk BCP-ALL were enrolled in the ALL-MB 2019 pilot study (NCT04723342). Patients received the usual risk-adapted induction therapy according to the ALL-MB 2015 protocol. Those who achieved a complete remission at the end of induction (EOI) received treatment with blinatumomab immediately after induction at a dose of 5 µg/m2/day for 7 days and 21 days at a dose of 15 µg/m2/day, followed by 12 months of maintenance therapy. MRD was measured using multicolor flow cytometry (MFC) at the EOI, then immediately after blinatumomab treatment, and then four times during maintenance therapy at 3-month intervals.All 177 patients successfully completed induction therapy and achieved a complete hematological remission. In 174 of these, MFC-MRD was measured at the EOI. 143 patients (82.2%) were MFC-MRD negative and the remaining 31 patients had varying degrees of MFC-MRD positivity.MFC-MRD was assessed in all 176 patients who completed the blinatumomab course. With one exception, all patients achieved MFC-MRD negativity after blinatumomab, regardless of the MFC-MRD score at EOI. One adolescent girl with high MFC-MRD positivity at EOI remained MFC-MRD positive. Of 175 patients who had completed 6 months of maintenance therapy, MFC-MRD data were available for 156 children. Of these, 155 (99.4%) were MFC-MRD negative. Only one boy with t(12;21) (p13;q22)/ETV6::RUNX1 became MFC-MRD positive again. The remaining 174 children had completed the entire therapy. MFC-MRD was examined in 154 of them, and 153 were MFC-MRD negative. A girl with hypodiploid BCP-ALL showed a reappearance of MFC-MRD with subsequent relapse.In summary, a single 28-day course of blinatumomab immediately after induction, followed by 12 months of maintenance therapy, is highly effective in achieving MRD-negativity in children with newly diagnosed non-high risk BCP-ALL and maintaining MRD-negative remission at least during the treatment period.

Laboratory characterization of the pediatric B/T subtype of mixed-phenotype acute leukemia: Report of a case series.

OBJECTIVES: Mixed-phenotype acute leukemia (MPAL) is a rare disease associated with difficulties in the correct lineage assignment of leukemic cells. One of the least common subtypes within this category is characterized by the simultaneous presence of B- and T-lineage-defining antigens. Each case of suspected B/T MPAL should be considered in light of all available laboratory and clinical data to avoid misdiagnosis. METHODS: In this study, we describe 6 pediatric patients who presented with leukemic blasts bearing B- and T-lineage antigens at diagnosis, including their clinical, immunophenotypic, morphologic, and cytogenetic characteristics. RESULTS: In 3 patients, more or less distinct populations of B- and T-lymphoid origin were found; the other 3 patients had a single mixed-phenotype blast population. All cases fulfilled the World Health Organization criteria, but not all of them turned out to be bona fide cases of B/T MPAL according to the available clinical and laboratory data. Found genetic lesions were helpful for the confirmation of MPAL instead of 2 concomitant tumors, but for a general B/T MPAL diagnosis, genetic studies provided the only descriptive data. CONCLUSIONS: The accurate diagnosis of B/T MPAL requires a multidisciplinary approach combining high-tech laboratory methods and close cooperation between treating physicians and pathologists.

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

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

Flow cytometric minimal residual disease measurement accounting for cytogenetics in children with non-high-risk acute lymphoblastic leukemia treated according to the ALL-MB 2008 protocol.

BACKGROUND: Quantitative measurement of minimal residual disease (MRD) is the "gold standard" for estimating the response to therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP-ALL, in terms of genetic subgroups with relation to clinically defined risk groups. METHODS: A total of 485 children with non-high-risk BCP-ALL with available cytogenetic data and MRD studied at the end-of-induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard-risk (SR) of intermediate-risk (ImR) regimens of "ALL-MB 2008" reduced-intensity protocol. RESULTS AND DISCUSSION: Among all study group patients, 203 were found to have low-risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC-MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC-MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low-(0.03%) and intermediate (0.01%) cytogenetic risk groups. CONCLUSION: Our data show that combining clinical risk factors with MFC-MRD measurement is the most useful tool for risk group stratification of children with BCP-ALL in the reduced-intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.

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

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

Technological aspects of manufacturing and analytical control of biological nanoparticles.

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