Long-term Expression Research Articles

VWA3A-derived ependyma promoter drives increased therapeutic protein secretion into the CSF

Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and confer long-term transgene expression. Despite an ever-growing library of capsid variants, widespread delivery of AAVs in the adult central nervous system remains a challenge. We have previously demonstrated successful distribution of secreted proteins by infection of the ependyma, a layer of post-mitotic epithelial cells lining the ventricles of the brain and central column of the spinal cord, and subsequent protein delivery via the cerebrospinal fluid (CSF). Here we define a functional ependyma promoter to enhance expression from this cell type.Using RNA sequencing on human autopsy samples, we identified disease- and age-independent ependyma gene signatures. Associated promoters were cloned and screened aslibraries in mouse and rhesus macaque to reveal cross-species function of a human DNA-derived von Willebrand factordomain containing 3A (VWA3A) promoter. When tested in mice, our VWA3A promoter drove strong, ependyma-localized expression of eGFP and increased secreted ApoE protein levels in the CSF by 2-12× over the ubiquitous iCAG promoter.

Lentiviral delivered aflibercept OXB-203 for treatment of neovascular AMD

Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness in the aging population, with vascular endothelial growth factor (VEGF) playing a key role. Treatment with recombinant anti-VEGFs is the current standard of care; however, it is only effective for 1-2months at a time and requires re-administration. Gene therapy could pave the way for stable, long-term expression of therapeutic anti-VEGF with a single dose, reducing the frequency of treatment and potentially improving clinical outcomes. As such, we have developed OXB-203, a lentiviral-based gene therapy encoding the anti-VEGF protein aflibercept. Aflibercept derived from OXB-203 exhibited comparable invitro binding characteristics to VEGF as recombinant aflibercept. Furthermore, its biological potency was demonstrated by the equivalent inhibition of VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and tubule formation as recombinant aflibercept. In a rat choroidal neovascularization (CNV) model of nAMD, a single subretinal administration of OXB-203 reduced laser-induced CNV lesion areas analogous to an intravitreal bolus of recombinant aflibercept. Finally, in a head-to-head comparative study, aflibercept derived from OXB-203 was shown to be expressed at significantly higher levels in ocular tissues than from an AAV8-aflibercept vector following a single subretinal delivery to rats. These findings support the therapeutic potential of OXB-203 for the management of nAMD.

Nerve growth factor receptor (Ngfr) induces neurogenic plasticity by suppressing reactive astroglial Lcn2/Slc22a17 signaling in Alzheimer’s disease

Neurogenesis, crucial for brain resilience, is reduced in Alzheimer’s disease (AD) that induces astroglial reactivity at the expense of the pro-neurogenic potential, and restoring neurogenesis could counteract neurodegenerative pathology. However, the molecular mechanisms promoting pro-neurogenic astroglial fate despite AD pathology are unknown. In this study, we used APP/PS1dE9 mouse model and induced Nerve growth factor receptor (Ngfr) expression in the hippocampus. Ngfr, which promotes neurogenic fate of astroglia during the amyloid pathology-induced neuroregeneration in zebrafish brain, stimulated proliferative and neurogenic outcomes. Histological analyses of the changes in proliferation and neurogenesis, single-cell transcriptomics, spatial proteomics, and functional knockdown studies showed that the induced expression of Ngfr reduced the reactive astrocyte marker Lipocalin-2 (Lcn2), which we found was sufficient to reduce neurogenesis in astroglia. Anti-neurogenic effects of Lcn2 was mediated by Slc22a17, blockage of which recapitulated the pro-neurogenicity by Ngfr. Long-term Ngfr expression reduced amyloid plaques and Tau phosphorylation. Postmortem human AD hippocampi and 3D human astroglial cultures showed elevated LCN2 levels correlate with reactive gliosis and reduced neurogenesis. Comparing transcriptional changes in mouse, zebrafish, and human AD brains for cell intrinsic differential gene expression and weighted gene co-expression networks revealed common altered downstream effectors of NGFR signaling, such as PFKP, which can enhance proliferation and neurogenesis in vitro when blocked. Our study suggests that the reactive non-neurogenic astroglia in AD can be coaxed to a pro-neurogenic fate and AD pathology can be alleviated with Ngfr. We suggest that enhancing pro-neurogenic astroglial fate may have therapeutic ramifications in AD.

Safety and feasibility of the gene transfer of hemopexin for conditions with increased free heme.

Heme is a fundamental molecule for several biological processes, but when released in the extracellular space such as in hemolytic diseases, it can be toxic to cells and tissues. Hemopexin (HPX) is a circulating protein responsible for removing free heme from the circulation, whose levels can be severely depleted in conditions such as sickle cell diseases. Accordingly, increasing HPX levels represents an attractive strategy to mitigate the deleterious effects of heme in these conditions. Gene transfer of liver-produced proteins with adeno-associated virus (AAV) has been shown to be an effective and safety strategy in animal and human studies mainly in hemophilia. Here, we report the feasibility of increasing HPX levels using an AAV8 vector expressing human HPX (hHPX). C57Bl mice were injected with escalating doses of our vector, and expression was assessed by enzyme immunoassay (ELISA), Western blot, and quantitative polymerase chain reaction (qPCR). In addition, the biological activity of transgenic hHPX was confirmed using two different models of heme challenge consisting of serial heme injections or phenylhydrazine-induced hemolysis. Sustained expression of hHPX was confirmed for up to 26 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. hHPX levels were significantly reduced by heme infusions and phenylhydrazine-induced hemolysis. No clinical toxicity or laboratory signs of liver damage were observed in preliminary short-term heme challenge studies. Our results confirm that long-term expression of hHPX is feasible and safe in mice, even in the presence of heme overload. Additional studies are needed to explore the effect of transgenic HPX protein in animal models of chronic hemolysis.

MRNA: Vaccine or Gene Therapy? The Safety Regulatory Issues

COVID-19 vaccines were developed and approved rapidly in response to the urgency created by the pandemic. No specific regulations existed at the time they were marketed. The regulatory agencies therefore adapted them as a matter of urgency. Now that the pandemic emergency has passed, it is time to consider the safety issues associated with this rapid approval. The mode of action of COVID-19 mRNA vaccines should classify them as gene therapy products (GTPs), but they have been excluded by regulatory agencies. Some of the tests they have undergone as vaccines have produced non-compliant results in terms of purity, quality and batch homogeneity. The wide and persistent biodistribution of mRNAs and their protein products, incompletely studied due to their classification as vaccines, raises safety issues. Post-marketing studies have shown that mRNA passes into breast milk and could have adverse effects on breast-fed babies. Long-term expression, integration into the genome, transmission to the germline, passage into sperm, embryo/fetal and perinatal toxicity, genotoxicity and tumorigenicity should be studied in light of the adverse events reported in pharmacovigilance databases. The potential horizontal transmission (i.e., shedding) should also have been assessed. In-depth vaccinovigilance should be carried out. We would expect these controls to be required for future mRNA vaccines developed outside the context of a pandemic.

Gene expression dynamics in input-responsive engineered living materials programmed for bioproduction.

Engineered living materials (ELMs) fabricated by encapsulating microbes in hydrogels have great potential as bioreactors for sustained bioproduction. While long-term metabolic activity has been demonstrated in these systems, the capacity and dynamics of gene expression over time is not well understood. Thus, we investigate the long-term gene expression dynamics in microbial ELMs constructed using different microbes and hydrogel matrices. Through direct gene expression measurements of engineered E.coli in F127-bisurethane methacrylate (F127-BUM) hydrogels, we show that inducible, input-responsive genetic programs in ELMs can be activated multiple times and maintained for multiple weeks. Interestingly, the encapsulated bacteria sustain inducible gene expression almost 10 times longer than free-floating, planktonic cells. These ELMs exhibit dynamic responsiveness to repeated induction cycles, with up to 97% of the initial gene expression capacity retained following a subsequent induction event. We demonstrate multi-week bioproduction cycling by implementing inducible CRISPR transcriptional activation (CRISPRa) programs that regulate the expression of enzymes in a pteridine biosynthesis pathway. ELMs fabricated from engineered S.cerevisiae in bovine serum albumin (BSA) - polyethylene glycol diacrylate (PEGDA) hydrogels were programmed to express two different proteins, each under the control of a different chemical inducer. We observed scheduled bioproduction switching between betaxanthin pigment molecules and proteinase A in S.cerevisiae ELMs over the course of 27 days under continuous cultivation. Overall, these results suggest that the capacity for long-term genetic expression may be a general property of microbial ELMs. This work establishes approaches for implementing dynamic, input-responsive genetic programs to tailor ELM functions for a wide range of advanced applications.

Liver lobe-specific hydrodynamic gene delivery to baboons: A preclinical trial for hemophilia gene therapy

Hydrodynamics-based gene transfer has been successfully employed for invivo gene delivery to the liver of small animals by tail vein injection and of large animals using a computer-assisted and image-guided protocol. In an effort to develop a hydrodynamic gene delivery procedure clinically applicable for gene therapy, we have evaluated the safety and effectiveness of a lobe-specific hydrodynamic delivery procedure for hepatic gene delivery in baboons. Reporter plasmid was used to assess the gene delivery efficiency of the lobe-specific hydrodynamic gene delivery, and plasmid-carrying human factor IX gene was used to examine the pattern of long-term gene expression. The results demonstrated liver lobe-specific gene delivery, therapeutic levels of human factor IX gene expression lasting for >100days, and the efficacy of repeated hydrodynamic gene delivery into the same liver lobes. Other than a transient increase in blood concentration of liver enzymes right after the injection, no significant adverse events were observed in animals during the study period. The results obtained from this first non-human primate study support the clinical applicability of the procedure for lobe-specific hydrodynamic gene delivery to liver.

Identification of a TuMV isolate (TuMV-ZR) from Pseudostellaria heterophylla and its development into a viral expression vector

Pseudostellaria heterophylla (P. heterophylla) is a popular Chinese medicinal herb that is cultivated widely in China. Viral infection is commonly encountered during the production of P. heterophylla. To identify viruses causing P. heterophylla disease, sRNA and mRNA libraries were built for 2 sets of P. heterophylla plants, one set that was planted only once (FGP) and one that was planted three consecutive three times (TGP) in a field, using virus-free tuberous roots as reproductive materials. A comprehensive procedure, including assembling virus-derived sRNA (vsRNA), assessing and cloning the full-length viral genome, building an infectious cloning vector and constructing a virus-based expression vector, was performed to identify viruses infecting P. heterophylla. Ultimately, 48 contig-related viruses were mined from 6 sRNA and 6 mRNA P. heterophylla libraries. A 9762-bp fragment was predicted to be the complete genome of TuMV virus. This sequence was cloned from P. heterophylla, and its infectivity was evaluated using the virus-infection model plant Nicotiana benthamiana (N. benthamiana) and host plant P. heterophylla. The resulting 9839-bp viral genome was successfully obtained from P. heterophylla and identified as a new P. heterophylla TuMV-ZR isolate. Simultaneously, TuMV-ZR infectious clones were shown to effectively infect P. heterophylla. Furthermore, TuMV-ZR expression vectors were developed, and the ability of a TuMV-ZR-based vector to express foreign genes was determined by analysis with the reporter gene EGFP. TuMV-ZR-based vectors were found to continuously express foreign genes in different organs of P. heterophylla throughout the whole vegetative period. In addition, TuMV-ZR vectors carrying EGFP accumulated in the tuberous roots of P. heterophylla, confirming that tuberous roots are key targets for viral infection and transmission. This study revealed the core pathogenicity of P. heterophylla mosaic virus and developed a new TuMV-ZR-based expression tool that led to long-term protein expression in P. heterophylla, laying the foundation for the identification of the mechanisms of P. heterophylla infection with mosaic viruses and developing tools to express value proteins in the tuberous roots of the medicinal plant P. heterophylla.

Rescue of GM3 synthase deficiency by spatially controlled, rAAV-mediated ST3GAL5 delivery.

GM3 synthase deficiency (GM3SD) is an infantile-onset epileptic encephalopathy syndrome caused by biallelic loss-of-function mutations in ST3GAL5. Loss of ST3GAL5 activity in humans results in systemic ganglioside deficiency and severe neurological impairment. No disease-modifying treatment is currently available. Certain recombinant adeno-associated viruses (rAAVs) can cross the blood-brain barrier to induce widespread, long-term gene expression in the CNS and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector using a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived induced pluripotent stem cell neurons and brain tissue from St3gal5-KO mice but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either the intracerebroventricular or i.v. route at P1, allowed for safe and effective rescue of lethality and behavior impairment in symptomatic GM3SD mice up to a year. These results support further clinical development of ST3GAL5 gene therapy.

IL-2 rescues impaired T cell function upon loss of Itk via metabolic reprogramming

Abstract IL-2 inducible T cell kinase (Itk) is a critical component of T cell receptor (TCR) signaling required for activation of PLCg and downstream transcription factors, that regulate expression of effector molecules. Loss of Itk impairs T-cell activation by modulating the strength of TCR signal. Itk −/−mice have defects in T cell development with decreased proliferation and impaired differentiation to multiple CD4 T helper lineages. Most severe defects are seen in differentiation of Th9 cells, which produce the cytokine, IL-9. Under Th9 differentiation conditions, Itk −/−cells initially appear similar to WT cells but at 72hrs fail to sustain long-term expression of effector molecules. Th9 differentiation could be rescued by inclusion of IL-2. Itk −/−CD4 T cells show decreased mTORC1 signaling and do not maintain expression of IRF4, c-Myc, pRb and multiple nutrient transporters, including the Glucose transporter 1 (GLUT1), transferrin receptor (CD71), and amino acid co-transporter (CD98), all of which were rescued by IL-2. IL-2 is not required initially but can be added as late as 48hrs into culture. Using SCENITH—a flow cytometry-based technique that profiles metabolism—we found that WT differentiated Th9 cells were primarily glycolytic. In contrast, Itk −/−Th9 cells were less synthetic and more dependent on mitochondrial respiration. Inclusion of IL-2 in Itk −/−cultures rescued metabolic profiles, comparable to WT cells. Our results suggest that metabolic flexibility is central to dictating the functional/effector fate of CD4 T cells and that the Tec kinase Itk and IL-2-mediated pathways are required for maintenance of metabolic integrity, providing novel insight into mechanisms by which IL-2 can rescue T cell functional defects. This work was supported by the Intramural Research Program of NIAID, NIH.

Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells.

Abstract 124: Autocrine signaling extends PD-L1 expression in NSCLC following an active immune response

Abstract Checkpoint inhibitors disrupting the PD-1/PD-L1 axis are used in multiple cancer subtypes. However, not all patients respond, and predictive biomarkers are needed to better stratify patients. PD-L1, the most studied biomarker, has given mixed results. The complexity of the immune response and plastic nature of PD-L1 expression on tumor and immune cells contributes to the heterogeneity of success. Not all malignant epithelial cells express PD-L1: some express it only when the correct signal is present, while others have constitutive expression. Additionally, the PD-L1 protein has multiple functions outside immune regulation, making its dynamics difficult to predict. In this study, we investigate these complex PD-L1 dynamics through a unique combination of in vitro studies and mathematical modeling simulations. IFN-γ, a pleotropic cytokine released by activated immune cells, induces PD-L1 expression in some tumor cells. Chronic IFN-γ results in long-term PD-L1 expression. However, relaxation dynamics following removal of IFN-γ remains unexplored. We show that PD-L1 relaxation depends on autocrine signaling, demonstrated by multiple in vitro assays seeded with varying population densities. Following 48h IFN-γ treatment, sub-confluent tumor cell populations demonstrated increased duration of PD-L1 expression, linking cell density to PD-L1 expression. Conditioned-media from A549 cells previously treated with IFN-γ induced PD-L1 on IFN-γ naïve tumor cell populations, indicative of autocrine signaling not previously identified. To reinforce our in vitro findings, we develop a hybrid agent-based model (ABM) to study PD-L1 dynamics. Tumor cells have variable PD-L1 expression and IFN-γ is modeled via reaction-diffusion. IFN-γ triggers tumor cells to secrete a diffusible factor that promotes PD-L1 expression. Initial fits of the ABM capture in vitro data dynamics for different conditions. In this ongoing project, we plan to identify the underlying mechanism responsible for extended PD-L1 expression in NSCLC cells, including investigation into specific molecular pathways. Identifying the cause for this phenomenon could help clinicians improve targeted therapy and introduce a potential avenue to enhance treatment in patients responsive to checkpoint inhibitors. Citation Format: Taylor M. Bursell, Sandhya Prabhakaran, Kimberly Luddy, Julian Pineriro, Rafael Bravo, Jeffrey West, Mark Robertson-Tessi, Aner Beg, Jhanelle Gray, Scott Antonia, Robert Gatenby, Alexander Anderson. Autocrine signaling extends PD-L1 expression in NSCLC following an active immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 124.

Stable Expression of Coagulation Factors by RPS6 Promoter

To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia. Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene. The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups. After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.

Human Cytomegalovirus IE2 Disrupts Neural Progenitor Development and Induces Microcephaly in Transgenic Mouse

Human cytomegalovirus (HCMV) is a significant contributor to congenital birth defects. Limited by the lack of animal models, the pathogenesis of neurological damage in vivo caused by HCMV infection and the role of individual viral genes remain to be elucidated. Immediate early (IE2) protein may play a function in neurodevelopmental problems caused by HCMV infection. Here, this study intended to investigate IE2’s long-term effects on development of the brain in IE2-expressing transgenic mice (Rosa26-LSL-IE2+/−, Camk2α-Cre) aimed to observe the phenotype of postnatal mice. The expression of IE2 in transgenic mice was confirmed by PCR and Western blot technology. We collected mouse brain tissue at 2, 4, 6, 8, and 10 days postpartum to analyze the developmental process of neural stem cells by immunofluorescence. We discovered that transgenic mice (Rosa26-LSL-IE2+/−, Camk2α-Cre) can reliably produce IE2 in the brain at various postpartum phases. Furthermore, we also observed the symptoms of microcephaly in postnatal transgenic mice, and IE2 can damage the amount of neural stem cells, prevent them from proliferating and differentiating, and activate microglia and astrocytes, creating an unbalanced environment in the brain’s neurons. In conclusion, we demonstrate that long-term expression of HCMV-IE2 can cause microcephaly through molecular mechanisms affecting the differentiation and development of neural stem cells in vivo. This work establishes a theoretical and experimental foundation for elucidating the molecular mechanism of fetal microcephaly brought by HCMV infection in throughout the period of neural development of pregnancy.Graphical

In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector

Recent therapeutic strategies for hemophilia include long-term therapeutic gene expression using adeno-associated virus (AAV) and rebalancing therapy via the downregulation of anticoagulant pathways. However, these approaches have limitations in immune responses or insufficiency to control acute bleeding. Thus, we developed a therapeutic strategy for hemophilia B by a combined rebalancing and human factor 9 (hF9) gene knockin (KI) using a lipid nanoparticle (LNP) and AAV. Antithrombin (AT; Serpin Family C Member 1 [Serpinc1]) was selected as the target anticoagulation pathway for the gene KI. First, the combined use of LNP-clustered regularly interspaced short palindromic repeats (CRISPR) and AAV donor resulted in 20% insertions or deletions (indels) in Serpinc1 and 67% reduction of blood mouse AT concentration. Second, hF9 coding sequences were integrated into approximately 3% of the target locus. hF9 KI yielded approximately 1,000ng/mL human factor IX (hFIX) and restored coagulation activity to a normal level. LNP-CRISPR injection caused sustained AT downregulation and hFIX production up to 63weeks. AT inhibition and hFIX protein-production ability could be maintained by the proliferation of genetically edited hepatocytes in the case of partial hepatectomy. The co-administration of AAV and LNP showed no severe side effects except random integrations. Our results demonstrate hemophilia B therapy by a combination of rebalancing and hF9 KI using LNP and AAV.

An optimized SpCas9 high-fidelity variant for direct protein delivery

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

Relationship between the deep features of the full-scan pathological map of mucinous gastric carcinoma and related genes based on deep learning

BackgroundLong-term differential expression of disease-associated genes is a crucial driver of pathological changes in mucinous gastric carcinoma. Therefore, there should be a correlation between depth features extracted from pathology-based full-scan images using deep learning and disease-associated gene expression. This study tried to provides preliminary evidence that long-term differentially expressed (disease-associated) genes lead to subtle changes in disease pathology by exploring their correlation, and offer a new ideas for precise analysis of pathomics and combined analysis of pathomics and genomics. MethodsFull pathological scans, gene sequencing data, and clinical data of patients with mucinous gastric carcinoma were downloaded from TCGA data. The VGG-16 network architecture was used to construct a binary classification model to explore the potential of VGG-16 applications and extract the deep features of the pathology-based full-scan map. Differential gene expression analysis was performed and a protein-protein interaction network was constructed to screen disease-related core genes. Differential, Lasso regression, and extensive correlation analyses were used to screen for valuable deep features. Finally, a correlation analysis was used to determine whether there was a correlation between valuable deep features and disease-related core genes. ResultThe accuracy of the binary classification model was 0.775 ± 0.129. A total of 24 disease-related core genes were screened, including ASPM, AURKA, AURKB, BUB1, BUB1B, CCNA2, CCNB1, CCNB2, CDCA8, CDK1, CENPF, DLGAP5, KIF11, KIF20A, KIF2C, KIF4A, MELK, PBK, RRM2, TOP2A, TPX2, TTK, UBE2C, and ZWINT. In addition, differential, Lasso regression, and extensive correlation analyses were used to screen eight valuable deep features, including features 51, 106, 109, 118, 257, 282, 326, and 487. Finally, the results of the correlation analysis suggested that valuable deep features were either positively or negatively correlated with core gene expression. ConclusionThe preliminary results of this study support our hypotheses. Deep learning may be an important bridge for the joint analysis of pathomics and genomics and provides preliminary evidence for long-term abnormal expression of genes leading to subtle changes in pathology.

Development and characterization of a chronic hepatitis B murine model with a mutation in the START codon of an HBV polymerase.

Chronic hepatitis B (CHB) is caused by the Hepatitis B virus (HBV) and affects millions of people worldwide. Developing an effective CHB therapy requires using in vivo screening methods, such as mouse models reflecting CHB based on hydrodynamic delivery of plasmid vectors containing a replication-competent HBV genome. However, long-term expression of HBV proteins is accompanied by production of progeny virions, thereby requiring a Biosafety Level (BSL) 3 animal facility. In the present study, we introduced a point mutation in the START codon of the HBV polymerase to develop a mouse model reflecting chronic hepatitis B infection without formation of viral progeny. We induced the mouse model by hydrodynamic injection of adeno-associated virus plasmid vector (pAAV) and minicircle plasmid (pMC) constructs into C57Bl/6 and C3H/HeN mouse strains, monitoring HBV antigens and antibodies in blood by enzyme-linked immunosorbent assay and analyzing liver expression of HBV core antigen by immunohistology. Persisting expression of viral antigens over 140 days (study endpoint) was observed only in the C3H/HeN mouse strain when using pAAV/1.2HBV-A and pMC/1.0HBV-D with pre-C and pre-S recombination sites. In addition, pAAV/1.2HBV-A in C3H/HeN sustained HBV core antigen positivity up to the study endpoint in C3H/HeN mice. Moreover, introducing the point mutation in the START codon of polymerase effectively prevented the formation of viral progeny. Our study establishes an accessible and affordable experimental paradigm for developing a robust mouse model reflecting CHB suitable for preclinical testing of anti-HBV therapeutics in a BSL2 animal facility.

Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A.

Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD50) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD50 of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.

Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression.

Transcranial ultrasound combined with intravenous microbubbles can be used to increase blood-brain barrier permeability or, at lower pressures, to mediate sonoselective gene delivery to endothelial cells. Previously, sonoselective gene delivery with plasmid-coated microbubbles as gene carriers resulted in transient transgene expression in the brain endothelium. We investigated the potential of recombinant adeno-associated virus 9 (rAAV9), a serotype known for its efficient transduction and long-term transgene expression, for sonoselective gene delivery to endothelial cells of the brain. We found that rAAV9 led to gene delivery to brain endothelial cells following intravenous administration at a dosage of 1 × 1011 GC/g. However, the sonoselective gene delivery approach with intravenous rAAV9, using the same parameters as previously used for plasmid delivery, did not increase transgene expression in brain endothelial cells targeted. These results suggest that intravenous rAAV9 are using mechanisms of entry into the cerebrovasculature that are not significantly influenced by sonoselective treatments known to facilitate endothelial cell entry of plasmids coated onto microbubbles.