Insertion Of Gene Research Articles

Transferring mouse Emx1 and Emx2 lentiviruses into the chicken embryonic brain and their implication to the organization and evolution of the amniote pallium.

Homeobox genes are highly conserved and play critical roles in brain development. Recently we have found that mammals have an additional fragment of approximately 20 amino acids in Emx1 and a poly-(Ala)6-7 in Emx2, compared to other amniotes. It has been shown that Emx1 and Emx2 have synergistic actions in the brain development. These reports raise an interesting issue whether the differences of Emx1 and Emx2 between mammals and non-mammals are concerned with the organization and evolution of amniote pallium. Lentiviruses expressing mouse Emx1 and Emx2 (mEmx1/2) with additional fragments were injected into the ventricle of the chick telencephalon at embryonic day 3 to study the effects of mEmx1/2 on the development of chick pallium, whereas injections of lentiviruses containing chick Emx1 and Emx2 (cEmx1/2), no targeted gene insert or saline were as controls. The expressions of reelin, vimentin, GABA and MAP2, neurogenesis patterns for calbindin and parvalbumin neurons and the sizes of anterior commissure were then studied by immuohistochemical staining, and open-field tests were performed to assess locomotor activities and curious or exploratory behaviors of the chicks. Following the injections of lentiviruses expressing mEmx1/2, the expressions of reelin, vimentin, GABA and MAP2 increased in most parts of Wulst (W) and mesopallium (M), but not of nidopallium (N). Neurogenesis patterns for calbindin (CB) and parvalbumin (PV) neurons changed towards mammalian inside-out one, and the sizes of anterior commissure staining for neurofilament were significantly larger. In addition, posthatchling chicks showed higher rates of passive avoidance after training, but no significant differences in the total distance traveled and the percentage of time spent in the central rectangle, compared to those in the control group. The present study indicated that mEmx1/2 had effects on the development of chick pallium, suggesting that they are probably involved in the organization and evolution of amniote pallium.

CRISPR knock-in of a chimeric antigen receptor into GAPDH 3'UTR locus generates potent B7H3-specific NK-92MI cells.

CAR-NK therapy is becoming a promising approach to treat solid tumors. However, the random insertion of the CAR gene and inflexible CAR expression caused by common preparation methods significantly impact its efficacy and safety. Here we successfully established a novel type of CAR-NK cells by integrating CAR sequences into the GAPDH 3'UTR locus of NK-92MI cells (CRISPR-CAR-NK), achieving site-specific integration of the CAR gene and allowing endogenous regulatory components to govern CAR expression. CRISPR-CAR-NK cells had comparable growth capacity but displayed superior anti-tumor activity compared with their lentiviral counterparts. They activated and degranulated more effectively when co-cultured with tumor cells, due to increased expression of activating receptors and decreased expression of inhibitory molecules. They also enhanced the production of Granzyme B and IFN-γ, and more effectively triggered the IFN-γ pathway. Moreover, CRISPR-CAR-NK cells demonstrated distinct properties from conventional CAR-NK concerning metabolic features and signal dependence. Notably, CRISPR-CAR-NK cells exhibited lower metabolic levels without compromising antitumor activity, and their function was less reliant on the PI3K-AKT pathway, implying that the CRISPR-CAR-NK cells have significant potential for enhanced synergy with AKT inhibitors and adaptation to nutrient stress within the tumor microenvironment. These findings provide a novel potential strategy for cancer immunotherapy and an experimental foundation and paradigm for optimizing CAR-NK cells utilizing CRISPR technology, highlighting the potential of CRISPR to advance immunotherapies.

The IclR-family transcriptional regulator XyrR controls flotation, motility, antibiotic production and virulence in Serratia sp. ATCC 39006

The opportunistic pathogen Serratia sp. ATCC 39006 (S39006) is a rod-shaped, motile, Gram-negative bacterium that produces a 𝛽-lactam antibiotic (a carbapenem) and a bioactive red-pigmented tripyrrole antibiotic, prodigiosin. It is also the only known enterobacterium that naturally produces intracellular gas vesicles (GVs), enabling cells to float in static water columns. Regulation of GVs and secondary metabolites in S39006 can be coordinated but such pleiotropy is still poorly understood. To uncover novel inputs to this complex regulatory network, we used transposon mutagenesis to identify a mutant with an insertion in an IclR-type transcriptional regulator gene. The iclR mutant showed diminished production of carbapenem, prodigiosin, GVs and cellulase. Furthermore, the mutant also showed increased swimming and swarming motilities but exhibited attenuated virulence in planta and ability to kill the nematode C. elegans. Using differential expression analysis of the intracellular proteomes of the wild type and iclR mutant, we confirmed that the mutation negatively impacted expression of the corresponding GV, carbapenem and prodigiosin gene clusters. In contrast, flagellar and chemotaxis proteins were overexpressed, consistent with the increased motility of the mutant. We also found that the proteins encoded by a putative yagEF-yjhF operon, involved in xylonate catabolism and transport, showed a 5- to 7-fold increase in expression. Finally, we show that IclR is a repressor of xylonate catabolism in S39006 and suggest that xylonate is potentially involved in controlling carbapenem and prodigiosin biosynthesis. Our results indicate that IclR is a global regulator that controls antibiotic biosynthesis, flotation through modulating GV assembly, and has pleiotropic impacts on the physiology and virulence of S39006. Based on these findings, we propose the designation of this IclR-family transcriptional regulator as XyrR (Xylonate response Regulator).

CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery.

To address a wide range of genetic diseases, genome editing tools that can achieve targeted delivery of large genes without causing double-strand breaks (DSBs) or requiring DNA templates are necessary. Here, we introduce CRISPR-Enabled Autonomous Transposable Element (CREATE), a genome editing system that combines the programmability and precision of CRISPR/Cas9 with the RNA-mediated gene insertion capabilities of the human LINE-1 (L1) element. CREATE employs a modified L1 mRNA to carry a payload gene, and a Cas9 nickase to facilitate targeted editing by L1-mediated reverse transcription and integration without relying on DSBs or DNA templates. Using this system, we demonstrate programmable insertion of a 1.1 kb gene expression cassette into specific genomic loci of human cell lines and primary T cells. Mechanistic studies reveal that CREATE editing is highly specific with no observed off-target events. Together, these findings establish CREATE as a programmable, RNA-based gene delivery technology with broad therapeutic potential.

DUO1 Activated Zinc Finger (AtDAZ) protein role in the generative cell body morphogenesis.

Arabidopsis MYB transcription factor, AtDUO1 regulates generative cell body (GC) morphogenesis from round to semi and fully elongated forms before pollen mitosis-II (PM II). It was hypothesised that DUO1 might regulate morphogenesis through any of its direct target genes or components of the DUO1-DAZ1 network. The developmental analysis of plants harbouring T-DNA insertions in some DUO1 target genes using light and fluorescence microscopy revealed abnormal GC morphogenesis only in daz1 and daz2, but gcs1, trm16, mapkkk10, mapkkk20, tet11, and tip1 all undergo normal elongation indicating that these target genes have no important roles in morphogenesis or may be redundant. The important regulatory role of DUO1 was confirmed through the observed incomplete rescue of morphogenesis of mutant duo1-1 GCs by DAZ1 and independently by a C-terminally deleted version of DUO1 (DUO1∆C3) lacking activation sequences. The evidence supports the important role of DAZ1 in GC shape partial morphogenesis. The C-terminus of DUO1 may regulate some target genes that affect GC body elongation. Furthermore, an intact DUO1 is shown to be indispensable for GC shape and nucleus elongation and subsequently for timely division and sperm cell morphogenesis. The development of the GC cytoplasmic projection is regulated independently of DUO1, and all its target genes were able to form it.

Functional analysis of quorum sensing-mediated pathogenicity in Burkholderia contaminans SK875 using transposon mutagenesis.

Burkholderia contaminans SK875, a member of Burkholderia cepacia complex (Bcc), are known to cause lung infections in cystic fibrosis patients. To gain deeper insights into its quorum sensing (QS)-mediated pathogenicity, we employed a transposon (Tn) insertion-based random mutagenesis approach. A Tn mutant library comprising of 15,000 transconjugants was generated through conjugation between wild-type (WT) recipient B. contaminans SK875 and the donor E. coli BW20767 carrying pRL27 plasmid. From this library, 26 mutants were initially screened using the reporter strain Agrobacterium tumefaciens NT1, identified as blue-colored indicator colonies. These mutants were further analyzed for phenotypic variations related to autoinducer (AI) production, morphological changes, motility, biofilm formation, protease activity, and virulence in Caenorhabditis elegans. The Tn insertion sites in the mutants were sequenced and aligned with the reference genome of B. contaminans SK875 (PRJNA439184). Sequence analysis revealed the Tn5 insertion in genes encoding Ribonuclease P protein, a hypothetical protein, gamma-glutamyltranspeptidase 1, GCN5-related N-acetyltransferase (DUF1311), cytochrome C oxidase assembly protein, glutamyl-Q tRNA synthetase, AFG1-like ATPase, chorismate synthase, and aldehyde oxidase. Compared to wild-type (WT) strain B. contaminans SK875, the mutants (SK1917, SK1925, SK1926, SK1927, SK1935) exhibited attenuated AI production, impaired swimming and swarming motility, reduced biofilm formation and protease activity, and decreased virulence in C. elegans. We suggest that these genes are likely involved in the QS-dependent pathogenicity of B. contaminans. This study also introduces a visual color-screening method for identifying novel gene functions related to QS-dependent pathogenicity in Burkholderia species.

CRISPR/Cas9-mediated homology-directed repair for precise insertion of constitutive promoter: Overexpression of OsNRAMP7 in TBR225 rice

Precise gene insertion using homology-directed repair (HDR) in Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has emerged as a powerful tool for plant genome modification, offering unprecedented control over genetic alterations. This study successfully applied this technology to TBR225, a major rice variety in Vietnam, aiming to overexpress the OsNRAMP7 gene by inserting a 35S constitutive promoter. Our research addressed the need for efficient methods to enhance specific gene expression in crop plants, with a focus on improving this economically important rice variety. We designed and implemented a single vector system comprising gRNA and Cas9 expression constructs, along with a donor DNA repair template containing the 35S promoter. The system was delivered via Agrobacterium-mediated transformation to TBR225 rice. Our results demonstrated the successful insertion of the 35S promoter upstream of the OsNRAMP7 open reading frame in TBR225, leading to significantly increased OsNRAMP7 expression in edited lines. Notably, we obtained homozygous transgene-free OsNRAMP7-overexpressing TBR225 lines in the T1 generation, confirming stable transmission of the desired modification in this Vietnamese rice variety. These results validate the effectiveness of CRISPR/Cas9-mediated HDR for precise genetic modifications in TBR225 rice. This successful research on Vietnam's major rice variety offers a valuable approach for crop improvement and functional genomics studies, potentially accelerating the development of rice varieties with enhanced traits such as stress tolerance or nutrient use efficiency.

Isolation and characterization of a novel S1-gene insertion porcine epidemic diarrhea virus with low pathogenicity in newborn piglets

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380_Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.

Regulation of nucleus-encoded trans-acting factors allows orthogonal fine-tuning of multiple transgenes in the chloroplast of Chlamydomonas reinhardtii.

The green microalga Chlamydomonas reinhardtii is a promising host organism for the production of valuable compounds. Engineering the Chlamydomonas chloroplast genome offers several advantages over the nuclear genome, including targeted gene insertion, lack of silencing mechanisms, potentially higher protein production due to multiple genome copies and natural substrate abundance for metabolic engineering. Tuneable expression systems can be used to minimize competition between heterologous production and host cell viability. However, complex gene regulation and a lack of tight regulatory elements make this a challenge in the Chlamydomonas chloroplast. In this work, we develop two synthetic tuneable systems to control the expression of genes on the chloroplast genome, taking advantage of the properties of the vitamin B12-responsive METE promoter and a modified thiamine (vitamin B1) riboswitch, along with nucleus-encoded chloroplast-targeted regulatory proteins NAC2 and MRL1. We demonstrate the capacity of these systems for robust, fine-tuned control of several chloroplast transgenes, by addition of nanomolar levels of vitamins. The two systems have been combined in a single strain engineered to avoid effects on photosynthesis and are orthogonal to each other. They were then used to manipulate the production of an industrially relevant diterpenoid, casbene, by introducing and tuning expression of the coding sequence for casbene synthase, as well as regulating the metabolite flux towards casbene precursors, highlighting the utility of these systems for informing metabolic engineering approaches.

CULTIVARES E CARACTERÍSTICAS DO GRÃO DE MILHO

Maize (Zea mays L) is of global importance as a commodity because it is the main source of energy used in animal feed. The anatomy of corn is divided into endosperm (83%), pericarp (5%), germ (11%) and tip (2%). The starch granules and the protein matrix of the endosperm classifies corn into two types: farinaceous and vitreous. The relationship between hard endosperm (Flint) and floury (Dent) defines the vitreousness of a corn grain. Thus, the greater the vitreousness, the greater the amount of hard endosperm present in the grain. Today on the market there are specific corn cultivars for the production of grain, green corn, sweet corn, white corn (hominy), silage, popcorn, among others. The cultivars varieties are highly heterozygous, with greater productive stability, having greater genetic variability, less uniformity in the product and low productivity, caused by low heterosis, have great rusticity and adaptability, being indicated for low technology systems. Commercial hybrids are the result of the crossing between genetically distinct and homozygous individuals, for maximum heterosis response. Simple hybrids are potentially productive and suitable for systems that employ high technology. Transgenic hybrid corn is obtained by Genetic Engineering from the modification of DNA by introducing genes from other species through recombinant DNA techniques. Transgenics aims at three main targets, which can be classified as: herbicide tolerance; insertion of insect and disease resistance genes; and quality of products. White corn is a special cultivar that is characterized by having toothed and floury grain, it is widely used for the production of hominy, starch (cornstarch), flour and etc. Popcorn is a special cultivar of low productivity with rounded grains, fully vitreous endosperm, and thin film which allows spacing when the grain is heated. Green corn is an alternative to family horticulture because it adds more income, it refers to corn harvested while still in the milky stage, with 70 to 80% moisture. Sweet corn and super sweet corn are also included as green corn, differs from conventional corn in that it is intended for the canning industry and by the presence of mutant alleles that block the conversion of sugars into starch in the endosperm, giving it a sweet character, making sweet corn wrinkled and translucent when dry. Man has selected and developed several maize cultivars with distinct and special characteristics for each cropping system and purpose of use. The present study aims to address through a literature review the anatomical and nutritional characteristics of the maize grain and to address the different cultivars used in Brazilian agriculture.

Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory illness in humans and currently lacks an approved vaccine. The Newcastle disease virus (NDV) vector is a well-established, safe, and effective platform for vaccine development. With recent advancements in stabilizing coronavirus spike proteins to enhance their antigenicity, this study aimed to determine whether modifications to the MERS-CoV spike protein could improve its presentation on NDV particles, allowing the resulting virus to be used as an inactivated vaccine. We codon-optimized the gene encoding the ectodomain of the MERS-CoV spike protein and incorporated modifications at the S1/S2 and S2' cleavage sites, along with a proline substitution at residues V1060-L1061. This modified spike gene was inserted into the NDV genome to create the NDV-SMERS virus. After purification and inactivation, the vaccine's immunogenicity was assessed in mice. Mice immunized with the inactivated NDV-SMERS vaccine developed robust anti-spike IgGs, neutralizing antibodies, and cellular immune responses. The study demonstrated that modifications to the MERS-CoV spike protein were essential for its effective presentation on NDV particles. Additionally, the spike gene insert remained stable through five egg passages, confirming the vector's stability. Engineering the MERS-CoV spike protein is crucial for its successful display on NDV particles. The strong immune responses elicited by the NDV-SMERS vaccine in mice highlight that NDV is a promising, safe, and effective platform for MERS-CoV vaccination.

Insertion/deletion (I/D) polymorphisms of angiotensin-converting enzyme gene and their implications for susceptibility and severity of COVID-19: A systematic review and meta-analysis.

The insertion or deletion polymorphisms of the angiotensin-converting enzyme gene (ACE I/D) have been the subject of significant research related to coronavirus disease 2019 (COVID-19). Despite this, the findings have remained uncertain and debatable. The aim of this study was to determine the associations between the ACE I/D polymorphisms and the susceptibility as well as the severity of COVID-19. A meta-analysis study (PROSPERO: CRD42022384562) was conducted by searching the articles published on PubMed, Scopus, and Embase as of May 15, 2023. Information regarding the impact of ACE I/D variant on the susceptibility to COVID-19 and its severity was collected and analyzed utilizing the Mantel-Haenszel method with a random effects model or fixed effects model, depending on the presence or absence of heterogeneity. Out of 3,335 articles, 21 articles were included, of which 13 investigated the association between ACE I/D and the risk of COVID-19 infection and 18 of them examined its influence on disease severity. The D allele of ACE increased risk of COVID-19 infection (OR: 1.41; 95%CI: 1.08-1.85; p-Egger: 0.0676; p-Heterogeneity: <0.001; p=0.0120), while ACE I allele (OR: 0.71; 95%CI: 0.54-0.93; p-Egger: 0.0676; p-Heterogeneity: <0.001; p=0.012) and II genotype (OR: 0.55; 95%CI: 0.34-0.87; p-Egger: 0.200; p-Heterogeneity: <0.001; p=0.011) decreased the risk of infection. Additionally, there was a notable association between the ACE ID genotype and an elevated likelihood of experiencing severe COVID-19 within the Asian population (OR: 1.46; 95%CI: 1.15-1.84; p-Egger: 0.092; p-Heterogeneity: 0.116; p=0.002). The presence of ACE I/D polymorphisms significantly influences the likelihood of being susceptible to and experiencing the severity of COVID-19.

Insights into the dynamics and evolution of Rummeliibacillus stabekisii prophages in extreme environments: from Antarctic soil to spacecraft floors.

Since prophages can play a multifaceted role in bacterial evolution, this study aims to characterize the virome of Rummeliibacillus stabekisii, a bacterium isolated from different environments, including Antarctic soil and NASA spacecraft floors. From the analyses, it was found that the Antarctic strain, PP9, had the largest number of prophages, including intact ones, indicating potential benefits for survival in adverse conditions. In contrast, other strains harbored predominantly degenerate prophages, suggesting a dynamic process of gene gain and loss during evolution. Furthermore, strain PP9 exhibited polylysogeny, a strategy capable of increasing its competitive advantage by providing a broader spectrum of defensive mechanisms. In addition, evidence demonstrates that prophage regions in PP9 act as hotspots for recombination events, favoring the insertion of different phages and possible antimicrobial resistance genes. Finally, lytic cycle induction experiments revealed at least two intact prophages active in PP9. In this way, understanding the interaction between viruses and bacteria can provide valuable information about microbial evolution and adaptation in extreme environments, such as Antarctica.

Reframing thalassaemia syndrome as a benign haematopoietic stem cell disorder.

Thalassaemia, caused by over 250 mutations in the beta globin gene, changes the haematopoietic stem cell (HSC) differentiation, leading to ineffective erythropoiesis. This Wider Perspective article overlooks its underlying nature as a benign HSC disorder with a significant impact on the erythroid cell lineage. The simplicity of managing symptoms through transfusions and iron chelation therapy has shifted the focus away from the development of cell-based treatments. The identification of the beta039 mutation by Chang and Kan in 1979 marked a turning point, suggesting as main approach the molecular level by correcting the beta globin chain imbalances through gene insertion and editing. However, challenges of technology have delayed the implementation of these strategies for over four decades. In contrast, the past two decades have witnessed significant advances in the treatment of HSC disorders of the myeloid clone which are driven by a 'target cell strategy'. Many current and innovative treatments for thalassaemia are now adopting this approach, highlighting the importance of identifying suitable candidates through risk stratification. This manuscript explores the evolving understanding of thalassaemia syndromes as congenital HSC disorders of the erythroid clone and examines the implications of this perspective for the development of future treatments.

Precise kilobase-scale genomic insertions in mammalian cells using PASTE.

Programmable gene integration technologies are an emerging modality with exciting applications in both basic research and therapeutic development. Programmable addition via site-specific targeting elements (PASTE) is a programmable gene integration approach for precise and efficient programmable integration of large DNA sequences into the genome. PASTE offers improved editing efficiency, purity and programmability compared with previous methods for long insertions into the mammalian genome. By combining the specificity and cargo size capabilities of site-specific integrases with the programmability of prime editing, PASTE can precisely insert cargoes of at least 36 kb with efficiencies of up to 60%. Here we outline best practices for design, execution and analysis of PASTE experiments, with protocols for integration of EGFP at the human NOLC1 and ACTB genomic loci and for readout by next generation sequencing and droplet digital PCR. We provide guidelines for designing and optimizing a custom PASTE experiment for integration of desired payloads at alternative genomic loci, as well as example applications for in-frame protein tagging and multiplexed insertions. To facilitate experimental setup, we include the necessary sequences and plasmids for the delivery of PASTE components to cells via plasmid transfection or in vitro transcribed RNA. Most experiments in this protocol can be performed in as little as 2 weeks, allowing for precise and versatile programmable gene insertion.

LysR-Type Transcriptional Regulator Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Regulating Type Three Secretion System

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. In a previous study, we screened for reduced virulence using Tn5 transposon mutants and identified a LysR-type transcriptional regulator (LTTR) as a potential virulence factor in Pcal. However, the role of LTTR in Pcal virulence has not been thoroughly investigated. In this study, we demonstrated that the Pcal NN14 mutant (with Tn5 insertion in the LTTR-encoding gene) showed reduced disease symptoms and bacterial populations in cabbage, indicating that LTTR contributes to Pcal virulence. RNA-seq analysis identified 39 LTTR-dependent genes. Genes associated with 13 of the type three secretion system (T3SS), two of flagellar apparatus, ABC transporters, and transcription factors were expressed at lower levels in the NN14 mutant compared to the wild type. Conversely, tssH and hcp, type six secretion system (T6SS)-related genes, showed higher expression in NN14. Furthermore, these differences in gene expression were observed in minimal medium, but not in nutrient-rich medium, suggesting that LTTR acts as a global regulator responsive to nutrient conditions. Additionally, LTTR activated the expression of T3SS-related genes during Pcal infection. We also demonstrated that NN14 showed a reduced ability to induce hypersensitive reaction (HR) cell death in non-host plants. Collectively, these results suggest that LTTR contributes to Pcal virulence by regulating T3SS in response to environmental changes.

Unveiling the Future of Cardiac Care: A Review of Gene Therapy in Cardiomyopathies.

For years, the treatment of many cardiomyopathies has been solely focused on symptom management. However, cardiomyopathies have a genetic substrate, and directing therapy towards the pathophysiology rather than the epiphenomenon of the disease may be a winning strategy. Gene therapy involves the insertion of genes or the modification of existing ones and their regulatory elements through strategies like gene replacement and gene editing. Recently, gene therapy for cardiac amyloidosis and Duchenne muscular dystrophy has received approval, and important clinical trials are currently evaluating gene therapy methods for rare heart diseases like Friedreich's Ataxia, Danon disease, Fabry disease, and Pompe Disease. Furthermore, favorable results have been noted in animal studies receiving gene therapy for hypertrophic, dilated, and arrhythmogenic cardiomyopathy. This review discusses gene therapy methods, ongoing clinical trials, and future goals in this area.

Antibiotics-Free Steady Bioproduction of Valuable Chemicals from Organic Wastes by Engineered Vibrio natriegens through Targeted Gene Integration.

Bioproduction of chemicals by using engineered bacteria is promising for a circular economy but challenged the instability of the introduced plasmid by conventional methods. Here, we developed a two-plasmid INTEGRET system to reliably integrate the targeted gene into the Vibrio natriegens genome, making it a powerful strain for efficient and steady bioproduction without requiring antibiotic addition. The INTEGRET system allows for gene insertion at over 75% inserting efficiency and flexibly controllable gene dosages. Additionally, simultaneous gene insertion at four genomic sites was achieved at 54.3% success rate while maintaining stable inheritance of exogenous sequences across multiple generations. The engineered strain could efficiently synthesize PHB from the fermentation of diverse organic wastes, with an efficiency comparable to those with overexpressed plasmid. When the mixture of seawater and molasses was used as the feedstock, it achieved a high PHB yield of 39.41 wt %. An extended application of the INTEGRET system for imparting the riboflavin production ability to the bacterium was also demonstrated. Our work presents a reliable and efficient genomic editing tool to facilitate the development of sustainable and environmentally benign biological platforms for converting biomass wastes into valuable chemicals.

Structures of vertebrate R2 retrotransposon complexes during target-primed reverse transcription and after second strand nicking.

R2 retrotransposons are model site-specific eukaryotic non-LTR retrotransposons that copy-and-paste into gene loci encoding ribosomal RNAs. Recently we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not characterized. Here, we report high resolution cryo-electron microscopy structures of two vertebrate A-clade R2 proteins, avian and testudine, at the initiation of target-primed reverse transcription and one structure after cDNA synthesis and second strand nicking. Using biochemical and cellular assays we discover the basis for high selectivity of template use and unique roles for each of the expanded A-clade zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif in vertebrate A-clade R2 proteins. Our work brings first insights to A-clade R2 protein structure during gene insertion and enables further improvement and adaptation of R2-based systems for precise transgene insertion.

Optimization of HITI-Mediated Gene Insertion for Rhodopsin and Peripherin-2 in Mouse Rod Photoreceptors: Targeting Dominant Retinitis Pigmentosa.

Among the genome-editing methods for repairing disease-causing mutations resulting in autosomal dominant retinitis pigmentosa, homology-independent targeted integration (HITI)-mediated gene insertion of the normal form of the causative gene is useful because it allows the development of mutation-agnostic therapeutic products. In this study, we aimed for the rapid optimization and validation of HITI-treatment gene constructs of this approach in developing HITI-treatment constructs for various causative target genes in mouse models of retinal degeneration. We constructed the Cas9-driven HITI gene cassettes in plasmid vectors to treat the mouse Rho gene. A workflow utilizing in vivo electroporation was established to validate the efficacy of these constructs. Single-cell genotyping was conducted to evaluate allelic donor gene insertion. The therapeutic potency of HITI-treatment plasmid and adeno-associated virus (AAV) vectors was examined by section immunohistochemistry and optomotor response (OMR) in Rho+/P23H mutant mice. We also targeted mouse Prph2 to examine the workflow. The optimized HITI-treatment constructs for mouse Rho genes achieved gene insertion in 80% to 90% of transduced mouse rod photoreceptor cells. This construct effectively suppressed degeneration and induced visual restoration in mutant mice. HITI-treatment constructs for the Rhodopsin gene demonstrated efficacy in AAV vectors and are adaptable for the mouse Prph2 gene locus. The study showcases a workflow for the rapid optimization and validation of highly effective HITI-treatment gene constructs against dominant-negative inheritance in inherited retinal dystrophy. These findings suggest the potential utility of this approach in developing HITI-treatment constructs for various target genes, advancing gene therapy products for diverse genetic disorders.