Precise Gene Therapy Research Articles

O102 RELATIONSHIP BETWEEN AGTR1 (c.1166 A>C) POLYMORPHISMS AND KIDNEY INJURY IN HYPERTENSION

Background: High blood pressure is the main cause of cardiovascular diseases. Kidney damage is one of the most common organ secondary damage to hypertension. The study of hypertension gene polymorphisms is an important means of precision treatment of primary hypertension. Objectives: The objective of this study was to explore the relationship between AGTR1 (c.1166 A>C) gene polymorphisms and hypertension combined with kidney damage, while exploring the relationship between codominant, dominant and recessive gene model and hypertension with kidney injury and the susceptibility of different genotypes to hypertension with kidney injury. Methods: The distribution of AGTR1 polymorphism in the AGTR1 in hypertensive patients (hypertension group, 292 patients) and hypertension with kidney injury patients (44 patients) were detected and compared by PCR-melting curve method. Results: The genotype distribution of hypertension and combined groups met Hardy-Weinberg equilibrium (p > 0.05); the distribution difference between the three genotypes was statistically significant (p < 0.05), the codominant, dominant and recessive distribution frequency of genotypes (p < 0.05), and no difference between A allele and C allele (p > 0.05). Conclusions: Our study identified the relationship of AGTRA (c.1166 A>C) with hypertension combined with renal injury, and compared the susceptibility of different genetic models, which may provide novel targets for precision gene therapy of hypertension.

Unlocking precision gene therapy: harnessing AAV tropism with nanobody swapping at capsid hotspots.

Adeno-associated virus (AAV) has been remarkably successful in the clinic, but its broad tropism is a practical limitation of precision gene therapy. A promising path to engineer AAV tropism is the addition of binding domains to the AAV capsid that recognize cell surface markers present on a targeted cell type. We have recently identified two previously unexplored capsid regions near the 2/5-fold wall and 5-fold pore of the AAV capsid that are amenable to insertion of larger protein domains, including nanobodies. Here, we demonstrate that these hotspots facilitate AAV tropism switching through simple nanobody replacement without extensive optimization in both VP1 and VP2. Our data suggest that engineering VP2 is the preferred path for maintaining both virus production yield and infectivity. We demonstrate highly specific targeting of human cancer cells expressing fibroblast activating protein (FAP). Furthermore, we found that the combination of FAP nanobody insertion plus ablation of the heparin binding domain can reduce off-target infection to a minimum, while maintaining a strong infection of FAP receptor-positive cells. Taken together, our study shows that nanobody swapping at multiple capsid locations is a viable strategy for nanobody-directed cell-specific AAV targeting.

Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets.

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.

Reversible Control of Gene Expression by Guest-Modified Adenosines in a Cell-Free System via Host-Guest Interaction.

Gene expression technology has become an indispensable tool for elucidating biological processes and developing biotechnology. Cell-free gene expression (CFE) systems offer a fundamental platform for gene expression-based technology, in which the reversible and programmable control of transcription can expand its use in synthetic biology and medicine. This study shows that CFE can be controlled via the host-guest interaction of cucurbit[7]uril (CB[7]) with N6-guest-modified adenosines. These adenosine derivatives were conveniently incorporated into the DNA strand using a post-synthetic approach and formed a selective and stable base pair with complementary thymidine in DNA. Meanwhile, alternate addition of CB[7] and the exchanging guest molecule induced the reversible formation of a duplex structure through the formation and dissociation of a bulky complex on DNA. The kinetics of the reversibility was fine-tuned by changing the size of the modified guest moieties. When incorporated into a specific region of the T7 promoter sequence, the guest-modified adenosines enabled tight and reversible control of in vitro transcription and protein expression in the CFE system. This study marks the first utility of the host-guest interaction for gene expression control in the CFE system, opening new avenues for developing DNA-based technology, particularly for precise gene therapy and DNA nanotechnology.

Fabrication of Metal Nuclear Acid Framework to Enable Carrier‐Free MNAzyme Self‐Delivery for Gastric Cancer Treatment

AbstractMulti‐component deoxyribozymes (MNAzymes) have shown extraordinary potential in precise gene therapy in vitro, however, the in vivo application is limited by complicated delivery systems. Herein, a novel DNA‐metal binding mechanism is discovered, and metal‐nucleic acid frameworks (MNFs) are built composed of MNAzymes and metal ions, which enable the carrier‐free self‐delivery of MNAzymes. Metal ions have a high affinity to DNA, however, the binding of metals with DNA at 20–30 base pair long (that normally a MNAzyme has) to form MNF structure is challenged by stringent high‐temperature synthesis conditions, poor stability of the products, and lack of targeting capabilities. While, it is discovered that through folding and entanglement of the MNAzyme with an aptamer tail, and prolonging the sequence to 71 base pair, the metal MNAzymes binding is significantly improved and stabilized to MNF structure even at room temperature. Moreover, the aptamer tail also endows MNFs with targeting capabilities. As proof of concept, a carrier‐free Ca/MNAzyme delivery system at room temperature, loaded with the model imaging protein BSA‐Cy5 is synthesized. This system can effectively target Her‐2 positive gastric cancer cells with the Her‐2 responsive aptamer tail and initiate dual gene regulation, thereby inducing energy depletion in cancer cells.

A smart mRNA-initiated DNAzyme nanogenerator for precise imaging and gene therapy in vivo

Despite the significant potential of gene therapy mediated by deoxyribozyme (DNAzyme) in various diseases, the success of clinical cancer treatment remains challenging due to its low selectivity and efficiency. In this study, a novel DNAzyme nanogenerator (NG) triggered by mRNA was developed for in vivo tumour-enhanced imaging and precise gene therapy. After entering target cells, the NG could initiate a DNAzyme catalytic reaction in response to tumour-associated mRNA targets within the cells. The DNAzyme catalytic reaction continuously cleaved the DNA substrate sandwiched by labeled dyes, resulting in significantly amplified fluorescence release and continuous generation of another effective DNAzyme, silencing VEGF-related genes. The detection limit of NG for TK1 mRNA reached 3.62 pM. Additionally, NG could selectively image at tumour sites and specifically generate DNAzyme in tumor cells. NG accurately identified tumors in vivo and significantly inhibited tumour growth with negligible toxicity, thus expanding the toolbox for on-demand gene delivery and precise therapy.

NIR light-controlled DNA nanodevice for amplified mRNA imaging and precise gene therapy

Accurately delivering antisense oligonucleotides (ASOs) to tumor cells for gene therapy poses a significant challenge. To address this issue, we develop a NIR light-activable DNA nanodevice to target survivin mRNA and simultaneously release ASOs in tumor cells, which allows high-precision spatiotemporal imaging and efficient gene therapy. The concept is based on upconversion nanoparticles, which can convert NIR light into UV emissions to activate an entropy-driven DNA walking system for mRNA recognition in tumor cells. The intramolecular toehold-mediated entropy-driven catalytic reaction generates a large amount of Bcl-2 ASOs, which allows accurate delivery of ASOs for gene therapy due to the specific targeting of mRNA in tumor cells. The results from in vitro and in vivo experiments show that the NIR light-activated DNA nanodevice can detect mRNA with high sensitivity by DNA walking amplification and high precision in gene delivery. Moreover, the released ASOs from DNA walking system down-regulate the expression of Bcl-2 anti-apoptosis protein and induce tumor cell apoptosis, thereby suppressing tumor growth without a transfection reagent. The NIR-light-controlled DNA nanodevice holds great potential for precise gene therapy in clinical applications.

Abstract 17260: Endothelium-Targeted Nanoparticle Delivery of FOXM1 Reactivates Aging-Impaired Endothelial Regeneration for Vascular Repair and Inflammatory Lung Injury Resolution: Precision Gene Therapy of Acute Respiratory Distress Syndrome

Introduction: Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS). However, the underlying molecular mechanisms remain elusive and there is no effective therapy for ARDS. Hypothesis: Impaired endothelial regeneration and vascular repair in aged lungs causes persistent lung injury leading to high incidence of ARDS and high mortality in elderly patients following sepsis challenge. Methods: Genetic lineage tracing was employed to determine the cell origin of endothelial regeneration. Aged Foxm1Tg mice was used for assessing the role of FoxM1 in activating endothelial regeneration. Endothelium-targeted nanoparticles were employed to deliver FOXM1 gene selectively to endothelial cells (ECs) in aged mice. Methylation analysis was employed to determine how aging impairs FoxM1 expression. Results: Genetic lineage tracing study showed that endothelial regeneration was mediated by lung endothelial proliferation in adult mice whereas this intrinsic regenerative program was impaired in aged mice. Expression of FoxM1, an important mediator of endothelial regeneration in young adult mice, was not induced in lungs of aged mice due to aging-induced hypermethylation of the Foxm1 promoter. Transgenic FOXM1 expression or in vivo endothelium-targeted nanoparticle delivery of FOXM1 gene reactivated endothelial regeneration, normalized vascular repair and promoted survival of aged mice following sepsis. Furthermore, nanoparticle delivery of FoxM1 gene selectively in lung ECs also reactivated endothelial regeneration in aged mice, supporting the novel concept of precision gene therapy. In COVID-19 lung autopsy samples, FOXM1 expression was not induced in vascular ECs of elderly patients in contrast to mid-age patients, validating the clinical relevance of our findings. Conclusions: We have demonstrate that persistent lung injury and high mortality in aged mice following sepsis challenge is ascribed to impaired endothelial regeneration and vascular repair. Therapeutic reactivation of FoxM1-dependent endothelial regeneration and vascular repair represents a potential effective therapy for elderly ARDS patients by endothelium-targeted nanoparticle delivery of FOXM1 gene.

Association between AGTR1 (c.1166 A>C) Polymorphisms and Kidney Injury in Hypertension.

High blood pressure is the main cause of cardiovascular diseases. Kidney damage is one of the most common organ secondary damage to hypertension. The study of hypertension gene polymorphisms is an important means of precision treatment of primary hypertension. The objective of this study was to explore the relationship between AGTR1 (c.1166 A>C) gene polymorphisms and hypertension combined with kidney damage, while exploring the relationship between codominant, dominant and recessive gene model and hypertension with kidney injury and the susceptibility of different genotypes to hypertension with kidney injury. The distribution of AGTR1 polymorphism in the AGTR1 in hypertensive patients (hypertension group, 292 patients) and hypertension with kidney injury patients (44 patients) were detected and compared by PCR-melting curve method. The genotype distribution of hypertension and combined groups met Hardy-Weinberg equilibrium (p > 0.05); the distribution difference between the three genotypes was statistically significant (p < 0.05), the codominant, dominant and recessive distribution frequency of genotypes (p < 0.05), and no difference between A allele and C allele (p > 0.05). Our study identified the relationship of AGTRA (c.1166 A>C) with hypertension combined with renal injury, and compared the susceptibility of different genetic models, which may provide novel targets for precision gene therapy of hypertension. URL: https://www.chictr.org.cn/indexEN.html; Unique identifier: ChiCTR2100051472.

Stimuli‐Responsive Dendritic Supramolecular Vector for Tumor‐Specific Gene Delivery

To simplify the preparation of dendritic materials, host–guest molecular recognition and self‐assembly are utilized to form a supramolecular dendritic gene vector (DNCVP). DNCVP is constructed from an amino dendron‐conjugated naphthol, viologen containing pH‐sensitive hydrazone‐bond‐linked PEG, and CB[8] with a molar ratio of 1:1:1. The pH‐ and reducing‐sensitivity of DNCVP is verified, and the stimuli‐responsive capacity enables the vector tumor targeting gene delivery ability. Owing to the protection of surface PEG, the supramolecular engineering endows the delivery vector with low cytotoxicity and good biocompatibility that are confirmed by the MTT assay. The excellent delivery ability of genes is investigated by in vitro transfection of pEGFP, pGL3, and silencing of siGAPDH. In vivo studies demonstrate promoted tumor accumulation of genes mediated by the dual‐responsive DNCVP and the transfection efficiency at the tumor site is greatly improved benefiting from the dynamic nature of noncovalent interactions. This study reveals DNCVP is a promising supramolecular dendritic gene delivery vector, providing a sophisticated strategy for precise gene therapy.

Abstract 14938: Endothelial Cell-Targeted Nanoparticle Delivery of Crispr/Cas9 Plasmid Dna Disrupts Endothelial Epas1 in Adult Rats and Inhibits Experimental Pah

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling that leads to right heart failure and death. HIF-2α (hypoxia-inducible factor-2α) activation in lung endothelial cells is obligatory to obliterative pulmonary vascular remodeling in PAH patients and animal PAH models. Hypothesis: Endothelial cell-targeted nanoparticle can deliver plasmid DNA expressing the CRISPR/Cas9 system to disrupt Epas1 (encoding HIF-2α) selectively in endothelial cells in adult rats and inhibit PAH in monocrotaline rats. Methods: To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether- block -poly(lactide-co-glycolide) (PEG- b -PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by rat Cdh5 promoter and guide RNAs targeting Epas1 driven by U6 promoter induced highly efficient genome editing in endothelial cells in 6-weeks old SD rats. Rat Cdh5 -Cas9 plasmid DNA with scramble guide RNA were injected as a control group. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, hemodynamic measurement of right ventricular systolic pressure RVSP and weight ratio of right ventricular versus left ventricular plus septum (RV/LV+S), an indicator of RV hypertrophy were determined. Results: Single administration of mixture of nanoparticles and CRISPR Cdh5 plasmid DNA caused diminished HIF-2α protein expression selectively in endothelial cells indicating highly efficient genome editing. Genomic editing-mediated disruption of Epas1 in endothelial cells reduced RVSP and right ventricular hypertrophy as well as obliterative pulmonary vascular remodeling in MCT-induced PAH rats. Conclusions: These findings demonstrate robust genome editing of Epas1 via EC-targeted nanoparticle delivery of CRISPR-Cas9 plasmid DNA in adult rats leading to attenuated PAH in MCT rats, suggesting EC-targeted nanoparticle delivery of genome editing system to disrupt PAH-causing gene(s) in ECs is a promising precision gene therapy of PAH in patients.

Hybrid Polyelectrolyte Nanocomplexes for Non-Viral Gene Delivery with Favorable Efficacy and Safety Profile.

The development of nanovectors for precise gene therapy is increasingly focusing on avoiding uncontrolled inflammation while still being able to effectively act on the target sites. Herein, we explore the use of non-viral hybrid polyelectrolyte nanocomplexes (hPECs) for gene delivery, which display good transfection efficacy coupled with non-inflammatory properties. Monodisperse hPECs were produced through a layer-by-layer self-assembling of biocompatible and biodegradable polymers. The resulting nanocomplexes had an inner core characterized by an EGFP-encoding plasmid DNA (pDNA) complexed with linear polyethyleneimine or protamine (PEI or PRM) stabilized with lecithin and poly(vinyl alcohol) (PVA) and an outer layer consisting of medium-molecular-weight chitosan (CH) combined with tripolyphosphate (TPP). PEI- and PRM-hPECs were able to efficiently protect the genetic cargo from nucleases and to perform a stimuli-responsive release of pDNA overtime, thus guaranteeing optimal transfection efficiency. Importantly, hPECs revealed a highly cytocompatible and a non-inflammatory profile in vitro. These results were further supported by evidence of the weak and unspecific interactions of serum proteins with both hPECs, thus confirming the antifouling properties of their outer shell. Therefore, these hPECs represent promising candidates for the development of effective, safe nanotools for gene delivery.

Principles of translational gene therapy for neuromuscular diseases

In recent years the theoretical hope has become reality and the first hereditary neuromuscular diseases have become causally treatable. Neuromuscular diseases have thus become the pacemaker of this form of therapy for the whole of neurology. This article describes the principles of precision gene therapy for neurogenetic diseases using examples of neuromuscular diseases. Various strategies of gene therapy have become established and are being tested in preclinical and clinical trials and evaluated as approved forms for long-term efficacy. The aim of every gene therapy is the modification or introduction of the target gene with initiation of adegradation of dysfunctional proteins. Various techniques, such as gene transfer, gene substitution or gene editing in vivo and ex vivo are now usable. For example, amodification of the pre-mRNA using antisense oligonucleotides or RNA interference (siRNA) can be used for exon skipping. An initiation of gene expression to produce the target protein can be based on amodification of the DNA by means of gene replacement, cell-based therapy (iPS cells), regulation by compensatory proteins or pharmacological treatment with so-called small molecules. Each method has advantages and complex disadvantages that must be individually evaluated. Phenotypic peculiarities of arare disease often only become apparent through specific translational therapy. It is already becoming obvious that avery early point in timing of gene therapy is probably the most effective. Newborn screening is therefore gaining additional importance as early diagnosis can achieve the best possible success of therapies, possibly even preventively.

CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome.

Defects in the DOCK8 gene causes combined immunodeficiency termed DOCK8 immunodeficiency syndrome (DIDS). DIDS previously belonged to the disease category of autosomal recessive hyper IgE syndrome (AR-HIES) but is now classified as a combined immunodeficiency (CID). This genetic disorder induces early onset of susceptibility to severe recurrent viral and bacterial infections, atopic diseases and malignancy resulting in high morbidity and mortality. This pathological state arises from impairment of actin polymerization and cytoskeletal rearrangement, which induces improper immune cell migration-, survival-, and effector functions. Owing to the severity of the disease, early allogenic hematopoietic stem cell transplantation is recommended even though it is associated with risk of unintended adverse effects, the need for compatible donors, and high expenses. So far, no alternative therapies have been developed, but the monogenic recessive nature of the disease suggests that gene therapy may be applied. The advent of the CRISPR/Cas gene editing system heralds a new era of possibilities in precision gene therapy, and positive results from clinical trials have already suggested that the tool may provide definitive cures for several genetic disorders. Here, we discuss the potential application of different CRISPR/Cas-mediated genetic therapies to correct the DOCK8 gene. Our findings encourage the pursuit of CRISPR/Cas-based gene editing approaches, which may constitute more precise, affordable, and low-risk definitive treatment options for DOCK8 deficiency.

Identification of Chronic Hypersensitivity Pneumonitis Biomarkers with Machine Learning and Differential Co-expression Analysis.

This study aims to identify the biomarkers for chronic hypersensitivity pneumonitis (CHP) and facilitate the precise gene therapy of CHP. Chronic hypersensitivity pneumonitis (CHP) is an interstitial lung disease caused by hypersensitive reactions to inhaled antigens. Clinically, the task of differentiating CHP and other interstitial lung diseases, especially idiopathic pulmonary fibrosis (IPF), was challenging. In this study, we analyzed the publically available gene expression profile of 82 CHP patients, 103 IPF patients, and 103 control samples to identify the CHP biomarkers. The CHP biomarkers were selected with advanced feature selection methods: Monte Carlo Feature Selection (MCFS) and Incremental Feature Selection (IFS). A Support Vector Machine (SVM) classifier was built. Then, we analyzed these CHP biomarkers through functional enrichment analysis and differential co-expression analysis. There were 674 identified CHP biomarkers. The co-expression network of these biomarkers in CHP included more negative regulations and the network structure of CHP was quite different from the network of IPF and control. The SVM classifier may serve as an important clinical tool to address the challenging task of differentiating between CHP and IPF. Many of the biomarker genes on the differential coexpression network showed great promise in revealing the underlying mechanisms of CHP.

Smart Manganese Dioxide-Based Lanthanide Nanoprobes for Triple-Negative Breast Cancer Precise Gene Synergistic Chemodynamic Therapy.

Small interfering RNA (siRNA)-based gene therapy has been widely studied as a promising treatment for malignant triple-negative breast cancer (TNBC), but efficient delivery of siRNA still remains a challenge. In this study, a smart manganese dioxide (MnO2)-based lanthanide nanoprobe therapeutic nanoplatform (ErNPs@MnO2-siS100A4-RGD) was developed for tumor imaging and precise stimuli-responsive S100A4 siRNA (siS100A4)-mediated gene therapy in synergism with chemodynamic therapy (CDT) of TNBC. ErNPs@MnO2-siS100A4-RGD has a tumor microenvironment-responsive capability attributed to the presence of MnO2, which can be degraded by glutathione (GSH) in the tumor region while releasing siRNA and generating Mn2+ to achieve precise gene therapy and a Fenton-like reaction-mediated CDT effect on TNBC. Subsequently, the lanthanide nanoprobes (ErNPs) are exposed to the second near-infrared region (NIR-II) fluorescence emission to realize the precise tumor location. Both the in vitro and in vivo results demonstrated that the smart nanoplatform possessed high siRNA delivery efficiency and GSH-responsive precise siRNA releasing ability, and compared with individual gene therapy, the GSH-depletion-enhanced CDT effect further reinforced TNBC inhibition, demonstrating excellent GSH-responsive-enhanced NIR-II precise tumor imaging therapy. These results indicate that the nanoplatform provides a crucial foundation for further research on theranostic systems of TNBC.

Lesion-Targeted Anti-Cancer Gene Delivery with Clinical Survival Benefits: A Molecular “Rosetta Stone” for Veterinary Hematology-Oncology and Precision Gene Therapy

For love of companion animals and a deep respect for the distinguished professors and colleagues, who guided a veterinary school graduate student...

CRISPR/Cas9-Mediated Gene Correction in Newborn Rabbits with Hereditary Tyrosinemia Type I

Patients with hereditary tyrosinemia type I (HT1) present acute and irreversible liver and kidney damage during infancy. CRISPR-Cas9-mediated gene correction during infancy may provide a promising approach to treat patients with HT1. However, all previous studies were performed on adult HT1 rodent models, which cannot authentically recapitulate some symptoms of human patients. The efficacy and safety should be verified in large animals to translate precise gene therapy to clinical practice. Here, we delivered CRISPR-Cas9 and donor templates via adeno-associated virus to newborn HT1 rabbits. The lethal phenotypes could be rescued, and notably, these HT1 rabbits reached adulthood normally without 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione administration and even gave birth to offspring. Adeno-associated virus (AAV)-treated HT1 rabbits displayed normal liver and kidney structures and functions. Homology-directed repair-mediated precise gene corrections and non-homologous end joining-mediated out-of-frame to in-frame corrections in the livers were observed with efficiencies of 0.90%-3.71% and 2.39%-6.35%, respectively, which appeared to be sufficient to recover liver function and decrease liver and kidney damage. This study provides useful large-animal preclinical data for rescuing hepatocyte-related monogenetic metabolic disorders with precise gene therapy.

Adeno-Associated Virus Serotype 8-Mediated Genetic Labeling of Cholangiocytes in the Neonatal Murine Liver.

Determination of the cellular tropism of viral vectors is imperative for designing precise gene therapy. It has been widely accepted that transduction of hepatocytes using adeno-associated virus serotype 8 (AAV8) is a promising approach to correct inborn errors in neonates, but the type of neonatal hepatic cells transduced by AAV8 has not been thoroughly investigated. To address this question, we used a reporter mouse that carries Cre recombinase (Cre)-inducible yellow fluorescent protein (YFP). Our analysis primarily focused on cholangiocytes, given their pivotal roles in normal liver function and disease. We treated RosaYFP/+ mice at postnatal day 2 (P2) with AAV8-cytomegalovirus (CMV) promoter-Cre and analyzed livers at P10 and P56. The vast majority of HNF4α+ hepatocytes were labeled with YFP at both time points, and 11.6% and 24.4% of CK19+ cholangiocytes were marked at P10 and P56, respectively. We also detected YFP+ cells devoid of hepatocyte and cholangiocyte markers, and a subset of these cells expressed the endothelial and fibroblast marker CD34. Next, we used the hepatocyte-specific thyroxine-binding globulin (TBG) promoter. Surprisingly, AAV8-TBG-Cre marked 6.8% and 30.9% of cholangiocytes at P10 and P56, respectively. These results suggest that AAV8 can be a useful tool for targeting cholangiocytes in neonatal livers.

Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences.

In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision.