Method For Gene Therapy Research Articles

Unravelling the Role of Tyrosine and Tyrosine Hydroxylase in Parkinson's Disease: Exploring Nanoparticle-based Gene Therapies.

Parkinson's disease (PD) is a neurodegenerative disorder that results from the progressive loss of neurons in the brain followed by symptoms such as slowness and rigidity in movement, sleep disorders, dementia and many more. The different mechanisms due to which the neuronal degeneration occurs have been discussed, such as mutation in PD related genes, formation of Lewy bodies, oxidation of dopamine. This review discusses current surgical treatment and gene therapies with novel developments proposed for PD. Gene therapy based on novel approaches will possess more potential advantages over the conventional methods. Currently, gene therapy for such disorders is still under the process of clinical trials and approval. The pathogenesis comes from the breakdown of dopaminergic neurons within substantia nigra (SN) by the action of tyrosinase enzyme and subsequent accumulation of α-synuclein within the neurons. These dopaminergic neurons are the main source of dopamine, the decline of which is responsible for the symptoms. So, gene therapy can possibly provide more stable supplementation and regulate the expression of tyrosinase enzyme, providing better symptomatic relief and lesser side effects. Dopamine replacement therapy is a wellstudied gene therapy method for PD. Another approach involves introducing functional genes for enzymes such as tyrosine hydroxylase, cyclohydrolases, and decarboxylases with the help of engineered vectors such as AAV and LV. Further, the potential application of nanoparticles in gene therapy as an efficient gene delivery and imaging system has been discussed. Among these, lipidbased nanoparticles such as PILs offer important benefits in terms of enhanced bioavailability, permeability to the cells, and solubility. So, this review paper summarizes some of the advanced gene therapy approaches for PD and the current status of clinical research in the development of gene therapy using nanoparticles.

Development of an in vitro model of dysferlinopathy via crispr/cas-mediated transcriptional activation of the dysf gene

Scientists need cell models from human tissues to develop methods of gene therapy and genome editing for monogenic diseases. It is preferable to use minimally invasive methods to obtain samples; these tissues can be applied for further screening in order to select the most effective approach to restore the synthesis of the target protein. We used the CRISPR/Cas9-SAM transcriptional activation system, which ensures expression of the DYSF gene in HEK293Т cells, as well as in fibroblasts from patients with dysferlinopathy (c.2779delG (Ala927LeufsX21)). After targeted activation of DYSF, it was possible to detect the main gene products: mRNA and protein (HEK293Т_ТА) and mRNA (fibroblasts). Transcriptionally activated dysferlin-deficient fibroblasts and HEK293 cells can be used to evaluate the in vitro efficacy of gene therapy for dysferlinopathies.

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.

Comparison of Gene-based and Hormonal Contraception Methods for Female Domestic Cats (Felis silvestris catus)

Abstract. with the growing population of stray and feral cats, traditional contraception methods like spaying and neutering, though effective, present limitations for Trap-Neuter-Return (TNR) programs in terms of cost, feasibility, and health risks. This article compares and contrasts gene-based and hormonal contraception methods in female domestic cats (Felis silvestris catus). The article delves into alternative non-invasive methods, particularly focusing on AAV9-fcMISv2 gene therapy and various hormonal methods such as Megestrol Acetate, GnRH agonists, and melatonin, and reviewing the advantages and drawbacks of these methods. The comparative analysis highlights the promising potential of gene-based therapies as a long-term solution for controlling stray and feral cat populations.

TP53 mutations in cancer: Molecular features and therapeutic opportunities (Review).

The tumour suppressor factor p53 plays an essential role in regulating numerous cellular processes, including the cell cycle, DNA repair, apoptosis, autophagy, cell metabolism and immune response. TP53 is the most commonly mutated gene in human cancers. These mutations are primarily non‑synonymous changes that produce mutant p53 proteins characterized by loss of function, a dominant negative effect on p53 tetramerisation and gain of function (GOF). GOF mutations not only disrupt the tumour‑suppressive activities of p53 but also endow the mutant proteins with new oncogenic properties. Recent studies analysing different pathogenic features of mutant p53 in cancer‑derived cell lines have demonstrated that restoring wild‑type p53, rather than removing GOF mutations, reduces cancer cell growth. These findings suggest that therapeutic strategies for reactivating wild‑type p53 function in cancer cells may bring a greater benefit than approaches halting mutant p53. This approach could involve the use of small molecules, gene therapy and other methods to re‑establish wild‑type p53 activity. This review describes the complexity of the biological activities of different p53 mutants and summarizes the current therapeutic approaches to restore p53 function.

A dual-signal biosensor based on surface-enhanced Raman spectroscopy for high-sensitivity quantitative detection and imaging of circRNA in living cells

Circular RNAs (circRNAs) are non-coding RNAs that play key roles in the development and progression of cancer through various mechanisms of action, making them promising biomarkers for cancer diagnosis, prognosis, and treatment. In the present study, a biosensor based on surface-enhanced Raman spectroscopy (SERS) was developed for rapid, simple, and sensitive quantitative detection of intracellular circRNAs for the first time. A dual-signal SERS nanoprobe with a 4MBN and ROX signal molecule was fabricated, and the ROX signal intensity was used to determine the concentration of target circSATB2. 4MBN was used as an internal standard to calibrate the ROX signal, thereby achieving highly sensitive and reliable detection of the target circRNA with a limit of detection of 0.043 pM. Furthermore, the relatively high expression of circSATB2 in lung cancer cells compared to that in normal lung epithelial cells was successfully characterized by the proposed SERS imaging method, which is consistent with the results of standard reverse transcription-polymerase chain reaction (RT-PCR). Monitoring of specific circRNAs using this SERS-based biosensor is a promising method for cancer diagnosis and gene therapy.

Advances in glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with significant research value due to its profound effects on dopaminergic, cholinergic, and motor neurons. Initially identified for its neuroprotective role in dopaminergic neurons, GDNF has been found to support various neuronal populations and plays a crucial role in neural development, maintenance, and repair. This review provides a comprehensive overview of the molecular characteristics, tissue distribution, physiological function of GDNF and its protective effect on a series of diseases, emphasizing its potential therapeutic applications. Meanwhile, this study discusses the challenges in delivering GDNF across the blood-brain barrier and explores current strategies to enhance its clinical efficacy, including the use of gene therapy and innovative delivery methods. In summary, the review underscores the promise of GDNF as a therapeutic agent for neurodegenerative diseases and nerve injuries, highlighting the need for further research to translate these findings into clinical practice.

Current Landscape of Gene Therapy for the Treatment of Cardiovascular Disorders.

Cardiovascular disorders (CVD) are the primary cause of death worldwide. Multiple factors have been accepted to cause cardiovascular diseases; among them, smoking, physical inactivity, unhealthy eating habits, age, and family history are flag-bearers. Individuals at risk of developing CVD are suggested to make drastic habitual changes as the primary intervention to prevent CVD; however, over time, the disease is bound to worsen. This is when secondary interventions come into play, including antihypertensive, anti-lipidemic, anti-anginal, and inotropic drugs. These drugs usually undergo surgical intervention in patients with a much higher risk of heart failure. These therapeutic agents increase the survival rate, decrease the severity of symptoms and the discomfort that comes with them, and increase the overall quality of life. However, most individuals succumb to this disease. None of these treatments address the molecular mechanism of the disease and hence are unable to halt the pathological worsening of the disease. Gene therapy offers a more efficient, potent, and important novel approach to counter the disease, as it has the potential to permanently eradicate the disease from the patients and even in the upcoming generations. However, this therapy is associated with significant risks and ethical considerations that pose noteworthy resistance. In this review, we discuss various methods of gene therapy for cardiovascular disorders and address the ethical conundrum surrounding it.

Current Status of Biomedical Products for Gene and Cell Therapy of Recessive Dystrophic Epidermolysis Bullosa.

This detailed review describes innovative strategies and current products for gene and cell therapy at different stages of research and development to treat recessive dystrophic epidermolysis bullosa (RDEB) which is associated with the functional deficiency of collagen type VII alpha 1 (C7) caused by defects in the COL7A1 gene. The use of allogenic mesenchymal stem/stromal cells, which can be injected intradermally and intravenously, appears to be the most promising approach in the field of RDEB cell therapy. Injections of genetically modified autologous dermal fibroblasts are also worth mentioning under this framework. The most common methods of RDEB gene therapy are gene replacement using viral vectors and gene editing using programmable nucleases. Ex vivo epidermal transplants (ETs) based on autologous keratinocytes (Ks) have been developed using gene therapy methods; one such ET successively passed phase III clinical trials. Products based on the use of two-layer transplants have also been developed with both types of skin cells producing C7. Gene products have also been developed for local use. To date, significant progress has been achieved in the development of efficient biomedical products to treat RDEB, one of the most severe hereditary diseases.

Review of Gene Therapy on Myocardial Ischemia-Reperfusion Injury Treatment

This article aims to summarize decade research which utilizing gene therapy method on myocardial ischemia-reperfusion injury. Relative articles are collected from CNKI retrieval. By analyzing and comparing the relative research articles published decade, the recent research conclusions are summarized. Methods and conclusions are compared on each upside and downside. The recent research is center on the gene transduction or stem cell transplantation. The adenovirus and adeno-associated virus are introduced into the gene transfer. The pretreatment of stem cell transplantation is also researched in recent research. All research conclusions claim that positive results are observed after treatment, including injury reduced or reversed, myocardial protect effects. Due to the complexity of mechanism in myocardial ischemia-reperfusion injury, the works may show conflicting results at the conclusion part. However, the results are roughly positive at the practical application in treatment. Still, the conclusions are limited at laboratory research on pigs and mice, and the safety evaluates are absence in some research, which confined the clinical research and further utilizing.

Therapeutic Strategy and Clinical Path of Facioscapulohumeral Muscular Dystrophy: Review of the Current Literature

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant genetic disease, which is caused by the mistaken expression of double homeobox protein 4 protein 4 (DUX4) in skeletal muscle. Patients with FSHD are usually accompanied by degenerative changes in the face, shoulders, and upper muscles, gradually accumulating in the lower limb muscles. The severity of patients is quite different, and most patients end up using wheelchairs and losing their self-care ability. At present, the exploration of treatment strategies for FSHD has shifted from relieving symptoms to gene therapy, which brings hope to the future of patients, but the current gene therapy is only in the clinical trial stage. Here, we conducted a comprehensive search of the relevant literature using the keywords FSHD, DUX4, and gene therapy methods including ASOs, CRISPR, and RNAi in the PubMed and Web of Science databases. We discussed the current advancements in treatment strategies for FSHD, as well as ongoing preclinical and clinical trials related to FSHD. Additionally, we evaluated the advantages and limitations of various gene therapy approaches targeting DUX4 aimed at correcting the underlying genetic defect.

Surfactants as a means of delivering a reporter genetic construct based on binase suicide gene to tumor cells

Among modern gene therapy methods for combating oncology, suicidal gene therapy based on the delivery of a cytotoxic agent to target cells is of particular importance and promise. As one of such genes, the gene for ribonuclease of Bacillus pumilus 7P, binase, can be considered; the enzyme has a high antitumor potential and low immunogenicity. In addition to the choice of a transgene, another factor influencing the effectiveness of gene therapy is the method of delivering the nucleic acid to target cells. Surfactants have high functional activity and are promising means of delivering therapeutic nucleic acids. The aim of this work was to evaluate the possibility of using geminal surfactants as a means of delivering a genetic construct based on the cytotoxic binase gene into tumor cells. To optimize the transfection conditions, a reporter genetic construct carrying the binase gene fused to the gene for the green fluorescent protein TurboGFP was created, which made it possible to evaluate the delivery efficiency by the fluorescence intensity. To eliminate the toxic effect of binase on recipient cells, the RNase inhibitor gene, barstar, was introduced into the genetic construct. A high complexing ability of geminal surfactants in relation to the reporter system was shown by methods of dynamic light scattering and fluorescence spectroscopy. For surfactant 16-6-16OH, the highest transfecting activity together with a low level of cytotoxicity was found. Thus, the study proved the possibility of using geminal surfactants for the delivery of therapeutic nucleic acids to target cells.

Catecholaminergic polymorphic ventricular tachycardia (CPVT): an insidious disease that can often lead to sudden cardiac death in young people

The first symptoms of catecholaminergic polymorphic ventricular tachycardia (CPVT) usually occur in childhood and adolescence. 60% of patients experience syncope before the age of 40. Sudden cardiac death (SCD) is the first symptom of the disease in 30-50% of patients with CPVT. Early diagnosis is therefore crucial for the patient's prognosis. The diagnosis of CPVT is confirmed by a normal resting ECG, exclusion of structural heart disease, detection of bidirectional or polymorphic ventricular tachycardia (VT) in the stress ECG and/or detection of a pathogenic mutant in a gene associated with CPVT. Up to 60% of CPVT patients carry changes in the RYR2 gene. This gene encodes the cardiac ryanodine receptor, the most important Ca2+-releasing channel of the sarcoplasmic reticulum, which plays a central role in the contraction and relaxation of the heart muscle. If the function of the ryanodine receptor is impaired, too much calcium enters the cells, which triggers life-threatening arrhythmias. The overactive ryanodine receptor is therefore the main target for gene therapy methods. Even though the development of gene therapy is progressing, there is still no causal therapy available and it is all the more important to make a diagnosis as early as possible, which enables appropriate behavior and adequate symptomatic therapy. The decisive factor here is the evaluation of the genetic analysis in the context of the clinical findings. Based on this, recommendations can be made for preventive measures and the avoidance of specific triggers that could lead to life-threatening arrhythmias.

Current Landscape of Various Techniques and Methods of Gene Therapy through CRISPR Cas9 along with its Pharmacological and Interventional Therapies in the Treatment of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is frequently referred to as a "lifestyle illness". In 2000, India (31.7 million) had the greatest global prevalence of diabetes mellitus, followed by China (20.8 million), the United States (17.7 million), and other countries. In recent years, the treatment of gene therapy (T2DM) has attracted intensive interest. We aimed to critically review the literature on the various techniques and methods, which may be a possible novel approach through the gene therapy CRISPR Cas9 and some other gene editing techniques for T2DM. Interventional and pharmacological approaches for the treatment of T2DM were also included to identify novel therapies for its treatment. An extensive literature survey was done on databases like PubMed, Elsevier, Science Direct and Springer. It can be concluded from the study that recent advancements in gene-editing technologies, such as CRISPR Cas9, have opened new avenues for the development of novel therapeutic approaches for T2DM. CRISPR Cas9 is a powerful tool that enables precise and targeted modifications of the genome.

Somatic Gene Therapy in the Prevention of Toxic Effects of Organophosphate Agents

Medical intervention in poisoning by organophosphate toxic agents (OPA) using atropine sulfate, 2-pyridinaldoxymethyl chloride (2-PAM), diazepam and other similar drugs can prevent the fatal outcome of poisoning. These drugs do not protect in case of sudden chemical attack and against post-exposure complications associated with permanent brain damage. The U.S. Department of Defense is funding research that can significantly simplify the protection of military personnel from OPA damage in the future. Their essence is in the use of gene therapy technologies, which allow experimental animals to produce their own proteins that destroy OPA and provide them with protection for several months. The aim of the work is to identify the achieved level of knowledge in the research using gene therapy technologies to create living objects resistant to OPA. The research method is analytical. The source base of the research are publications in scientific journals and descriptions of patents. Discussion of the results. As an enzyme that breaks down OPA in such experiments, genetically modified paraoxanase 1 (PON1) showed the greatest efficiency. PON1 hydrolyzes G-type OPAs, paraoxone, chlorpyrifosoxone, diazoxone and several other organophosphates. Adenoassociated virus vectors (AAV8, etc.) were used to introduce the gene encoding PON1 into the animal's body. A single injection of AAV8 carrying the recombinant PON1-IF11 gene (AAV8-PON1-IF11) resulted in high expression and secretion of the recombinant PON1-IF11 protein into the bloodstream and provided asymptomatic protection against multiple lethal doses of G-type OPA for at least 5 months. These studies are still in their early stage. An analysis of the affiliation of the authors of publications and patents showed a high involvement of the U.S. military department and its cooperating organizations (DTRA, etc.) in such research. Conclusion. Given the fascination in the West with the ideas of human modification using gene therapy methods, this direction will be intensively developed for military purposes. At the same time, the idea of pre-created resistance to OPA is in demand by the widespread use of organophosphates in agriculture. The author believes that it would be safer to use allogeneic mesenchymal stem cells transfected with genetically modified PON1 variants with enhanced enzyme activity. This resistance to OP agents can be health protective and lifesaving in soldiers in real combat when the enemy uses these agents. However, this approach must be based on a strong experimental background. The door is open, the technologies are available.

Modified mRNA-Mediated CCN5 Gene Transfer Ameliorates Cardiac Dysfunction and Fibrosis without Adverse Structural Remodeling.

Modified mRNAs (modRNAs) are an emerging delivery method for gene therapy. The success of modRNA-based COVID-19 vaccines has demonstrated that modRNA is a safe and effective therapeutic tool. Moreover, modRNA has the potential to treat various human diseases, including cardiac dysfunction. Acute myocardial infarction (MI) is a major cardiac disorder that currently lacks curative treatment options, and MI is commonly accompanied by fibrosis and impaired cardiac function. Our group previously demonstrated that the matricellular protein CCN5 inhibits cardiac fibrosis (CF) and mitigates cardiac dysfunction. However, it remains unclear whether early intervention of CF under stress conditions is beneficial or more detrimental due to potential adverse effects such as left ventricular (LV) rupture. We hypothesized that CCN5 would alleviate the adverse effects of myocardial infarction (MI) through its anti-fibrotic properties under stress conditions. To induce the rapid expression of CCN5, ModRNA-CCN5 was synthesized and administrated directly into the myocardium in a mouse MI model. To evaluate CCN5 activity, we established two independent experimental schemes: (1) preventive intervention and (2) therapeutic intervention. Functional analyses, including echocardiography and magnetic resonance imaging (MRI), along with molecular assays, demonstrated that modRNA-mediated CCN5 gene transfer significantly attenuated cardiac fibrosis and improved cardiac function in both preventive and therapeutic models, without causing left ventricular rupture or any adverse cardiac remodeling. In conclusion, early intervention in CF by ModRNA-CCN5 gene transfer is an efficient and safe therapeutic modality for treating MI-induced heart failure.

Exploring translational approaches for fully biological cardiac rhythm restoration through ion channel gene therapy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (Starting grant 716509) to D.A. Pijnappels Background Recently it was demonstrated how the heart itself could be enabled to quickly restore its rhythm by realizing a biologically-integrated cardiac defibrillator (BioICD) through modification and subsequent expression of ion channels in cardiomyocytes [1]. By incorporating these frequency-dependent depolarizing ion channels, abnormal cardiac rhythm could be rapidly detected and terminated to restore sinus rhythm in a fully biological and shock-free manner. However, from a translational point of view, it remains unclear how such rhythm restoration can be realized via ion channel gene therapy. Purpose To explore and understand the importance of the distribution and number of BioICD-expressing cardiomyocytes in realizing fully biological restoration of cardiac rhythm. Methods To this purpose, two different realistic gene therapy configurations, i.e. those corresponding to systemic and local transgene delivery, were tested in a digital twin of human ventricular cardiac monolayers. In the systemic delivery group, BioICD-expressing cells were uniformly distributed over the tissue at different total expression percentages. In the local delivery group, circular areas were populated with BioICD-expressing cells, randomly arranged in a Gaussian distribution. Spiral waves were initiated in both groups and studied for 10 seconds. An additional set of simulations for the systemic delivery group involved an adapted BioICD ion channel with a slower opening and faster closing rate. Results Upon comparing both gene therapy methods, the systemic approach showed a gradually increasing arrhythmia termination chance with increasing BioICD-expression percentage, while local delivery did not result in termination. Instead, local delivery resulted in islands of ionic heterogeneity, causing attraction and anchoring of the spiral waves in a size and distance-dependent manner. Building on the results of the systemic approach, different ion channel parameters were tried, which resulted in normal rhythm being restored in all cases for >50% BioICD expressing cells. Time till termination was inversely related to the percentage, resulting in only 4.3s and 2.5s for 50% and 100%, respectively. Regarding termination, it was observed that conduction blocks appeared throughout the tissue and subsequently connected to force arrhythmic waves to terminate, while this process remained incomplete in the <50% groups. Conclusion This study reveals that wide-spread distribution of BioICD-expressing cardiomyocytes is required for the realization of fully biological self-restoration of cardiac rhythm, of which the efficiency is ion-channel-dynamics- and dosage-dependent. Local expression, however, results in stabilization of spiral wave activity. Further exploration of this emerging concept of biological cardioversion may not only expand our understanding of cardiac arrhythmias, but also pave the way to breakthrough advances in arrhythmia management.

Translational exploration of fully biological restoration of cardiac rhythm via ion channel gene therapy

Abstract Background Recently it was demonstrated how the heart itself could be enabled to quickly restore its rhythm by realizing a biologically-integrated cardiac defibrillator (BioICD) through modification and subsequent expression of ion channels in cardiomyocytes [1]. By incorporating these frequency-dependent depolarizing ion channels, abnormal cardiac rhythm could be rapidly detected and terminated to restore sinus rhythm in a fully biological and shock-free manner. However, from a translational point of view, it remains unclear how such rhythm restoration can be realized via ion channel gene therapy. Purpose To explore and understand the importance of the distribution and number of BioICD-expressing cardiomyocytes in realizing fully biological restoration of cardiac rhythm. Methods To this purpose, two different realistic gene therapy configurations, i.e. those corresponding to systemic and local transgene delivery, were tested in a digital twin of human ventricular cardiac monolayers. For the systemic delivery group, BioICD-expressing cells were homogeneously distributed over the tissue with fixed total expression percentage. For the local delivery group, circular areas were given BioICD-expressing cells, randomly patterned in a Gaussian distribution. In both groups spiral waves were initiated and subsequently studied for 10 seconds. For systemic delivery, an additional set of simulations was performed for an adapted BioICD ion channel with a slower opening and faster closing rate. Results Upon comparing both gene therapy methods, the systemic approach showed a gradually increasing arrhythmia termination chance with increasing BioICD-expression percentage, while local delivery did not result in termination. Instead, local delivery resulted in islands of ionic heterogeneity, causing attraction and anchoring of the spiral waves in a size and distance-dependent manner. Building on the results of the systemic approach, different ion channel parameters were tried, which resulted in normal rhythm being restored in all cases for >50% BioICD expressing cells. Time till termination was inversely related to the percentage, resulting in only 4.3s and 2.5s for 50% and 100%, respectively. Regarding termination, it was observed that conduction blocks appeared throughout the tissue and subsequently connected to force arrhythmic waves to terminate, while this process remained incomplete in the <50% groups. Conclusion This study reveals that wide-spread distribution of BioICD-expressing cardiomyocytes is required for the realization of fully biological self-restoration of cardiac rhythm, of which the efficiency is ion-channel-dynamics- and dosage-dependent. Local expression, however, results in stabilization of spiral wave activity. Further exploration of this emerging concept of biological cardioversion may not only expand our understanding of cardiac arrhythmias, but also pave the way to breakthrough advances in arrhythmia management.

#1118 Direct delivery of novel gene therapy to podocytes enables pathway to clinical translation for the treatment of glomerular diseases

Abstract Background and Aims Adeno associated vector (AAV) gene therapy has the potential to treat a wide range of kidney diseases via the targeted delivery of genes to kidney cells to repair cellular and renal function. Despite the potential to impact many kidney conditions, kidney AAV gene therapy has proven challenging due to technical and physiological hurdles limiting the ability of AAV to be effectively delivered to podocytes. We have developed a novel AAV gene therapy, PS-001, and local method of administration, to effectively and safely deliver the podocin gene NPHS2 to podocytes. Biallelic NPHS2 mutations are the most common cause of monogenic childhood nephrotic syndrome. Curative efficacy and resolution of disease phenotype was demonstrated in transgenic murine models following PS-001 gene therapy treatment. The potential for translation of this approach to NPHS2 nephropathy patients in the clinic was also demonstrated in pre-clinical studies in Gottingen Minipigs. Method Transgenic murine models representative of NPHS2-driven nephropathy were treated with an AAV gene therapy encoding NPHS2, including a knock in mouse model (Nphs2R140Q/−) which introduces a R140Q mutation in the podocin gene. Mice were assessed for improvement in disease phenotype via analysis of proteinuria, serum albumin, renal histopathology and survival. Mice were treated with an analogue of PS-001, as the PS-001 capsid is not permissive in rodents. Gottingen minipigs were treated with PS-001 and followed for 4 weeks. Clinically relevant doses and a procedure for local administration specifically to the kidney were established. In-life safety assessments from blood and urine were monitored and at study end pig tissues were assessed for podocin biodistribution using multiple assays (qPCR, RT-qPCR, ELISA, RNAScope and IF) and for toxicological effects via histopathological analysis. Results Using our AAV gene therapy platform, which includes a capsid that is highly effective at transducing human podocytes and promoter that drives gene expression specifically in the podocyte, the gene therapy product PS-001 encoding NPHS2 was generated. Using a murine analogue of PS-001, in vivo proof of concept studies in a translationally relevant murine disease model (Nphs2R140Q/−) showed NPHS2 delivery and podocin expression in glomeruli and efficacy in terms of renal function rescue. This was demonstrated by resolution of severe proteinuria (mean ACR ug/mg 6049 ± 2803 vs 192 ± 51 in PS-001 treated animals, n = 11), improved serum albumin (g/L 27.50 ± 3.33 g/L vs 30.75 ± 2.46 in PS-001 treated animals, P = .021) and reduction in glomerulosclerosis (mean sclerosed glomeruli 81% ± 19 vs 18.75% ± 12 in PS-001 treated animals, P < .0001). The methods to translate the approach to the clinical setting using a local administration were established in pigs. Administration of PS-001 resulted in transgene mRNA expression across 89% ± 11% of kidney glomeruli as assessed by RNAScope (n = 3 pigs), and transgene derived podocin could be specifically detected in podocytes as assessed by IF analysis. Using qPCR, RT-PCR and ELISA it was shown that using local administration, podocin gene expression was restricted exclusively to the kidney glomerulus, with gene expression undetectable in off-target organs including the liver. Additional toxicological assessments of renal and other organ function from urine and blood, and post-study histopathological assessments revealed no pre-clinical treatment-related safety concerns. Conclusion We present data showing development of an AAV gene therapy and method of administration for the delivery of transgenes specifically to podocytes. We have shown transgenes can be efficiently targeted to podocytes to replace defective genes or to use the podocyte as a protein factory to modulate glomerular biology. This technology therefore provides an important novel modality for potentially treating a broad range of glomerular disease. PS-001 is being developed as a novel approach to treat steroid resistant nephrotic syndrome caused by mutated NPHS2, and may become the first podocyte targeted gene therapy to enter clinical development.

Chromosomal aberration analysis: Novel noninvasive techniques for early-stage cancer screening

ObjectiveChromosome breakage is a catastrophic event that leads to the progressive development and progression of cancer. In order to analyze the changes of peripheral blood microenvironment of tumor patients, to explore the indicators of non-specific non-invasive tumor early screening. This paper presents a new idea of whether the gene sequence near the DNA damage break point is the gene sequence that controls the unrestricted growth of normal cells. MethodsThe chromosomal aberrations of peripheral blood lymphocytes were analysed in 60 healthy adult and 49 cancer patients before radiotherapy. ResultsThe detection rate of chromosomal aberrations was high in tumor patients, and “dicentric + translocations” of chromosomes were detected in 36 patients (73.47 %). The chi-square test showed statistically significant differences (P < 0.01), and chromosome adhesion and dissolution were observed. Conclusions“Dicentric + Translocation” chromosome can be used as a nonspecific early screening indicator for cancer. This is worthy of further study. This index can be used to determine the genetic basis of various cancers at the gene level to modify the base sequence and prevent the occurrence of cancer. It is worthy of further study, and it can provide a new method for gene therapy of tumors.