Paying attention to the natural course of disease for a development of gene therapy of inherited retinal diseases / 中华实验眼科杂志

Most inherited retinal diseases (IRDs) severely impair vision and lack effective treatments.With the approval of Luxturna, the world's first gene therapy drug for IRDs in 2017 by the U. S.FDA, gene therapy has brought new hope for the treatment of the disease.With an early onset and a relatively small number of patients, the understanding of the natural course of IRDs is limited in the past.The research on gene therapy of IRDs is mainly based on the in-depth understanding of the pathogenesis and natural course of disease, and the selection of the optimal treatment window for the implementation of gene therapy is the premise of successful treatment.At the same time, the main vector for gene therapy is recombinant virus vector, and its tissue-immunogenicity, tumorigenicity, safety of its integration with host cells and effectiveness determine the outcome of therapy, so the evaluation technology of IRDs gene therapy needs to be established.Gene therapy for ophthalmic diseases also involves the consideration of laws and regulations, ethics, product process, races and regional environment, disease progression, gene mutation types, patient benefit and risk ratio, and other factors.Therefore, it is of great significance to take full account of the differences in IRDs population, especially the particularity of children patients, and actively carry out the study on the natural course of IRDs in China for the scientific and normative development of clinical trials of gene therapy, the effective establishment of endpoint and outcome indicators for clinical studies of gene therapy, and the compliance with international norms of ethics.

Cystic fibrosis gene therapy / 国际儿科学杂志

Cystic fibrosis(CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator(CFTR) gene that encodes a cAMP-regulated anion channel.Although CF is a multi-organ system disease, most people with CF die of progressive lung disease that begins early in childhood and is characterized by chronic bacterial infection and inflammation.Nearly 90% of people with CF have at least one copy of the ΔF508 mutation, but there are hundreds of CFTR mutations that result in a range of disease severities.A CFTR gene replacement approach would be efficacious regardless of the disease-causing mutation.After the discovery of the CFTR gene in 1989, the in vitro proof-of-concept for gene therapy for CF was quickly established in 1990.In 1993, the first of many gene therapy clinical trials attempted to rescue the CF defect in airway epithelia.Despite the initial enthusiasm, there is still no FDA-approved gene therapy for CF.Here we discuss the history of CF gene therapy, from the discovery of the CFTR gene to current state-of-the-art gene delivery vector designs.While implementation of CF gene therapy has proven more challenging than initially envisioned; thanks to continued innovation, it may yet become a reality.

Current Trends in Viral Gene Therapy for Human Orthopaedic Regenerative Medicine

BACKGROUND: Viral vector-based therapeutic gene therapy is a potent strategy to enhance the intrinsic reparative abilities of human orthopaedic tissues. However, clinical application of viral gene transfer remains hindered by detrimental responses in the host against such vectors (immunogenic responses, vector dissemination to nontarget locations). Combining viral gene therapy techniques with tissue engineering procedures may offer strong tools to improve the current systems for applications in vivo. METHODS: The goal of this work is to provide an overview of the most recent systems exploiting biomaterial technologies and therapeutic viral gene transfer in human orthopaedic regenerative medicine. RESULTS: Integration of tissue engineering platforms with viral gene vectors is an active area of research in orthopaedics as a means to overcome the obstacles precluding effective viral gene therapy. CONCLUSION: In light of promising preclinical data that may rapidly expand in a close future, biomaterial-guided viral gene therapy has a strong potential for translation in the field of human orthopaedic regenerative medicine.

Cystic fibrosis gene therapy / 国际儿科学杂志

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cAMP-regulated anion channel.Although CF is a multi-organ system disease,most people with CF die of progressive lung disease that begins early in childhood and is characterized by chronic bacterial infection and inflammation.Nearly 90％ of people with CF have at least one copy of the △F508 mutation,but there are hundreds of CFTR mutations that result in a range of disease severities.A CFTR gene replacement approach would be efficacious regardless of the diseasecausing mutation.After the discovery of the CFTR gene in 1989,the in vitro proof-of-concept for gene therapy for CF was quickly established in 1990.In 1993,the first of many gene therapy clinical trials attempted to rescue the CF defect in airway epithelia.Despite the initial enthusiasm,there is still no FDA-approved gene therapy for CF.Here we discuss the history of CF gene therapy,from the discovery of the CFTR gene to current state-of-the-art gene delivery vector designs.While implementation of CF gene therapy has proven more challenging than initially envisioned;thanks to continued innovation,it may yet become a reality.

Los vectores virales y su relación con la bioseguridad en el laboratorio / Viral vectors and their relationship with biosafety in the laboratory / Vetores virais e sua relação com a biossegurança no laboratório

Los vectores virales constituyen un amplio grupo de agentes usados en los laboratorios de biología molecular y microbiología, para transferir ácido nucleico externo dentro de una célula "blanco". Estos laboratorios requieren medidas de bioseguridad para proteger al personal, el ambiente de trabajo y la población en general de exposiciones no intencionales a estos agentes biológicos. Esta revisión incluye definiciones y generalidades sobre los vectores más usados, así como consideraciones sobre los niveles de bioseguridad necesarios, teniendo en cuenta tanto la constitución genética del vector original, como el tipo de inserto que se quiera clonar y expresar en una determinada célula. Además se describen diferentes propiedades de los vectores como el tropismo, las distintas formas de transmisión, la estabilidad del agente viral y su persistencia, que resultan claves para determinar el grupo de riesgo. Dentro de las condiciones de trabajo adecuadas se incluyen medidas de contención, ensayos de riesgo ambiental y una breve descripción de la biocustodia. Finalmente se destaca el papel de los Comités de Bioseguridad Institucional, como elemento crítico en las actividades necesarias para impedir las exposiciones y proteger al personal del laboratorio y al medio ambiente.

Viral vectors are a broad group of agents used in molecular biology and microbiology laboratories to transfer exogen nucleic acid. These labs require biosafety measures designed to protect their staff, the population and the environment that may be unintentionally exposed to hazardous organisms. This review includes general properties of known vectors, variations in biosafety levels in relation to original vector genetics and the type of insert to be cloned and expressed in a determined cell. It is also important to consider other issues such as transmission, stability and persistence to determine the vector risk group. Under adequate working conditions, containment measures, risk assays and a short description on biosecurity are included. Finally the function of Institutional Biosafety Committees is a critical element to advise on measures needed to prevent exposures and protect laboratory workers and the environment.

Os vetores virais constituem um amplo grupo de agentes utilizados nos laboratórios de biologia molecular e microbiologia para transferir ácido nucleico externo para uma célula "alvo". Esses laboratórios exigem medidas de biossegurança para proteger o pessoal, o ambiente de trabalho e a população em geral de exposições não intencionais a esses agentes biológicos. Esta revisão inclui definições e generalidades sobre os vetores mais comumente usados, bem como considerações sobre os níveis de biossegurança necessários, levando em consideração a constituição genética do vetor original e o tipo de enxerto a ser clonado e expresso numa célula específica. Além disso, são descritas diferentes propriedades dos vetores, como o tropismo, as diferentes formas de transmissão, a estabilidade do agente viral e a sua persistência, dados-chave para a determinação do grupo de risco. As condições de trabalho adequadas incluem medidas de contenção, testes de risco ambiental e uma breve descrição da biocustódia. Finalmente, o papel das Comissões Institucionais de Biossegurança é destacado como um elemento crítico nas atividades necessárias para prevenir exposições e para proteger o pessoal de laboratório e o meio ambiente.

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Cholesterol improves the transfection efficiency of polyallylamine as a non-viral gene delivery vector

ABSTRACT Cationic polymers such as polyallylamine (PAA) having primary amino groups are poor transfection agents and possess a high cytotoxicity index when used without any chemical modification. In this study, PAA was modified with cholesterol in order to improve transfection efficiency and to reduce cytotoxicity. PAA polymers with molecular weights of 15 and 65 kDa were selected and grafted with cholesterol at percentages of 5, 10, 15, 30, and 50. After purification, the efficacy of the synthetic vectors was evaluated in terms of DNA condensation using the ethidium bromide test, buffering capacity, particle size, zeta potential, transfection efficiency, and cytotoxicity assay in Neuro2A cell lines. According to the ethidium bromide test, these vectors can condense DNA at moderate and high carrier to plasmid (C/P) ratios. The buffering capacity of the prepared vector in both molecular weights was less than unmodified PAA. Particle size measurements demonstrated that modified PAAs were able to form nanoparticles ranging in size from 125 to 530 nm. The vectors based on PAA 15 kDa demonstrated a better transfection efficiency than the vectors made of PAA 65 kDa. Cytotoxicity studies showed that toxicity of all vectors was less than PAA. Some cholesterol modified polymers composed of PAA (15 kDa) were suitable vectors for gene delivery with low cytotoxicity.

The design of non-viral nanometer vectors for delivery of small interfering RNA / 国际药学研究杂志

RNA interference (RNAi) is a method that allows double-stranded RNA (dsRNA) to induce the target gene mRNA to degrade in the body and lead to different levels of gene silencing. Small interfering RNA (siRNA) is the effector molecules of RNAi, and needs the delivery vehicle into the cells to play a therapeutic role. Nanoscale carrier can adjust the rate of drug release, increase permeability of biofilm, change distribution in the body, and improve bioavailability. In this manuscript, materials, structure and biological characteristics of existing non-viral nanometer vectors delivery for siRNA are summarized.

The design of non-viral nanometer vectors for delivery of small interfering RNA / 国际药学研究杂志

RNA interference(RNAi)is a method that allows double-stranded RNA(dsRNA)to induce the target gene mRNA to degrade in the body and lead to different levels of gene silencing. Small interfering RNA(siRNA)is the effector molecules of RNAi, and needs the delivery vehicle into the cells to play a therapeutic role. Nanoscale carrier can adjust the rate of drug release ,increase permeability of biofilm,change distribution in the body,and improve bioavailability. In this manuscript,materials,structure and bio?logical characteristics of existing non-viral nanometer vectors delivery for siRNA are summarized.

Non-viral vectors for targeted therapy of prostate cancer: Recent advance / 第二军医大学学报

The current therapies for treating prostate cancer, in many cases, also cause damage to normal tissues and organs at the same time when killing cancer cells. The recent development of targeted therapy to some extent, may be a good solution to this problem. As vectors in targeted therapy, viral vectors have strong immunogenicity, toxicity and other disadvantages, and non-viral vectors for targeted therapy of prostate cancer may overcome these drawbacks. This review summarized the recent application of cationic polymers, liposomes and polymeric chitosan as non-viral vectors of gene drug for targeted therapy of prostate cancer.

Gene therapy in India: A focus.

Gene therapy refers to the treatment of genetic diseases using normal copies of the defective genes. It has the potential to cure any genetic disease with long-lasting therapeutic benefits. It remained an enigma for a long period of time, which was followed by a series of setbacks in the late 1990s. Gene therapy has re-emerged as a therapeutic option with reports of success from recent clinical studies. The United States and Europe has been pioneers in this field for over two decades. Recently, reports of gene therapy have started coming in from Asian countries like China, Japan and Korea. This review focuses on the current status of gene therapy in India.

Delivery systems for gene therapy.

The structure of DNA was unraveled by Watson and Crick in 1953, and two decades later Arber, Nathans and Smith discovered DNA restriction enzymes, which led to the rapid growth in the field of recombinant DNA technology. From expressing cloned genes in bacteria to expressing foreign DNA in transgenic animals, DNA is now slated to be used as a therapeutic agent to replace defective genes in patients suffering from genetic disorders or to kill tumor cells in cancer patients. Gene therapy provides modern medicine with new perspectives that were unthinkable two decades ago. Progress in molecular biology and especially, molecular medicine is now changing the basics of clinical medicine. A variety of viral and non-viral possibilities are available for basic and clinical research. This review summarizes the delivery routes and methods for gene transfer used in gene therapy.

HSV-1-derived amplicon vectors: recent technological improvements and remaining difficulties: a review

Amplicons are defective and non-integrative vectors derived from herpes simplex virus type 1. As the vector genome carries no virus genes, amplicons are both non-toxic for the infected cells and non-pathogenic for the inoculated organisms. In addition, the large transgenic capacity of amplicons, which allow delivery of up to 150 Kbp of foreign DNA, makes these vectors one of the most powerful, interesting and versatile gene delivery platforms. We present here recent technological developments that have significantly improved and extended the use of amplicons, both in cultured cells and in living organisms. In addition, this review also discusses the many difficulties still pending to be solved, in order to achieve stable and physiologically regulated transgene expression.

A potencialidade dos lentivetores na terapia gênica / The potentiality of lentivectors in gene therapy

Os lentivírus, bem como os oncovírus e os spumavírus, pertencem à família Retroviridae. Essa família possui genoma composto por duas fitas simples de RNA e enzima transcriptase reversa. Os vírus de imunodeficiência humana (HIV), felina (FIV) e simiana (SIV) são os representantes mais bem conhecidos dos lentivírus, devido aos seus efeitos patológicos. O genoma dos lentivírus apresenta uma organização mais complexa do que dos oncovírus e muitos dos seus processos moleculares já estão descritos OU já são conhecidos. Além disso, são capazes de infectar inclusive células quiescentes, características essa que se opõe ao tropismo dos oncovírus. Essas propriedades levaram ao desenvolvimento de vetores lentivirais para terapia gênica e a perspectiva do emprego terapêutico dos mesmos na clínica – por exemplo, para síndrome da imunodeficiência adquirida (SIDA). Uma série de modificações de engenharia genética no lentivetor baseado em HIV levou ao desenvolvimento do primeiro protocolo clínico de terapia gênica lentiviral contra a SIDA, nos EUA. Atualmente, existem vários protocolos clínicos com lentivetores em andamento, seguindo a tendência de empregá-los também em outras doenças.(AU)

Lentivirus, oncovirus and spumavirus belong to the Retroviridae family. The genome of this family is composed of two single strands of RNA and reverse transcriptase enzyme. The human, feline and simian immunodeficiency virus (HIV, FIV, SIV) are well known lentivirus in our society due to their pathological effects. The Lentiviral genome is more complex than the oncovira and much of its molecular mechanisms are already known. In addition, they are able to infect also quiescent cells, in opposition to oncovirus tropism. These properties lead to the development of gene therapy lentiviral vectors and to their possible therapeutic use at clinical level – acquired immunodeficiency syndrome (AIDS), for example. After intense genetic engineering, a HIV based lentivector was first used in a clinical trial in a gene therapy protocol for AIDS, in USA. Currently, there are several clinical trials in progress using lenviral vectors, following the tendency of evaluating their potential to treat many other diseases.(AU)

Terapia genica:pperspectivas y consideraciones eticas en relacion con su aplicacion / Genetic therapy: Perspectives and ethical considerations of its application

La Terapia Génica es una metodología que aborda la inserción de material genético en un individuo para tratar una enfermedad ya sea de forma directa (in vivo) o indirectamente, a través del uso de células como vehículo de liberación (ex vivo). La aplicación de este procedimiento conlleva la aparición de riesgos, por lo cual ha despertado un gran dilema ético. Hasta el momento, en humanos, solo se ha practicado la terapia somática y a pesar de que se ha avanzado considerablemente en las investigaciones y ensayos, aún existen problemas que limitan su uso. Tal es el caso del empleo de vectores virales que pueden causar inflamación y toxicidad en el tejido hospedero. La posibilidad de aplicación de la Terapia Génica depende de múltiples factores como son:tipo y patrón de herencia, tipo de mutación, tamaño del gen,control génico ytejido donde se manifieste la enfermedad.A pesar de las dificultades que aún se presentan, existe un consenso a favor de la aplicación de este procedimiento, siempre y cuando los beneficios sean mayores que los riesgos. La terapia germinal ha sido rechazada por muchos científicos porque sus ventajas no compensan los peligros asociados a la misma, además de que existen alternativas terapéuticas con el mismo potencial y que no comparten los mismos riesgos. Hasta el momento, se han realizado varios protocolos clínicos de Terapia Génica y los resultados han sido prometedores lo que posibilita la continuación de su aplicación en un futuro inmediato.

Genetic therapy is a methodology that approaches the incorporation of genetic material in an individual to treat a disease by direct way (in vivo) or in an indirect way through the use of cells as a vehicle of liberation (ex vivo). This procedure's application carries on the onset of risks; therefore it has awakened a great ethical dilemma. So far, in humans, only somatic practice therapy has been done and even when great advances have been achieved in research and assays, there are still limiting problems for its use. That is the case of viral vectors that can cause inflammation and toxicity in the host. The possibility of genetic therapy application, depend on numerous factors such as: type and pattern of inheritance, mutation type, gene size, genetic control and the tissue where the disease is patent. In spite of the still present difficulties, there is a consensus in favour of this procedure's application as long as the benefits are greater than risks. Germinal therapy has been rejected by many scientists because its advances do not compensate the associated dangers, besides; there are better therapeutic alternatives without the risks. So far, several clinical protocols of genetic therapy have been done and they show promising results, which allow the continuation of its application in an immediate future.