scholarly journals Gene Therapy in Rare Respiratory Diseases: What Have We Learned So Far?

2020 ◽  
Vol 9 (8) ◽  
pp. 2577 ◽  
Author(s):  
Lucía Bañuls ◽  
Daniel Pellicer ◽  
Silvia Castillo ◽  
María Mercedes Navarro-García ◽  
María Magallón ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Respiratory Diseases ◽  
Viral Vectors ◽  
Gene Editing ◽  
Alternative Therapy ◽  
Transcription Activator ◽  
Gene Therapy Approach ◽  
Gene Therapy Application ◽  
Alpha 1 Antitrypsin Deficiency

Gene therapy is an alternative therapy in many respiratory diseases with genetic origin and currently without curative treatment. After five decades of progress, many different vectors and gene editing tools for genetic engineering are now available. However, we are still a long way from achieving a safe and efficient approach to gene therapy application in clinical practice. Here, we review three of the most common rare respiratory conditions—cystic fibrosis (CF), alpha-1 antitrypsin deficiency (AATD), and primary ciliary dyskinesia (PCD)—alongside attempts to develop genetic treatment for these diseases. Since the 1990s, gene augmentation therapy has been applied in multiple clinical trials targeting CF and AATD, especially using adeno-associated viral vectors, resulting in a good safety profile but with low efficacy in protein expression. Other strategies, such as non-viral vectors and more recently gene editing tools, have also been used to address these diseases in pre-clinical studies. The first gene therapy approach in PCD was in 2009 when a lentiviral transduction was performed to restore gene expression in vitro; since then, transcription activator-like effector nucleases (TALEN) technology has also been applied in primary cell culture. Gene therapy is an encouraging alternative treatment for these respiratory diseases; however, more research is needed to ensure treatment safety and efficacy.

scholarly journals TALEN Mediated Gene Targeting for CF Gene Therapy

2018 ◽  
Author(s):  
Emily Xia ◽  
Yiqian Zhang ◽  
Huibi Cao ◽  
Jun Li ◽  
Rongqi Duan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Airway Epithelial Cells ◽  
In Vivo Studies ◽  
Lacz Reporter ◽  
Gene Correction ◽  
Transcription Activator ◽  
Stringent Requirement

Cystic Fibrosis (CF) is an inherited monogenic disorder, amenable to gene based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human CFTR minigene into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we have detected both CFTR mRNA and protein expression by qPCR and Wetern analysis, respectively. We have also confirmed CFTR function correction by flurometric Image Plate Reader (FLIPR) and iodide efflux assays. Taking together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.

scholarly journals Potential of helper-dependent Adenoviral vectors in CRISPR-cas9-mediated lung gene therapy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ranmal Avinash Bandara ◽  
Ziyan Rachel Chen ◽  
Jim Hu
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Lung Disease ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genetic Diseases ◽  
Adenoviral Vectors ◽  
The Status

AbstractSince CRISPR/Cas9 was harnessed to edit DNA, the field of gene therapy has witnessed great advances in gene editing. New avenues were created for the treatment of diseases such as Cystic Fibrosis (CF). CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Despite the success of gene editing with the CRISPR/Cas9 in vitro, challenges still exist when using CRISPR/Cas9 in vivo to cure CF lung disease. The delivery of CRISPR/Cas9 into lungs, as well as the difficulty to achieve the efficiency required for clinical efficacy, has brought forth new challenges. Viral and non-viral vectors have been shown to deliver DNA successfully in vivo, but the sustained expression of CFTR was not adequate. Before the introduction of Helper-Dependent Adenoviral vectors (HD-Ad), clinical trials of treating pulmonary genetic diseases with first-generation viral vectors have shown limited efficacy. With the advantages of larger capacity and lower immunogenicity of HD-Ad, together with the versatility of the CRISPR/Cas9 system, delivering CRISPR/Cas9 to the airway with HD-Ad for lung gene therapy shows great potential. In this review, we discuss the status of the application of CRISPR/Cas9 in CF gene therapy, the existing challenges in the field, as well as new hurdles introduced by the presence of CRISPR/Cas9 in the lungs. Through the analysis of these challenges, we present the potential of CRISPR/Cas9-mediated lung gene therapy using HD-Ad vectors with Cystic Fibrosis lung disease as a model of therapy.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

Gene editing as a promising approach for respiratory diseases

2018 ◽  
Vol 55 (3) ◽  
pp. 143-149 ◽  
Author(s):  
Yichun Bai ◽  
Yang Liu ◽  
Zhenlei Su ◽  
Yana Ma ◽  
Chonghua Ren ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Gene Editing ◽  
Gene Mutations ◽  
Well Being ◽  
Short Review ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Mortality And Morbidity ◽  
Chronic Respiratory Diseases ◽  
Difficulty Breathing

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations. Clinically, a single pulmonary disease can exhibit more than one phenotype or coexist with multiple organ disorders. To correct abnormal function or repair injured respiratory tissues, one of the most promising techniques is to correct mutated genes by gene editing, as some gene mutations have been clearly demonstrated to be associated with genetic or heterogeneous respiratory diseases. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are three innovative gene editing technologies developed recently. In this short review, we have summarised the structure and operating principles of the ZFNs, TALENs and CRISPR/Cas9 systems and their preclinical and clinical applications in respiratory diseases.

80 Biallelic CRISPR-Cas9 editing of gene associated with coat colour in microinjected bovine zygotes reaching the blastocyst stage

2019 ◽  
Vol 31 (1) ◽  
pp. 165
Author(s):  
M. Poirier ◽  
D. Miskel ◽  
F. Rings ◽  
K. Schellander ◽  
M. Hoelker
Keyword(s):  
Gene Editing ◽  
Fluorescent Protein ◽  
Survival Rates ◽  
Coat Colour ◽  
Blastocyst Stage ◽  
Transcription Activator ◽  
Guide Rna ◽  
Blastocyst Rate ◽  
Copa Gene

Successful genome editing of blastocysts using zygote microinjection with transcription activator-like effector nucleases has already been accomplished in cattle as well as a limited number of CRISPR-Cas9 microinjections of zygotes, mostly using RNA. Recent editing of the Pou5f1 gene in bovine blastocysts using CRISPR-Cas9, clarifying its role in embryo development, supports the viability of this technology to produce genome edited cattle founders. To further this aim, we hypothesise that editing of the coatomer subunit α (COPA) gene, a protein carrier associated with the dominant red coat colour phenotype in Holstein cattle, is feasible through zygote microinjection. Here, we report successful gene editing of COPA in cattle zygotes reaching the blastocyst stage, a necessary step in creating genome edited founder animals. A single guide RNA was designed to target the sixth exon of COPA. Presumptive zygotes derived from slaughterhouse oocytes by in vitro maturation and fertilization were microinjected either with the PX458 plasmid (Addgene #48138; n=585, 25ng µL−1) or with a ribonucleoprotein effector complex (n=705, 20, 50, 100, and 200ng µL−1) targeting the sixth exon of COPA. Plasmid injected zygotes were selected for green fluorescent protein (GFP) fluorescence at Day 8, whereas protein injected zygotes were selected within 24h post-injection based on ATTO-550 fluorescence. To assess gene editing rates, single Day 8 blastocysts were PCR amplified and screened using the T7 endonuclease assay. Positive structures were Sanger sequenced using bacterial cloning. For plasmid injected groups, the Day 8 blastocyst rate averaged 30.3% (control 18.1%). The fluorescence rate at Day 8 was 6.3%, with a GFP positive blastocyst rate of 1.6%, totaling 7 blastocysts. The T7 assay revealed editing in GFP negative blastocysts and morulae as well, indicating that GFP is not a precise selection tool for successful editing. In protein injection groups, the highest concentration yielded the lowest survival rates (200ng µL−1, 50.0%, n=126), whereas the lowest concentration had the highest survival rate (20ng µL−1, 79.5%, n=314). The Day 8 blastocyst rate reached an average of 25% across groups. However, no edited blastocysts were observed in the higher concentration groups (100,200ng µL−1). The highest number of edited embryos was found in the lowest concentration injected (20ng µL−1, 4/56). Edited embryos showed multiple editing events neighbouring the guide RNA target site ranging from a 12-bp insertion to a 9-bp deletion, as well as unedited sequences. Additionally, one embryo showed a biallelic 15-bp deletion of COPA (10 clones). One possible reason for the presence of only mosaic editing and this in-frame deletion could be that a working copy of COPA is needed for proper blastocyst formation and that a knockout could be lethal. Additional validation and optimization is needed to elucidate the functional role of COPA during early development and its modulation when creating founder animals.

Advances in therapeutic application of CRISPR-Cas9

2019 ◽  
Vol 19 (3) ◽  
pp. 164-174 ◽  
Author(s):  
Jinyu Sun ◽  
Jianchu Wang ◽  
Donghui Zheng ◽  
Xiaorong Hu
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Malignant Tumor ◽  
Gene Editing ◽  
Genome Engineering ◽  
Therapeutic Application ◽  
Adaptive Immune ◽  
Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Gene therapy for neurodegenerative and ocular diseases using lentiviral vectors

2005 ◽  
Vol 110 (1) ◽  
pp. 37-46 ◽  
Author(s):  
G. Scott Ralph ◽  
Katie Binley ◽  
Liang-Fong Wong ◽  
Mimoun Azzouz ◽  
Nicholas D. Mazarakis
Keyword(s):  
Gene Therapy ◽  
Nervous System ◽  
Viral Vectors ◽  
Viral Vector ◽  
Lentiviral Vectors ◽  
Great Promise ◽  
Ocular Diseases ◽  
Vector Systems ◽  
Gene Therapy Application ◽  
Wide Range

Gene therapy holds great promise for the treatment of a wide range of inherited and acquired disorders. The development of viral vector systems to mediate safe and long-lasting expression of therapeutic transgenes in specific target cell populations is continually advancing. Gene therapy for the nervous system is particularly challenging due to the post-mitotic nature of neuronal cells and the restricted accessibility of the brain itself. Viral vectors based on lentiviruses provide particularly attractive vehicles for delivery of therapeutic genes to treat neurological and ocular diseases, since they efficiently transduce non-dividing cells and mediate sustained transgene expression. Furthermore, novel routes of vector delivery to the nervous system have recently been elucidated and these have increased further the scope of lentiviruses for gene therapy application. Several studies have demonstrated convincing therapeutic efficacy of lentiviral-based gene therapies in animal models of severe neurological disorders and the push for progressing such vectors to the clinic is ongoing. This review describes the key features of lentiviral vectors that make them such useful tools for gene therapy to the nervous system and outlines the major breakthroughs in the potential use of such vectors for treating neurodegenerative and ocular diseases.

PREPARATION OF CALCIUM PHOSPHATE NANOPARTICLES AS NON-VIRAL VECTORS FOR GENE DELIVERY SYSTEM

2017 ◽  
Vol 29 (04) ◽  
pp. 1750027 ◽  
Author(s):  
Ko-Chung Yen ◽  
I-Hua Chen ◽  
Feng-Huei Lin
Keyword(s):  
Gene Therapy ◽  
Calcium Phosphate ◽  
Gene Delivery ◽  
Delivery System ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Molar Ratio ◽  
Gene Delivery System ◽  
Calcium Phosphate Nanoparticles

A major aim of gene therapy is the efficient and specific delivery of therapeutic gene into the desired target tissues. Development of reliable vectors is a major challenge in gene therapy. The aim of this study is to develop calcium phosphate nanoparticles as novel non-viral vectors for the gene delivery system. Calcium phosphate nanoparticles were prepared by water-in-oil microemulsion method with a water to surfactant molar ratio, Wo [Formula: see text] 2–10. This paper studies the design and synthesis of ultra-low size, highly monodispersed DNA doped calcium phosphate nanoparticles of size around 100[Formula: see text]nm in diameter. The structure of DNA-calcium phosphate nanocomplex observed by TEM was displayed as a shell-like structure. This study used pEGFP as a reporter gene. The encapsulating efficiency to encapsulate DNA inside the nanoparticles was greater than 80%. In the MTT test, both calcium phosphate nanoparticles and DNA-calcium phosphate nanocomplex have no negative effect for 293T cells. By gel electrophoresis of free and entrapped pEGFP DNA, the DNA encapsulated inside the nanoparticles was protected from the external DNaseI environment. In vitro transfection studies in 293T cell-line, the DNA-calcium phosphate nanocomplex could be used safely to transfer the encapsulated DNA into the 293T cells and expression green fluorescent protein. The characteristic of DNA-calcium phosphate nanocomplex to deliver DNA belongs to slow release. The property of DNA-calcium phosphate nanocomplex was fit in the requirement of non-viral vectors for the gene delivery system.

Advances in delivery vectors for gene therapy in liver cancer

2020 ◽  
Vol 11 (1) ◽  
pp. 833-850 ◽  
Author(s):  
Katherine E Redd Bowman ◽  
Phong Lu ◽  
Erica R Vander Mause ◽  
Carol S Lim
Keyword(s):  
Hepatocellular Carcinoma ◽  
Gene Therapy ◽  
Viral Vectors ◽  
Gene Editing ◽  
Cancer Death ◽  
Common Cause ◽  
The Third ◽  
Gene Delivery Vectors ◽  
Efficient Delivery ◽  
Therapy Delivery

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death globally, mainly due to lack of effective treatments – a problem that gene therapy is poised to solve. Successful gene therapy requires safe and efficient delivery vectors, and recent advances in both viral and nonviral vectors have made an important impact on HCC gene therapy delivery. This review explores how adenoviral, retroviral and adeno-associated viral vectors have been modified to increase safety and delivery capacity, highlighting studies and clinical trials using these vectors for HCC gene therapy. Nanoparticles, liposomes, exosomes and virosomes are also featured in their roles as HCC gene delivery vectors. Finally, new discoveries in gene editing technology and their impacts on HCC gene therapy are discussed.

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