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.

scholarly journals CRISPR Gene-Editing Models Geared Toward Therapy for Hereditary and Developmental Neurological Disorders

2021 ◽  
Vol 9 ◽  
Author(s):  
Poh Kuan Wong ◽  
Fook Choe Cheah ◽  
Saiful Effendi Syafruddin ◽  
M. Aiman Mohtar ◽  
Norazrina Azmi ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Neurological Disorders ◽  
Gene Editing ◽  
Fragile X ◽  
Genetic Diseases ◽  
Basic Principles ◽  
Short Palindromic Repeat

Hereditary or developmental neurological disorders (HNDs or DNDs) affect the quality of life and contribute to the high mortality rates among neonates. Most HNDs are incurable, and the search for new and effective treatments is hampered by challenges peculiar to the human brain, which is guarded by the near-impervious blood-brain barrier. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), a gene-editing tool repurposed from bacterial defense systems against viruses, has been touted by some as a panacea for genetic diseases. CRISPR has expedited the research into HNDs, enabling the generation of in vitro and in vivo models to simulate the changes in human physiology caused by genetic variation. In this review, we describe the basic principles and workings of CRISPR and the modifications that have been made to broaden its applications. Then, we review important CRISPR-based studies that have opened new doors to the treatment of HNDs such as fragile X syndrome and Down syndrome. We also discuss how CRISPR can be used to generate research models to examine the effects of genetic variation and caffeine therapy on the developing brain. Several drawbacks of CRISPR may preclude its use at the clinics, particularly the vulnerability of neuronal cells to the adverse effect of gene editing, and the inefficiency of CRISPR delivery into the brain. In concluding the review, we offer some suggestions for enhancing the gene-editing efficacy of CRISPR and how it may be morphed into safe and effective therapy for HNDs and other brain disorders.

scholarly journals Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Licia Totani ◽  
Concetta Amore ◽  
Antonio Piccoli ◽  
Giuseppe Dell’Elba ◽  
Angelo Di Santo ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lung Disease ◽  
Pde4 Inhibitor ◽  
Free Dna ◽  
Impaired Lung Function ◽  
Cellular Integrity ◽  
Net Release

Neutrophilic inflammation is a key determinant of cystic fibrosis (CF) lung disease. Neutrophil-derived free DNA, released in the form of extracellular traps (NETs), significantly correlates with impaired lung function in patients with CF, underlying their pathogenetic role in CF lung disease. Thus, specific approaches to control NETosis of neutrophils migrated into the lungs may be clinically relevant in CF. We investigated the efficacy of phosphodiesterase (PDE) type-4 inhibitors, in vitro, on NET release by neutrophils from healthy volunteers and individuals with CF, and in vivo, on NET accumulation and lung inflammation in mice infected with Pseudomonas aeruginosa. PDE4 blockade curbed endotoxin-induced NET production and preserved cellular integrity and apoptosis in neutrophils, from healthy subjects and patients with CF, challenged with endotoxin, in vitro. The pharmacological effects of PDE4 inhibitors were significantly more evident on CF neutrophils. In a mouse model of Pseudomonas aeruginosa chronic infection, aerosol treatment with roflumilast, a selective PDE4 inhibitor, gave a significant reduction in free DNA in the BALF. This was accompanied by reduced citrullination of histone H3 in neutrophils migrated into the airways. Roflumilast-treated mice showed a significant improvement in weight recovery. Our study provides the first evidence that PDE4 blockade controls NETosis in vitro and in vivo, in CF-relevant models. Since selective PDE4 inhibitors have been recently approved for the treatment of COPD and psoriasis, our present results encourage clinical trials to test the efficacy of this class of drugs in CF.

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.

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 Evolving AAV-delivered therapeutics towards ultimate cures

2021 ◽  
Author(s):  
Xiangjun He ◽  
Brian Anugerah Urip ◽  
Zhenjie Zhang ◽  
Chun Christopher Ngan ◽  
Bo Feng
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Treatment Options ◽  
Genetic Diseases ◽  
The United States ◽  
European Medicines Agency ◽  
Gene Therapies ◽  
Therapeutic Modalities ◽  
United States Food

AbstractGene therapy has entered a new era after decades-long efforts, where the recombinant adeno-associated virus (AAV) has stood out as the most potent vector for in vivo gene transfer and demonstrated excellent efficacy and safety profiles in numerous preclinical and clinical studies. Since the first AAV-derived therapeutics Glybera was approved by the European Medicines Agency (EMA) in 2012, there is an increasing number of AAV-based gene augmentation therapies that have been developed and tested for treating incurable genetic diseases. In the subsequent years, the United States Food and Drug Administration (FDA) approved two additional AAV gene therapy products, Luxturna and Zolgensma, to be launched into the market. Recent breakthroughs in genome editing tools and the combined use with AAV vectors have introduced new therapeutic modalities using somatic gene editing strategies. The promising outcomes from preclinical studies have prompted the continuous evolution of AAV-delivered therapeutics and broadened the scope of treatment options for untreatable diseases. Here, we describe the clinical updates of AAV gene therapies and the latest development using AAV to deliver the CRISPR components as gene editing therapeutics. We also discuss the major challenges and safety concerns associated with AAV delivery and CRISPR therapeutics, and highlight the recent achievement and toxicity issues reported from clinical applications.

scholarly journals Optimizations of In Vitro Mucus and Cell Culture Models to Better Predict In Vivo Gene Transfer in Pathological Lung Respiratory Airways: Cystic Fibrosis as an Example

Pharmaceutics ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 47
Author(s):  
Rosy Ghanem ◽  
Véronique Laurent ◽  
Philippe Roquefort ◽  
Tanguy Haute ◽  
Sophie Ramel ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Cell Culture ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Culture Models ◽  
Transfer Strategies ◽  
Cell Culture Models

The respiratory epithelium can be affected by many diseases that could be treated using aerosol gene therapy. Among these, cystic fibrosis (CF) is a lethal inherited disease characterized by airways complications, which determine the life expectancy and the effectiveness of aerosolized treatments. Beside evaluations performed under in vivo settings, cell culture models mimicking in vivo pathophysiological conditions can provide complementary insights into the potential of gene transfer strategies. Such models must consider multiple parameters, following the rationale that proper gene transfer evaluations depend on whether they are performed under experimental conditions close to pathophysiological settings. In addition, the mucus layer, which covers the epithelial cells, constitutes a physical barrier for gene delivery, especially in diseases such as CF. Artificial mucus models featuring physical and biological properties similar to CF mucus allow determining the ability of gene transfer systems to effectively reach the underlying epithelium. In this review, we describe mucus and cellular models relevant for CF aerosol gene therapy, with a particular emphasis on mucus rheology. We strongly believe that combining multiple pathophysiological features in single complex cell culture models could help bridge the gaps between in vitro and in vivo settings, as well as viral and non-viral gene delivery strategies.

Mesenchymal Stem Cells: A New Generation of Therapeutic Agents as Vehicles in Gene Therapy

2020 ◽  
Vol 20 (4) ◽  
pp. 269-284
Author(s):  
Mahmoud Gharbavi ◽  
Ali Sharafi ◽  
Saeed Ghanbarzadeh
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Therapeutic Agents ◽  
Targeted Gene Delivery ◽  
The Status

In recent years, mesenchymal stem cells (MSCs) as a new tool for therapeutic gene delivery in clinics have attracted much attention. Their advantages cover longer lifespan, better isolation, and higher transfection efficiency and proliferation rate. MSCs are the preferred approach for cell-based therapies because of their in vitro self-renewal capacity, migrating especially to tumor tissues, as well as anti-inflammatory and immunomodulatory properties. Therefore, they have considerable efficiency in genetic engineering for future clinical applications in cancer gene therapy and other diseases. For improving therapeutic efficiency, targeted therapy of cancers can be achieved through the sustained release of therapeutic agents and functional gene expression induction to the intended tissues. The development of a new vector in gene therapy can improve the durability of a transgene expression. Also, the safety of the vector, if administered systemically, may resolve several problems, such as durability of expression and the host immune response. Currently, MSCs are prominent candidates as cell vehicles for both preclinical and clinical trials due to the secretion of therapeutic agents in several cancers. In the present study, we discuss the status of gene therapy in both viral and non-viral vectors along with their limitations. Throughout this study, the use of several nano-carriers for gene therapy is also investigated. Finally, we critically discuss the promising advantages of MSCs in targeted gene delivery, tumor inhibition and their utilization as the gene carriers in clinical situations.

scholarly journals Efficient viral delivery of Cas9 into human safe harbor

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hideki Hayashi ◽  
Yoshinao Kubo ◽  
Mai Izumida ◽  
Toshifumi Matsuyama
Keyword(s):  
Viral Vectors ◽  
Gene Editing ◽  
Lentiviral Vector ◽  
Integration Site ◽  
Genetic Diseases ◽  
Safe Harbor ◽  
Efficient System ◽  
Guide Rnas ◽  
Aavs1 Locus

AbstractGene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient.

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