scholarly journals The Landscape of Non-Viral Gene Augmentation Strategies for Inherited Retinal Diseases

2021 ◽  
Vol 22 (5) ◽  
pp. 2318
Author(s):  
Lyes Toualbi ◽  
Maria Toms ◽  
Mariya Moosajee
Keyword(s):  
Viral Vectors ◽  
Matrix Attachment Region ◽  
Great Promise ◽  
Viral Gene ◽  
Retinal Diseases ◽  
Gene Copies ◽  
Augmentation Strategies ◽  
Viral Gene Therapy ◽  
Effective Option ◽  
Attachment Region

Inherited retinal diseases (IRDs) are a heterogeneous group of disorders causing progressive loss of vision, affecting approximately one in 1000 people worldwide. Gene augmentation therapy, which typically involves using adeno-associated viral vectors for delivery of healthy gene copies to affected tissues, has shown great promise as a strategy for the treatment of IRDs. However, the use of viruses is associated with several limitations, including harmful immune responses, genome integration, and limited gene carrying capacity. Here, we review the advances in non-viral gene augmentation strategies, such as the use of plasmids with minimal bacterial backbones and scaffold/matrix attachment region (S/MAR) sequences, that have the capability to overcome these weaknesses by accommodating genes of any size and maintaining episomal transgene expression with a lower risk of eliciting an immune response. Low retinal transfection rates remain a limitation, but various strategies, including coupling the DNA with different types of chemical vehicles (nanoparticles) and the use of electrical methods such as iontophoresis and electrotransfection to aid cell entry, have shown promise in preclinical studies. Non-viral gene therapy may offer a safer and effective option for future treatment of IRDs.

scholarly journals Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Eleana F. Stavrou ◽  
Emannuouil Simantirakis ◽  
Meletios Verras ◽  
Carlos Barbas ◽  
George Vassilopoulos ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Bone Marrow Cells ◽  
Globin Gene ◽  
K562 Cells ◽  
Replication Initiation ◽  
Matrix Attachment Region ◽  
Globin Mrna ◽  
Dna Replication Initiation ◽  
Episomal Vectors ◽  
Attachment Region

AbstractWe report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34+ cells, with transfection efficiencies of 46.3 ± 5.2%, 23.0 ± 2.1% and 24.2 ± 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of γ-globin mRNA by 3.3 ± 0.13, of γ-globin protein by 6.75 ± 3.25 and HbF protein by 2 ± 0.2 fold, while the vector remained episomal and non integrated. In murine β-YAC BMCs the vector mediated the activation of the silent human γ-globin gene and in CD34+ cells, increased γ-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of β-globin Replicator, transferred into CD34+ cells, produced CD34+ eGFP+ cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 ± 0.13 fold increased γ-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors.

scholarly journals Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism

2016 ◽  
Vol 90 (21) ◽  
pp. 9878-9888 ◽  
Author(s):  
Kenton T. Woodard ◽  
Katharine J. Liang ◽  
William C. Bennett ◽  
R. Jude Samulski
Keyword(s):  
Heparan Sulfate ◽  
Viral Vectors ◽  
Rational Design ◽  
Ex Vivo ◽  
Single Amino Acid ◽  
Subretinal Space ◽  
Great Promise ◽  
Viral Gene ◽  
Adeno Associated Virus ◽  
Inner Limiting Membrane

ABSTRACTMany adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescencein situhybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on humanex vivoretinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery.IMPORTANCEAdeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. The need for development of an easy, less invasive injection route for ocular gene therapy is met by intravitreal delivery, but delivery of AAV by this route results in poor transduction outcomes. The inner limiting membrane (ILM) creates a barrier separating the vitreous and the retina. Binding of AAV to heparan sulfate proteoglycan (HSPG) at the ILM may allow the virus to traverse this barrier for better retinal transduction. We show that HSPG binding is correlated with greater accumulation and penetration of AAV in the retina. We demonstrated that this accumulation is conserved across mouse and human retinas and that the addition of HSPG binding to other AAV capsids can increase the number of vectors accumulating at the ILM without dictating tropism.

scholarly journals CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1704
Author(s):  
Zelda Odé ◽  
Jose Condori ◽  
Nicolas Peterson ◽  
Sheng Zhou ◽  
Giedre Krenciute
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Vectors ◽  
Great Promise ◽  
Viral Gene ◽  
Specific Gene ◽  
Site Specific ◽  
Optimized Protocol ◽  
Starting Point ◽  
Gene Integration

T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci.

scholarly journals Viral Gene Therapy for Glioblastoma Multiforme: A Promising Hope for the Current Dilemma

2021 ◽  
Vol 11 ◽  
Author(s):  
Junsheng Li ◽  
Wen Wang ◽  
Jia Wang ◽  
Yong Cao ◽  
Shuo Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Glioblastoma Multiforme ◽  
Brain Tumors ◽  
Therapeutic Approach ◽  
Viral Vectors ◽  
Viral Gene ◽  
Target Cells ◽  
Antitumor Effects ◽  
Promising Therapeutic Approach ◽  
Viral Gene Therapy

Glioblastoma multiforme (GBM), as one of the most common malignant brain tumors, was limited in its treatment effectiveness with current options. Its invasive and infiltrative features led to tumor recurrence and poor prognosis. Effective treatment and survival improvement have always been a challenge. With the exploration of genetic mutations and molecular pathways in neuro-oncology, gene therapy is becoming a promising therapeutic approach. Therapeutic genes are delivered into target cells with viral vectors to act specific antitumor effects, which can be used in gene delivery, play an oncolysis effect, and induce host immune response. The application of engineering technology makes the virus vector used in genetics a more prospective future. Recent advances in viral gene therapy offer hope for treating brain tumors. In this review, we discuss the types and designs of viruses as well as their study progress and potential applications in the treatment of GBM. Although still under research, viral gene therapy is promising to be a new therapeutic approach for GBM treatment in the future.

An Update on Ocular Gene Therapy for Monogenic and Multifactorial Retinal Diseases

2019 ◽  
Vol 3 (5) ◽  
pp. 366-377
Author(s):  
Alice Y. Hou ◽  
Szilárd Kiss
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Gene Replacement ◽  
Age Related Macular Degeneration ◽  
Great Promise ◽  
Retinal Diseases ◽  
Multifactorial Diseases ◽  
Monogenic Disorders ◽  
Age Related ◽  
Augmentation Strategy

Monogenic and multifactorial retinal diseases are genetically and clinically diverse conditions that have historically shared an untreatable clinical course. In recent years, the advent of targeted treatments like gene therapy has enabled intraocular delivery of viral vectors, such as adeno-associated viruses and retroviruses, that establish long-term stable protein expression. Despite the innate distinctions between mainly monogenic disorders affecting the retina such as Leber congenital amaurosis (LCA), heterogeneous monogenic conditions like retinitis pigmentosa (RP), and multifactorial diseases such as age-related macular degeneration (AMD), gene therapy has shown great promise as a gene replacement or augmentation strategy to treat or halt these disorders. This review will present an overview of retinal gene therapy and provide a comprehensive analysis of past and current clinical trials for LCA, RP, and AMD. It will discuss the paradigm shift that has occurred in ophthalmic care thanks to the development of gene therapy, as well as its challenges for the future.

scholarly journals VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

2021 ◽  
Author(s):  
Arjun Khakhar ◽  
Cecily Wang ◽  
Ryan Swanson ◽  
Sydney Stokke ◽  
Furva Rizvi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Viral Vectors ◽  
Viral Vector ◽  
Tobacco Rattle Virus ◽  
Plant Size ◽  
Great Promise ◽  
Attractive Alternative ◽  
Gibberellin Biosynthesis ◽  
In Planta

Abstract Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work we developed a technology called VipariNama (ViN) in which vectors based on the Tobacco Rattle Virus (TRV) are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

scholarly journals How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

2021 ◽  
Vol 22 (14) ◽  
pp. 7545
Author(s):  
Myriam Sainz-Ramos ◽  
Idoia Gallego ◽  
Ilia Villate-Beitia ◽  
Jon Zarate ◽  
Iván Maldonado ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Clinical Practice ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Viral Vector ◽  
Clinical Success ◽  
Genetic Material ◽  
Viral Gene ◽  
Promising Alternative ◽  
Vector Systems

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

scholarly journals α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 771
Author(s):  
Julen Rodríguez-Castejón ◽  
Ana Alarcia-Lacalle ◽  
Itziar Gómez-Aguado ◽  
Mónica Vicente-Pascual ◽  
María Ángeles Solinís Aspiazu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fabry Disease ◽  
Viral Vector ◽  
Viral Gene ◽  
Lysosomal Storage ◽  
Wild Type ◽  
Dose Administration ◽  
Viral Gene Therapy ◽  
Liver Transaminases ◽  
Low Levels

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.

scholarly journals Screening of a Protein That Interacts with the Matrix Attachment Region-Binding Protein fromDunaliella salina

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Rui Yang ◽  
Zhaoxi Li ◽  
Yan Lin ◽  
Baosheng Yang ◽  
Tianyun Wang
Keyword(s):  
Binding Protein ◽  
Matrix Attachment Region ◽  
Regulatory Function ◽  
Glutathione S Transferase ◽  
Reading Frame ◽  
Cis Acting ◽  
Binding Partner ◽  
The Matrix ◽  
Attachment Region ◽  
Regulated Functions

We isolated the matrix attachment region-binding protein (MBP) DMBP-1 fromDunaliella salinain our previous studies. MBPs are part of the cis-acting protein family cluster. The regulatory function possibly works through the interaction of the MBPs with each other. In the present study, DMBP-1 was used as the bait in screening theD. salinacDNA library for DMBP-1 interactors that could potentially mediate the DMBP-1-regulated functions. A novel MBP, namely, DMBP-2, was identified as a DMBP-1 binding partner. The cDNA of DMBP-1 was 823 bp long and contained a 573 bp open reading frame, which encoded a polypeptide of 191 amino acids. The interaction between DMBP-2 and DMBP-1 was further confirmed through glutathione S-transferase pull-down assays.

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