Stabilizability of Vector Systems with Uniform Actuation Unpredictability

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

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Jumping Ahead with Sleeping Beauty: Mechanistic Insights into Cut-and-Paste Transposition

Viruses ◽

10.3390/v13010076 ◽

2021 ◽

Vol 13 (1) ◽

pp. 76

Author(s):

Matthias T. Ochmann ◽

Zoltán Ivics

Keyword(s):

Genetic Engineering ◽

Structural Data ◽

Sleeping Beauty ◽

Preclinical Studies ◽

Gene Vector ◽

Functional Information ◽

Vector Systems ◽

Novel Variants ◽

Insight Into ◽

Cellular Genome

Sleeping Beauty (SB) is a transposon system that has been widely used as a genetic engineering tool. Central to the development of any transposon as a research tool is the ability to integrate a foreign piece of DNA into the cellular genome. Driven by the need for efficient transposon-based gene vector systems, extensive studies have largely elucidated the molecular actors and actions taking place during SB transposition. Close transposon relatives and other recombination enzymes, including retroviral integrases, have served as useful models to infer functional information relevant to SB. Recently obtained structural data on the SB transposase enable a direct insight into the workings of this enzyme. These efforts cumulatively allowed the development of novel variants of SB that offer advanced possibilities for genetic engineering due to their hyperactivity, integration deficiency, or targeting capacity. However, many aspects of the process of transposition remain poorly understood and require further investigation. We anticipate that continued investigations into the structure–function relationships of SB transposition will enable the development of new generations of transposition-based vector systems, thereby facilitating the use of SB in preclinical studies and clinical trials.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22147545 ◽

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.

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Vector systems for the expression, analysis and cloning of DNA sequence inS. cerevisiae

Yeast ◽

10.1002/yea.320010202 ◽

1985 ◽

Vol 1 (2) ◽

pp. 83-138 ◽

Cited By ~ 129

Author(s):

Stephen A. Parent ◽

Carol M. Fenimore ◽

Keith A. Bostian

Keyword(s):

Dna Sequence ◽

Expression Analysis ◽

Vector Systems

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Host-vector systems

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1989.0061 ◽

1989 ◽

Vol 324 (1224) ◽

pp. 477-485 ◽

Cited By ~ 2

Keyword(s):

Genetic Manipulation ◽

Pharmaceutical Products ◽

Host Cells ◽

Animal Cells ◽

Vector Systems ◽

Wide Range ◽

Novel Host ◽

Basic Concepts ◽

Foreign Genes ◽

New Generation

In 1980 it was only possible to express foreign genes in bacteria and a few easily cultured animal cells. During the subsequent eight years specialized vectors have been developed to allow the genetic manipulation of a wide range of both prokaryotes and eukaryotes. One of the major goals of biotechnology in 1980 was to use host cells as ‘factories’ for the production of proteins that were only available in minute quantities from natural sources. This has already lead to a new generation of pharmaceutical products. Advances in our understanding of host-vector systems have defined new goals. The basic concepts of expression vector design will be illustrated. Some of the new goals are discussed with particular reference to the exploitation of novel host-vector systems to develop vaccines and anti-viral agents against AIDS.

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Conjugation of an antibody Fv fragment to a virus coat protein: cell-specific targeting of recombinant polyoma-virus-like particles

Biochemical Journal ◽

10.1042/bj3560867 ◽

2001 ◽

Vol 356 (3) ◽

pp. 867-873 ◽

Cited By ~ 20

Author(s):

Kay STUBENRAUCH ◽

Stefan GLEITER ◽

Ulrich BRINKMANN ◽

Rainer RUDOLPH ◽

Hauke LILIE

Keyword(s):

Coat Protein ◽

Mammalian Cells ◽

Recombinant Antibody ◽

Specific Antigen ◽

Antibody Fragment ◽

Fusion Peptides ◽

Virus Like Particles ◽

Vector Systems ◽

Virus Coat

The development of cell-type-specific delivery systems is highly desirable for gene-therapeutic applications. Current virus-based vector systems show broad cell specificity, which results in the need to restrict the natural tropism of these viral systems. Here we demonstrate that tumour-cell-specific virus-like particles can be functionally assembled in vitro from recombinant viral coat protein expressed in Escherichia coli. The insertion of a negatively charged peptide in the HI loop of polyoma VP1 interferes with the binding of VP1 to the natural recognition site on mammalian cells and also serves as an adapter for the coupling of antibody fragments that contain complementary charged fusion peptides. A recombinant antibody fragment of the tumour-specific anti-(Lewis Y) antibody B3 could be coupled to the mutant VP1 by engineered polyionic peptides and an additional disulphide bond. With this system an entirely recombinant cell-specific delivery system assembled in vitro could be generated that transfers genes preferentially to cells presenting the tumour-specific antigen on the cell surface.

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Bacteria in Cancer Therapy: Renaissance of an Old Concept

International Journal of Microbiology ◽

10.1155/2016/8451728 ◽

2016 ◽

Vol 2016 ◽

pp. 1-14 ◽

Cited By ~ 50

Author(s):

Sebastian Felgner ◽

Dino Kocijancic ◽

Michael Frahm ◽

Siegfried Weiss

Keyword(s):

Treatment Options ◽

Tumor Therapy ◽

The Past ◽

Novel Treatment ◽

Vector Systems ◽

Rising Incidence ◽

Solid Foundation ◽

Ancient Times ◽

Therapeutic Compounds ◽

The 19Th Century

The rising incidence of cancer cases worldwide generates an urgent need of novel treatment options. Applying bacteria may represent a valuable therapeutic variant that is intensively investigated nowadays. Interestingly, the idea to apply bacteria wittingly or unwittingly dates back to ancient times and was revived in the 19th century mainly by the pioneer William Coley. This review summarizes and compares the results of the past 150 years in bacteria mediated tumor therapy from preclinical to clinical studies. Lessons we have learned from the past provide a solid foundation on which to base future efforts. In this regard, several perspectives are discussed by which bacteria in addition to their intrinsic antitumor effect can be used as vector systems that shuttle therapeutic compounds into the tumor. Strategic solutions like these provide a sound and more apt exploitation of bacteria that may overcome limitations of conventional therapies.

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