scholarly journals CRISPR-Cas9 in gene therapy: much control on breaking, little control on repairing

2015 ◽  
Author(s):  
Kaveh Daneshvar
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Immune Responses ◽  
Genome Editing ◽  
Target Cells ◽  
Target Dna ◽  
Target Activity ◽  
In Vivo Delivery ◽  
Target Effects

Recent advances in CRISPR-Cas9 genome editing tool have made great promises to basic and biomedical research as well as gene therapy. Efforts to make the CRISPR-Cas9 system applicable in gene therapy are largely focused on two aspects: 1) increasing the specificity of this system by eliminating off-target effects, and 2) optimizing in vivo delivery of the CRISPR-Cas9 DNA constructs to target cells and limiting the expression of Cas9 and gRNA to prevent immune responses. Moreover, there is an unnoted but crucial consideration about the mode of DNA repair at the lesion caused by CRISPR-Cas9. In this commentary, I briefly highlight recent publications on in vivo use of the CRISPR-Cas9 system in gene therapy. I then discuss concerns about the off-target activity and immune responses triggered by the use CRISPR-Cas9 in gene therapy. Following this, I focus on the undesired on-target DNA repair events that can occur as a result of the activity of CRISPR-Cas9. This concise commentary sets itself apart from previous perspectives by focusing on the modes of DNA repair employed following a CRISPR-Cas9 induced genomic insult, and by carefully weighing the benefits of the outcomes. In particular, the present manuscript underscores the need for more study on controlled DNA repair in systems targeted with CRISPR-Cas9 genome editing tools.

scholarly journals CRISPR-Cas9 in gene therapy: much control on breaking, little control on repairing

2015 ◽  
Author(s):  
Kaveh Daneshvar
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Immune Responses ◽  
Genome Editing ◽  
Biomedical Research ◽  
Target Cells ◽  
Target Dna ◽  
In Vivo Delivery ◽  
Target Effects

Recent advances in CRISPR-Cas9 genome editing tool have made great promises to basic and biomedical research as well as gene therapy. Efforts to make the CRISPR-Cas9 system applicable in gene therapy are largely focused on two aspects: 1) increasing the specificity of this system by eliminating off-target effects, and 2) optimizing in vivo delivery of the CRISPR-Cas9 DNA constructs to target cells and limiting the expression of Cas9 and gRNA to prevent toxicity immune responses. However, there is an unnoted but crucial consideration about the mode of DNA repair at the lesion caused by CRISPR-Cas9. In this commentary, I briefly highlight recent publications on in vivo use of the CRISPR-Cas9 system in gene therapy. I then discuss the undesired on-target DNA repair events that can occur as a result of the activity of CRISPR-Cas9. Overall, this commentary underscores the need for more study on controlled DNA repair in systems targeted with CRISPR-Cas9 genome editing tools.

scholarly journals CRISPR-Cas9 in gene therapy: much control on breaking, little control on repairing

2015 ◽  
Author(s):  
Kaveh Daneshvar
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Immune Responses ◽  
Genome Editing ◽  
Biomedical Research ◽  
Target Cells ◽  
Target Dna ◽  
In Vivo Delivery ◽  
Target Effects

Recent advances in CRISPR-Cas9 genome editing tool have made great promises to basic and biomedical research as well as gene therapy. Efforts to make the CRISPR-Cas9 system applicable in gene therapy are largely focused on two aspects: 1) increasing the specificity of this system by eliminating off-target effects, and 2) optimizing in vivo delivery of the CRISPR-Cas9 DNA constructs to target cells and limiting the expression of Cas9 and gRNA to prevent toxicity immune responses. However, there is an unnoted but crucial consideration about the mode of DNA repair at the lesion caused by CRISPR-Cas9. In this commentary, I briefly highlight recent publications on in vivo use of the CRISPR-Cas9 system in gene therapy. I then discuss the undesired on-target DNA repair events that can occur as a result of the activity of CRISPR-Cas9. Overall, this commentary underscores the need for more study on controlled DNA repair in systems targeted with CRISPR-Cas9 genome editing tools.

scholarly journals CRISPR-Cas9 in gene therapy: much control on breaking, little control on repairing

2015 ◽  
Author(s):  
Kaveh Daneshvar
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Immune Responses ◽  
Genome Editing ◽  
Biomedical Research ◽  
Target Cells ◽  
Target Dna ◽  
In Vivo Delivery ◽  
Target Effects

Recent advances in CRISPR-Cas9 genome editing tool have made great promises to basic and biomedical research as well as gene therapy. Efforts to make the CRISPR-Cas9 system applicable in gene therapy are largely focused on two aspects: 1) increasing the specificity of this system by eliminating off-target effects, and 2) optimizing in vivo delivery of the CRISPR-Cas9 DNA constructs to target cells and limiting the expression of Cas9 and gRNA to prevent immune responses. However, there is an unnoted but crucial consideration about the mode of DNA repair at the lesion caused by CRISPR-Cas9. In this commentary, I briefly highlight recent publications on in vivo use of the CRISPR-Cas9 system in gene therapy. I then discuss the undesired on-target DNA repair events that can occur as a result of the activity of CRISPR-Cas9. Overall, this commentary underscores the need for more study on controlled DNA repair in systems targeted with CRISPR-Cas9 genome editing tools.

scholarly journals CRISPR-Cas9 in gene therapy: much control on breaking, little control on repairing

2015 ◽  
Author(s):  
Kaveh Daneshvar
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Immune Responses ◽  
Genome Editing ◽  
Biomedical Research ◽  
Target Cells ◽  
Target Dna ◽  
In Vivo Delivery ◽  
Target Effects

Recent advances in CRISPR-Cas9 genome editing tool have made great promises to basic and biomedical research as well as gene therapy. Efforts to make the CRISPR-Cas9 system applicable in gene therapy are largely focused on two aspects: 1) increasing the specificity of this system by eliminating off-target effects, and 2) optimizing in vivo delivery of the CRISPR-Cas9 DNA constructs to target cells and limiting the expression of Cas9 and gRNA to prevent toxicity immune responses. However, there is an unnoted but crucial consideration about the mode of DNA repair at the lesion caused by CRISPR-Cas9. In this commentary, I briefly highlight recent publications on in vivo use of the CRISPR-Cas9 system in gene therapy. I then discuss the undesired on-target DNA repair events that can occur as a result of the activity of CRISPR-Cas9. Overall, this commentary underscores the need for more study on controlled DNA repair in systems targeted with CRISPR-Cas9 genome editing tools.

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 Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

scholarly journals Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

2017 ◽  
Author(s):  
Philippe E. Mangeot ◽  
Valérie Risson ◽  
Floriane Fusil ◽  
Aline Marnef ◽  
Emilie Laurent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Target Cells ◽  
Mouse Bone Marrow ◽  
Primary Cells ◽  
Hematopoietic Stem

AbstractProgrammable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

Abstract 543: Clinical Development of Cardiac Reprogramming Gene Therapy

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Huanyu Zhou ◽  
Laura M Lombardi ◽  
Christopher A Reid ◽  
Jin Yang ◽  
Chetan Srinath ◽  
...  
Keyword(s):  
Heart Failure ◽  
Gene Therapy ◽  
Clinical Application ◽  
Cardiac Fibroblasts ◽  
Target Cells ◽  
Large Animal ◽  
Safety And Efficacy ◽  
Treat Heart Failure ◽  
Ability To Regenerate

Heart failure affects an estimated 38 million people worldwide and is typically caused by cardiomyocyte (CM) loss or dysfunction. Although CMs have limited ability to regenerate, a large pool of non-myocytes, including cardiac fibroblasts (CFs), exist in the postnatal heart. In vivo reprogramming of non-myocytes into functional CMs is emerging as a potential new approach to treat heart failure and substantial proof-of-concept has been achieved in this new field. However, challenges remain in terms of clinical application. First, reported human reprogramming cocktails often consist of five to seven factors that require multiple AAV vectors for delivery. Thus, a less complex cocktail that is able to fit into one AAV vector is needed for this technology to impact human health. Second, the lack of specificity in AAV tropism further complicates the safety and regulatory landscape. A means to limit the expression of reprogramming factors to target cells is critical for maximizing long-term safety. Lastly, although promising studies in small animals have already been reported, safety and efficacy results in large animal MI models are critical to justify cardiac reprogramming in human clinical trials. We have developed a novel human cardiac reprogramming cocktail that consists of only two transcription factors and one miRNA. This new cocktail has been engineered into a single AAV cassette to efficiently reprogram human CFs into cardiomyocytes. We also substantially improved transduction of hCFs through AAV capsid engineering and eliminated CMs expression through a microRNA de-targeting method. Moreover, our novel cardiac reprogramming gene therapy improved cardiac function in both rat and swine MI models upon delivery at various time-points after MI without inducing arrhythmias. Given these promising safety and efficacy results in larger animals, we endeavor to translate direct cardiac reprogramming for clinical application.

scholarly journals Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1318 ◽  
Author(s):  
Nadja Bischoff ◽  
Sandra Wimberger ◽  
Marcello Maresca ◽  
Cord Brakebusch
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Animal Models ◽  
Molecular Biology ◽  
Small Molecules ◽  
Genome Editing ◽  
Drug Targets ◽  
Targeted Mutagenesis ◽  
Dna Repair Mechanisms ◽  
Repair Mechanisms

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.

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