scholarly journals Design of time-delayed safety switches for CRISPR gene therapy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dashan Sun
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Time Delay ◽  
Gene Editing ◽  
Data Fitting ◽  
Elimination Process ◽  
Good Balance ◽  
Efficient Gene ◽  
Target Effect ◽  
Editing Process

AbstractCRISPR system is a powerful gene editing tool which has already been reported to address a variety of gene relevant diseases in different cell lines. However, off-target effect and immune response caused by Cas9 remain two fundamental problems. Inspired by previously reported Cas9 self-elimination systems, time-delayed safety switches are designed in this work. Firstly, ultrasensitive relationship is constructed between Cas9-sgRNA (enzyme) and Cas9 plasmids (substrate), which generates the artificial time delay. Then intrinsic time delay in biomolecular activities is revealed by data fitting and utilized in constructing safety switches. The time-delayed safety switches function by separating the gene editing process and self-elimination process, and the tunable delay time may ensure a good balance between gene editing efficiency and side effect minimization. By addressing gene therapy efficiency, off-target effect, immune response and drug accumulation, we hope our safety switches may offer inspiration in realizing safe and efficient gene therapy in humans.

scholarly journals Design of time-delayed safety switches for CRISPR gene therapy

2021 ◽  
Author(s):  
Dashan Sun
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Time Delay ◽  
Cell Lines ◽  
Gene Editing ◽  
Drug Accumulation ◽  
Work Time ◽  
Crispr System ◽  
Efficient Gene ◽  
Target Effect

CRISPR system is a powerful gene editing tool which has already been reported to address a variety of gene relevant diseases in different cell lines. However, off-target effect and immune response caused by Cas9 remain two fundamental problems. In our work, time-delayed safety switches are designed based on either artificial ultrasensitivity transmission module or intrinsic time delay in biomolecular activities. By addressing gene therapy efficiency, off-target effect, immune response and drug accumulation, we hope our safety switches may offer inspiration in realizing safe and efficient gene therapy in humans.

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.

scholarly journals Extensive adaptive immune response of AAVs and Cas proteins in non-human primates

2019 ◽  
Author(s):  
Puhao Xiao ◽  
Raoxian Bai ◽  
Ting Zhang ◽  
Yin Zhou ◽  
Zhigang Zhou ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Immune Responses ◽  
Gene Editing ◽  
Adaptive Immune Response ◽  
Gene Correction ◽  
Adaptive Immune ◽  
Correction System ◽  
Cas Proteins

AbstractThe CRISPR-mediated Cas system is the most widely used tool in gene editing and gene therapy for its convenience and efficiency. Delivery of the CRISPR system by adeno-associated viruses (AAVs) is currently the most promising approach to gene therapy. However, pre-existing adaptive immune responses against CRISPR nuclease (PAIR-C) and AAVs has been found in human serum, indicating that immune response is a problem that cannot be ignored, especially for in vivo gene correction. Non-human primates (NHPs) share many genetic and physiological traits with human, and are considered as the bridge for translational medicine. However, whether NHPs have same PAIR-C status with human is still unknown. Here, macaques (rhesus and cynomolgus), including normal housed and CRISPR-SpCas9 or TALENs edited individuals, were used to detect PAIR-C which covered SaCas9, SpCas9, AsCas12a and LbCas12a. Dogs and mice were also detected to expand the range of species. In addition, pre-existing adaptive antibodies to AAV8 and AAV9 were performed against macaques of different ages. The results showed that adaptive immunity was pre-existing in the macaques regardless of Cas proteins and AAVs. These findings indicate that the pre-existing adaptive immune of AAV-delivered CRISPR construction and correction system should be concerned for in vivo experiments.

Selective and Efficient Gene Delivery of CD40-Ligand by a Fiber-Modified Adenovirus Vector with Specific Antibody to Human Leukemia and Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5263-5263
Author(s):  
Kazunori Kato ◽  
Sachie Hirai ◽  
Yukari Masuta ◽  
Aiko Kuroishi ◽  
Kiminori Nakamura ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
B Cells ◽  
Gene Delivery ◽  
Tumor Cells ◽  
Specific Antibody ◽  
Adenovirus Vector ◽  
Cd40 Ligand ◽  
Myeloma Cells ◽  
Efficient Gene

Abstract The use of adenovirus vector for cancer gene therapy is limited by their low transduction efficiency for lymphoma, leukemia and myeloma. We previously reported that highly efficient gene delivery of CD40-ligand by a modified adenovirus vector with the integrin-binding motif, RGD, in the H1 loop of the fiber knob (AxCAhCD40L-F/RGD) could induce phenotypic alteration followed by T cell immune response to autologous leukemia cells. But the utility of adenovirus with RGD-motif is still limited by their lack of specificity on tumor cells. Recent studies revealed a novel strategy of targeting adenovirus using a bispecific single-chain antibody (scFv) specific for adenovirus and target molecules on tumor cell surface. However, this approach should permit the production of high quantities of active bispecific scFv for in vivo use. To target adenovirus to hematopoietic tumor cells efficiently, we herein constructed a modified adenovirus vector that contained a synthetic immunoglobulin G-binding domain (termed Z33) in H1 loop of the fiber knob. A recombinant adenovirus encoding EGFP, lacZ (as reporter gene; Ax3CAZ3-F/Z33 or Ax3EGFP-F/Z33) or CD40L (as a therapeutic gene; Ax3CD40L-F/Z33) with Z33-modified fiber were tested for gene transfer efficiency into human tumors such as lymphoma, leukemia and myeloma cells. By the treatment with various antibodies specific for CD20 (Rituximab), CD40, CD38, CCR2 or CXCR4 that are expressed on leukemic cells, we achieved 3 to 10-fold enhancement of gene expression in lymphoma/leukemia (Ramos, Daudi or THP-1) and myeloma cells (MM1S, IM-9 or KMS5) compared with control IgG-treated tumors. We also examined specific gene delivery to freshly isolated leukemia B cells from patients that also contains normal lymphocytes. By using antibody to CD20 or CD40, we could selectively deliver CD40L gene with Ax3CD40L-F/Z33 into leukemia B cells (>50% at 300 pu/cell), but not in T and monocytes, followed by the induction of immune costimulatory molecules that are important in anti-leukemia immune response. Overall, our results indicated that combination of Z33-modified adenovirus vector and tumor specific antibody can be used as a modality for the gene therapy of leukemia and myeloma.

Genetic Engineering of AAV Capsid Gene for Gene Therapy Application

2020 ◽  
Vol 20 (5) ◽  
pp. 321-332
Author(s):  
Yunbo Liu ◽  
Xu Zhang ◽  
Lin Yang
Keyword(s):  
Gene Therapy ◽  
Neutralizing Antibodies ◽  
Human Subjects ◽  
Genetic Diseases ◽  
Capsid Gene ◽  
Human Populations ◽  
Stable Gene ◽  
Efficient Gene ◽  
Gene Therapy Application

Adeno-associated virus (AAV) is a promising vector for in vivo gene therapy because of its excellent safety profile and ability to mediate stable gene expression in human subjects. However, there are still numerous challenges that need to be resolved before this gene delivery vehicle is used in clinical applications, such as the inability of AAV to effectively target specific tissues, preexisting neutralizing antibodies in human populations, and a limited AAV packaging capacity. Over the past two decades, much genetic modification work has been performed with the AAV capsid gene, resulting in a large number of variants with modified characteristics, rendering AAV a versatile vector for more efficient gene therapy applications for different genetic diseases.

scholarly journals Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 779
Author(s):  
Man Teng ◽  
Yongxiu Yao ◽  
Venugopal Nair ◽  
Jun Luo
Keyword(s):  
Biological Sciences ◽  
Gene Therapy ◽  
Genetic Engineering ◽  
Gene Function ◽  
Viral Genome ◽  
Gene Editing ◽  
Vaccine Development ◽  
Future Prospects ◽  
Dna Viruses ◽  
Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response

1997 ◽  
Vol 57 (1-3) ◽  
pp. 19-25 ◽  
Author(s):  
Marielle Christ ◽  
Monika Lusky ◽  
Fabienne Stoeckel ◽  
Dominique Dreyer ◽  
Annick Dieterlé ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Recombinant Adenovirus ◽  
Host Immune Response ◽  
Adenovirus Vectors

scholarly journals Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

2021 ◽  
Vol 7 (7) ◽  
pp. 505
Author(s):  
Ping Zhang ◽  
Yu Wang ◽  
Chenxi Li ◽  
Xiaoyu Ma ◽  
Lan Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fungal Pathogens ◽  
Gene Editing ◽  
Recombination Frequency ◽  
Target Sequence ◽  
Widespread Application ◽  
Genetic Studies ◽  
Efficient Gene ◽  
Cryptococcus Species ◽  
Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

Overcoming the Immune Response to Permit Ex Vivo Gene Therapy for Spine Fusion With Human Type 5 Adenoviral Delivery of the LIM Mineralization Protein-1 cDNA

Spine ◽  
2003 ◽  
Vol 28 (3) ◽  
pp. 219-226 ◽  
Author(s):  
Hak-Sun Kim ◽  
Manjula Viggeswarapu ◽  
Scott D. Boden ◽  
Yunshan Liu ◽  
Gregory A Hair ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Ex Vivo ◽  
Spine Fusion ◽  
Human Type ◽  
Adenoviral Delivery ◽  
Ex Vivo Gene Therapy

scholarly journals Hepatic DR5 Induces Apoptosis and Limits Adenovirus Gene Therapy Product Expression in the Liver

2002 ◽  
Vol 76 (11) ◽  
pp. 5692-5700 ◽  
Author(s):  
Huang-Ge Zhang ◽  
Jinfu Xie ◽  
Liang Xu ◽  
Pingar Yang ◽  
Xin Xu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Transgene Expression ◽  
Cell Activation ◽  
Nk Cell ◽  
Death Domain ◽  
Activated T Cells ◽  
Tnf Α ◽  
Product Expression ◽  
Ifn Γ

ABSTRACT A major limitation of adenovirus (Ad) gene therapy product expression in the liver is subsequent elimination of the hepatocytes expressing the gene therapy product. This elimination is caused by both necrosis and apoptosis related to the innate and cell-mediated immune response to the Ad. Apoptosis of hepatocytes can be induced by the innate immune response by signaling through death domain receptors on hepatocytes including the tumor necrosis factor alpha (TNF-α) receptor (TNFR), Fas, and death domain receptors DR4 and DR5. We have previously shown that blocking signaling through TNFR enhances and prolongs gene therapy product expression in the liver. In the present study, we constructed an Ad that produces a soluble DR5-Fc (AdsDR5), which is capable of neutralizing TNF-related apoptosis-inducing ligand (TRAIL). AdsDR5 prevents TRAIL-mediated apoptosis of CD3-activated T cells and decreases hepatocyte apoptosis after AdCMVLacZ administration and enhances the level and duration of lacZ transgene expression in the liver. In addition to blocking TRAIL and directly inhibiting apoptosis, AdsDR5 decreases production of gamma interferon (IFN-γ) and TNF-α and decreases NK cell activation, all of which limit Ad-mediated transgene expression in the liver. These results indicate that (i) AdsDR5 produces a DR5-Fc capable of neutralizing TRAIL, (ii) AdsDR5 can reduce activation of NK cells and reduce induction of IFN-γ and TNF-α after Ad administration, and (iii) administration of AdsDR5 can enhance Ad gene therapy in the liver.

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