lentiviral inspired editing
89 views | +0 today
Your new post is loading...
Your new post is loading...
Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells

Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells | lentiviral inspired editing | Scoop.it
Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because t

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, September 13, 3:57 AM

In this work, the authors report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, they detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not.

 

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing 

The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing  | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, September 5, 11:41 AM

Here, the authors developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. They show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well.

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells

Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells | lentiviral inspired editing | Scoop.it
Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because t

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, September 13, 3:57 AM

In this work, the authors report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, they detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not.

 

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing

Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, September 20, 11:07 AM

A significant recent advance in genome engineering is the development of the CRISPR/Cas9 system for nuclease based genome editing. However, several cell types are not easily transfected and in vivo delivery of the CRISPR system remains challenging. To enhance delivery efficiency, one solution is use of viral vectors such as lentiviral or adeno-associated viral systems. However, these viral vectors have the potential for undesired random integration into the host genome.

In this study, the scientists repurpose Sendai virus, an RNA virus with no viral DNA phase as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75–98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other RNA viral vectors as versatile and efficient tools for gene editing.

GEG Tech develops several lines of vectors based on lentivirus and offers to include CRISPR system within. For the moment, GEG Tech provides the CRISPR system into integrating lentiviral vectors and non-integrating lentiviral vectors which already offer a wide range of applications. The R&D team is working to include the CRISPR system in the specific line of vectors only developed by GEG Tech which is RNA lentiviral vector. In this way, the CRISPR system may benefit to the advantage of the lentiviral vector technology: efficiency, cell/tissue specificity, high cargo capacity and low immunogenicity while being expressed transiently and having high level of biosafety. We look forward to offering you this type of vector!

 

To know more about GEG Tech RNA lenti-ONE Trans vectors

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Development of Lentiviral Vectors for Targeted Integration and Protein Delivery 

Development of Lentiviral Vectors for Targeted Integration and Protein Delivery  | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, June 24, 8:47 AM

The method in this chapter describes the design of human immunodeficiency virus type 1 (HIV-1) integrase (IN)-fusion proteins which were developed to transport different proteins into the nuclei of lentiviral vector (LV)-transduced cells. The vectors can hence be used for various protein transduction needs. An outline of the necessary methods is also given to study the functionality of a chosen IN-fusion protein in a cell culture assay.

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice - Nature Biotechnology

A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice - Nature Biotechnology | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, February 3, 1:48 PM

Here the authors used AAV vectors for gene therapy of liver disease. They combine this vector with the CRISPR cas9 system to generate stable gene modification. They intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylaseThis resulted in reversion of the mutation in 10% (6.7–20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. 


www.geg-tech.com/Vectors



Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo - Nature Biotechnology

Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo - Nature Biotechnology | lentiviral inspired editing | Scoop.it
Cas9-mediated gene editing corrects hereditary tyrosinemia in a mouse model.

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, February 3, 1:39 PM

In this study, the authors combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in  mouse model of human hereditary tyrosinemia.Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.


www.geg-tech.com/Vectors

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Ex Vivo Gene Therapy Using Human Mesenchymal Stem Cells to Deliver Growth Factors in the Skeletal Muscle of a Familial ALS Rat Model

Ex Vivo Gene Therapy Using Human Mesenchymal Stem Cells to Deliver Growth Factors in the Skeletal Muscle of a Familial ALS Rat Model | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, January 11, 6:21 AM

In this chapter, the scientists describe a protocol of ex vivo delivery of growth factors via lentiviral vector-mediated genetic modification of human mesenchymal stem cells (hMSCs) transplantation into the skeletal muscle of a familial l amyotrophic lateral sclerosis (ALS)rat model. They show that intramuscular growth factor delivery via hMSCs can enhance neuromuscular innervation and motor neuron survival in a rat model of ALS 


www.geg-tech.com/Vectors

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Expression of Multiple Functional RNAs or Proteins from One Viral Vector

Expression of Multiple Functional RNAs or Proteins from One Viral Vector | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, December 1, 2015 11:02 AM

Here, Tomas Bjorklund review design choices for enabling expression of two functional protein or RNA sequences from a single viral vector. These type of tools are very useful in neuroscience-related field of neuronal control and modulation, e.g., using optogenetics or DREADDs, but are also desirable in applications of CRISPR/Cas9 in situ genome editing and more refined therapeutic approaches. 


www.geg-tech.com/Vectors

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, November 10, 2015 11:55 AM

Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Thanks to a sendai vector, the authors induced pluripotent stem cells (PKDiPSCs) from  PKD patient erythrocytes. Then, they edited PKDiPSCs integrating a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.


www.geg-tech.com/Vectors

Scooped by juan carlos ramirez
Scoop.it!

CRISPR Cas9: A novel approach to genetic engineering

Provides the basic mechanism behind the CRISPR Cas9 complex, while describing application and need.
more...
No comment yet.
Rescooped by juan carlos ramirez from TAL effector science
Scoop.it!

Targeted Myostatin Gene Editing in Multiple Mammalian Species Directed by a Single Pair of TALE Nucleases - Molecular Therapy Nucleic Acids

(via T. Lahaye, thx)

Xu et al, 2013

Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Strategies to block myostatin signaling pathway have been extensively pursued to increase muscle mass in various disease settings including muscular dystrophy. Here, we report a new class of reagents based on transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level. We designed a pair of MSTN TALENs to target a highly conserved sequence in the coding region of the myostatin gene. We demonstrate that codelivery of these MSTN TALENs induce highly specific and efficient gene disruption in a variety of human, cattle, and mouse cells. Based upon sequence analysis, this pair of TALENs is expected to be functional in many other mammalian species. Moreover, we demonstrate that these MSTN TALENs can facilitate targeted integration of a mCherry expression cassette or a larger muscular dystrophy gene (dysferlin) expression cassette into the MSTN locus in mouse or human cells. Therefore, targeted editing of the myostatin gene using our highly specific and efficient TALEN pair would facilitate cell engineering, allowing potential use in translational research for cell-based therapy.


Via dromius
more...
No comment yet.
Rescooped by juan carlos ramirez from TAL effector science
Scoop.it!

Manipulating mitochondrial genomes in the clinic: playing by different rules

Manipulating mitochondrial genomes in the clinic: playing by different rules | lentiviral inspired editing | Scoop.it

Moraes et al 2014

Recently, several publications have surfaced describing methods to manipulate mitochondrial genomes in tissues and embryos. With them, a somewhat sensationalistic uproar about the generation of children with ‘three parents’ has dominated the discussion in the lay media. It is important that society understands the singularities of mitochondrial genetics to judge these procedures in a rational light, so that this ongoing discussion does not preclude the helping of patients and families harboring mutated mitochondrial genomes.


Via dromius
more...
dromius's curator insight, March 27, 2014 8:55 AM

describes mitoTALENs

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Episomal Lentiviral Vector-Mediated miR-145 Overexpression Inhibits Proliferation and Induces Apoptosis of Human Esophageal Carcinomas Cells

Episomal Lentiviral Vector-Mediated miR-145 Overexpression Inhibits Proliferation and Induces Apoptosis of Human Esophageal Carcinomas Cells | lentiviral inspired editing | Scoop.it

Background: Gene therapy is a promising approach for the treatment of various cancers.


Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, September 6, 11:27 AM

In the present study the scientists constructed an episomal lentiviral vector using the β-interferon matrix attachment region to express the microRNA -145 (miR-145), and examining the effect of miR-145 overexpression on human esophageal carcinoma (EC) cells. They found that  miR-145 overexpression inhibited cell proliferation and induced apoptosis. Moreover, the lentiviral vector did not integrate into the host genome, but was maintained episomally at lower copy numbers. The m.iR-145-expressing episomal lentiviral vectors could be a promising tool for gene therapy in the treatment of EC.

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Lentiviral expression system for the
purification of secreted proteins from
human cell cultures

Lentiviral expression system for the<br/>purification of secreted proteins from<br/>human cell cultures | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, September 7, 10:09 AM

Recombinant proteins of therapeutic use are ideally produced in human cells to ensure appropriate co- and post-translational modifications. CHO cells are mostly used but recent studies highlight that proteins produced in this cell line have a different glycosylation pattern than the same proteins produced in HEK cells, which may affect their function. While the exact implications of differences in glycosylation are not yet fully understood, a strong argument for the production of therapeutic proteins in HEK cells is made by the finding that CHO cells can add terminal galactose-α-1,3-galactose (α-Gal) to proteins. α-Gal is a non-human antigenic glycan and its reaction with circulating antibodies present in most individuals can lead to anaphylaxis. This glycan is absent in proteins produced in HEK cells. In this related study, scientists describe a simple and effective method for the purification of a His-tagged human protein from the culture media of lentiviral vector-transduced HEK 293 T cells. All reagents are readily available at a relatively low cost and no specialized equipment is needed to complete the procedure. Using soluble CD4 (sCD4), a truncated version of the CD4 receptor that is secreted, they showed that lentiviral vector mediated over 10-fold higher secretion of the protein in comparison to 293 T cells. Furthermore, small-scale purification from 50 ml of culture media with reduced serum content yielded up to 1 mg of pure sCD4.

The procedure outlined in this study can be performed without the need for specialized reagents or equipment and could easily be adapted by any laboratory. This strategy can be adapted to a large-scale format without significantly increasing the cost to further improve the yield.

 

To know more about the Article

To know more about GEG Tech Lenti-ONE Vectors

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Elements of lentiviral vector design toward gene therapy for treating mucopolysaccharidosis

Elements of lentiviral vector design toward gene therapy for treating mucopolysaccharidosis | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, September 6, 11:55 AM

Lentiviral vector encoding correct IDUA cDNA could be used for treating MPS I. To optimize the lentiviral vector design, 9 constructs were designed by combinations of various promoters, enhancers, and codon optimization. After 30 days, one vector, CCEoIDW, achieved the highest IDUA levels and

is selected as the optimal lentiviral vector for treating MPS I disease and will be applied in large animal preclinical studies. Further, taken both in vitro and in vivo comparisons together, codon optimization, use of EF-1α promoter and woodchuck hepatitis virus posttranscriptional response element (WPRE) could enhance transgene expression. These results provided a better understanding of factors contributing efficient transgene expression in lentiviral gene therapies.

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection | Protocol

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection | Protocol | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, June 23, 10:29 AM

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

U.K. Aims at $15B Gene Therapy Industry

U.K. Aims at $15B Gene Therapy Industry | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, February 15, 6:48 AM

According to Stephen Ward, Ph.D., the U.K. means business when it says it has the goal of building a £10 billion ($15 billion) cell and gene therapy industry in that country.

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice - Nature Biotechnology

A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice - Nature Biotechnology | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, February 3, 1:48 PM

Here the authors used AAV vectors for gene therapy of liver disease. They combine this vector with the CRISPR cas9 system to generate stable gene modification. They intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylaseThis resulted in reversion of the mutation in 10% (6.7–20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. 


www.geg-tech.com/Vectors



Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Calimmune and St. Jude Expand Partnership to Improve Lentiviral Vector Manufacturing for Novel Gene Therapies

Calimmune and St. Jude Expand Partnership to Improve Lentiviral Vector Manufacturing for Novel Gene Therapies | lentiviral inspired editing | Scoop.it
Jude Children’s Research Hospital.

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, January 8, 5:47 AM

Calimmune Inc., a clinical-stage gene therapy company, has expanded its intellectual property estate covering Cytegrity™, an innovative lentiviral vector manufacturing technology, through a second licensing agreement with St. Jude Children’s Research Hospital. Financial terms of the deal were not disclosed.


www.geg-tech.com/Vectors

Rescooped by juan carlos ramirez from Vectorology - GEG Tech top picks
Scoop.it!

Genetic treatment of a molecular disorder: gene therapy approaches to sickle cell disease

Genetic treatment of a molecular disorder: gene therapy approaches to sickle cell disease | lentiviral inspired editing | Scoop.it

Via BigField GEG Tech
more...
BigField GEG Tech's curator insight, January 14, 8:23 AM

This review focuses on progress made toward achieving gene therapy, the current state of the field, consideration of factors that may determine clinical success, and prospects for future development.


www.geg-tech.com/Vectors

Scooped by juan carlos ramirez
Scoop.it!

Researcher discusses advances in gene therapy - Medical Xpress

Researcher discusses advances in gene therapy - Medical Xpress | lentiviral inspired editing | Scoop.it
After leading successful clinical trials of gene therapy in Milan, Roncarolo hopes to build on that success at Stanford through collaboration with colleagues in the fields of genetics and stem cell science.
more...
No comment yet.
Scooped by juan carlos ramirez
Scoop.it!

Cut & paste chomosomes: RGEN opens a new approach into cancer research

juan carlos ramirez's insight:

Using the novel technique,of CRISPR/Cas9 researchers have created chromosome translocations in a human cell line and in primary mesenchymal and umbilical cord blood cells, identical to those observed in two types of cancer, the  acute myeloid leukaemia and the Ewin's sracoma

more...
No comment yet.
Rescooped by juan carlos ramirez from CRISPR-Cas System for Eukaryotic Genome Engineering
Scoop.it!

TALEN or Cas9 -- rapid, efficient and specific choices for genomic modifications -Journal of Genetics and Genomics

TALEN or Cas9 -- rapid, efficient and specific choices for genomic modifications -Journal of Genetics and Genomics | lentiviral inspired editing | Scoop.it

Precise modifications of complex genomes at the single nucleotide level have been one of the big goals for scientists working in basic and applied genetics, including biotechnology, drug development, gene therapy and synthetic biology. However, the relevant techniques for making these manipulations in model organisms and human cells have been lagging behind the rapid high throughput studies in the post-genomic era with a bottleneck of low efficiency, time consuming and laborious manipulation, and off-targeting problems. Recent discoveries of TALEs (transcription activator-like effectors) coding system and CRISPR (clusters of regularly interspaced short palindromic repeats) immune system in bacteria have enabled the development of customized TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 to rapidly edit genomic DNA in a variety of cell types, including human cells, and different model organisms at a very high efficiency and specificity. In this review, we first briefly summarize the development and applications of TALENs and CRISPR/Cas9 mediated genome editing technologies; compare the advantages and constraints of each method; particularly, discuss the expected applications of both techniques in the field of site-specific genome modification and stem cell based gene therapy; finally, propose the future directions and perspectives for readers to make the choices.


Via dromius, Amir Taheri Ghahfarokhi
more...
No comment yet.