Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
Nat Biotechnol ; 42(3): 458-469, 2024 Mar.
Article in English | MEDLINE | ID: mdl-37127662

ABSTRACT

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.


Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , Gene Knock-In Techniques , Transgenes/genetics
2.
Nat Med ; 25(2): 229-233, 2019 02.
Article in English | MEDLINE | ID: mdl-30664785

ABSTRACT

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.


Subject(s)
Gene Editing , Leber Congenital Amaurosis/genetics , Leber Congenital Amaurosis/physiopathology , Animals , Cell Line , Gene Knock-In Techniques , Humans , Mice , Primates , Reproducibility of Results , Vision, Ocular
3.
BMC Genomics ; 19(1): 212, 2018 03 21.
Article in English | MEDLINE | ID: mdl-29562890

ABSTRACT

BACKGROUND: Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations. RESULTS: To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290, and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes. CONCLUSIONS: UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Gene Rearrangement , Genome, Human , INDEL Mutation , Osteosarcoma/diagnosis , Antigens, Neoplasm/genetics , Bone Neoplasms/diagnosis , Bone Neoplasms/genetics , Cell Cycle Proteins , Cells, Cultured , Cytoskeletal Proteins , Genomics/methods , Humans , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Osteosarcoma/genetics , Sequence Deletion , T-Lymphocytes/metabolism , T-Lymphocytes/pathology
4.
Genome Biol ; 13(3): R23, 2012.
Article in English | MEDLINE | ID: mdl-22455878

ABSTRACT

We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA.


Subject(s)
Escherichia coli/genetics , Microbial Consortia/genetics , Prochlorococcus/genetics , RNA, Bacterial/genetics , Rhodobacter sphaeroides/genetics , Chemical Fractionation , DNA, Complementary/analysis , DNA, Complementary/biosynthesis , Feces/microbiology , Gene Expression Regulation, Bacterial , High-Throughput Nucleotide Sequencing , Humans , RNA, Messenger/analysis , RNA, Messenger/biosynthesis
5.
Am J Respir Cell Mol Biol ; 40(6): 751-5, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19029017

ABSTRACT

The destruction of elastic fibers has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Emphysema has been described in autosomal dominant cutis laxa, which can be caused by mutations in the elastin gene. Previously, a rare functional mutation in the terminal exon of elastin was found in a case of severe, early-onset COPD. To test the hypothesis that other similar elastin mutations may predispose to COPD, we screened 90 probands from the Boston Early-Onset COPD Study and 90 smoking control subjects from the Normative Aging Study for mutations in elastin exons using high-resolution DNA melt analysis followed by resequencing. Rare nonsynonymous single-nucleotide polymorphisms (SNPs) seen only in cases were examined for segregation with airflow obstruction within pedigrees. Common nonsynonymous SNPs were tested for association with COPD in a family-based analysis of 949 subjects from the Boston Early-Onset COPD Study, and in a case-control analysis in 389 COPD cases from the National Emphysema Treatment Trial and 472 control subjects from the Normative Aging Study. Of 28 elastin variants found, 3 were nonsynonymous SNPs found only in cases. The previously described Gly773Asp mutation was found in another proband. The other two SNPs did not clearly segregate with COPD within families. Two common nonsynonymous SNPs did not demonstrate significant associations in either a family-based or case-control analysis. Exonic SNPs in the elastin gene do not appear to be common risk factors for severe COPD.


Subject(s)
Elastin/genetics , Elastin/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/metabolism , Adult , Aged , Case-Control Studies , Emphysema/genetics , Emphysema/metabolism , Exons , Female , Genetic Variation , Humans , Male , Middle Aged , Pedigree , Polymorphism, Genetic , Polymorphism, Single Nucleotide
SELECTION OF CITATIONS
SEARCH DETAIL
...