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Targeted non-viral DNA insertions

a dna insertion and non-viral technology, applied in the field of targeted non-viral dna insertions, can solve the problems of protein to be knocked out of the frame, many applications cannot be served by this method, and mass cell death, so as to reduce the loss of cell viability and reduce the off-target

Inactive Publication Date: 2020-11-19
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about editing the genome of a cell by inserting large nucleotide sequences into a specific region. The method reduces off-target effects and loss of cell viability. This can be useful for research and development of new treatments for genetic diseases.

Problems solved by technology

However, since these mutations are random and introduced by non-homologous end joining, they can cause a protein to be knocked out of frame (Schumann et al.
However, since the size of these oligonucleotides is limited to the length of DNA that can be chemically synthesized (<about 200 bps), and a large fraction of that is taken up by homology arms, many applications cannot be served by this method due to the limited size of integrations.
In addition to size limitations, it is well established that electroporation of naked DNA, in particular, naked DNA larger than about 200 bps, into cells often leads to massive cell death owing to the activation of intrinsic cellular defense mechanism (Cornu et al.
Although non-integrating viral vectors, such as integrase defective lentiviral vectors or adeno-associated viral (AAV) vectors, have been used to deliver large donor nucleic acid sequences to cells, these vectors require viral infection and cause off-target effects.

Method used

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  • Targeted non-viral DNA insertions
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  • Targeted non-viral DNA insertions

Examples

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examples

[0093]The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

example i

[0094]The data provided in Example I were generated as outlined in the protocol below.[0095]Clinical protocols / donor consent were established[0096]Isolation of PBMCs was performed with SepMate using the manufacturer's protocol.[0097]Isolation of Bulk T Cells was performed with EasySep using the manufacturer's protocol.[0098]Freezing was performed with Bambanker medium using the manufacturer's protocol.[0099]20 million cells per mL[0100]Thawing[0101]1 mL Roswell Park Memorial Institute Medium (RPMI) added on top of thawed cells, which were then combined and washed in media[0102]Cells rested in media only overnight prior to stimulation

Primary T Cell Culture

[0103]Media[0104]RPMI+10% FBS[0105]XVivo15+5% FBS; or[0106]Immunocult (Serum free)[0107]Useful for culturing cells in a serum free environment[0108]Stimulation[0109]1:1 CD3 / CD28 magnetic Dynabeads[0110]Ratios of 0.25:1 up to 2:1 can be used[0111]Magnetic bead removal prior to electroporation can improve efficiency[0112]Cytokines Pre...

example ii

Isolation of Human Primary T Cells for Gene Targeting

[0178]Primary human T cells were isolated from healthy human donors either from fresh whole blood samples, residuals from leukoreduction chambers after Trima Apheresis (Blood Centers of the Pacific), or leukapheresis products (StemCell). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by Ficoll centrifugation using SepMate tubes (STEMCELL, per manufacturer's instructions). T cells were isolated from PBMCs from all cell sources by magnetic negative selection using an EasySep Human T Cell Isolation Kit (STEMCELL, per manufacturer's instructions). Unless otherwise noted, isolated T cells were stimulated and used directly (fresh). When frozen cells were used, previously isolated T cells that had been frozen in Bambanker freezing medium (Bulldog Bio) per manufacturer's instructions were thawed, cultured in media without stimulation for 1 day, and then stimulated and handled as described for freshly iso...

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Abstract

Provided herein are methods and compositions for editing the genome of a cell. In some embodiments, a nucleotide sequence of at least 200 nucleotides in length is inserted into a target region in the genome of a cell.

Description

PRIOR RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 62 / 520,117 filed on Jun. 15, 2017 and U.S. Provisional Application No. 62 / 552,180 filed on Aug. 30, 2017, both of which are hereby incorporated by reference in their entireties.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT[0002]This invention was made with government support under grant no. P50 GM082250 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]The ability to introduce small mutations (indels) at targeted sites in the genome of cells by electroporating a Cas9-gRNA complex (RNP) into the cells has been developed. However, since these mutations are random and introduced by non-homologous end joining, they can cause a protein to be knocked out of frame (Schumann et al. PNAS 112(33): 10437-10442 (2015)). Other methods have been developed to introduce a...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/113C12N15/10C12N15/90C12N9/22
CPCC12N15/102C12N15/1138C12N9/22C12N15/90C12N2320/53C12N2310/20A61K48/0016
Inventor ROTH, THEODORE LEEMARSON, ALEXANDER
Owner RGT UNIV OF CALIFORNIA
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