Delivery and use of the crispr-cas systems, vectors and compositions for hepatic targeting and therapy

A composition, liver target technology, applied in the manufacture of gene therapy compositions, introduction of foreign genetic material using vectors, drug combinations, etc.

Active Publication Date: 2018-05-04
THE BROAD INST INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While genome editing technologies such as designer zinc fingers, transcriptional activator-like effectors (TALEs), or homing meganucleases are available for generating targeted ge

Method used

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  • Delivery and use of the crispr-cas systems, vectors and compositions for hepatic targeting and therapy
  • Delivery and use of the crispr-cas systems, vectors and compositions for hepatic targeting and therapy
  • Delivery and use of the crispr-cas systems, vectors and compositions for hepatic targeting and therapy

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0912] Example 1: CRISPR complex activity in the nucleus of eukaryotic cells

[0913] An exemplary type II CRISPR system is the type II CRISPR locus from S. pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1 and two non-coding RNA elements tracrRNA and a short sequence composed of non-repetitive sequences. A characteristic array of repeated sequences (direct repeats) spaced apart by segments (spacers, about 30 bp each). In this system, targeted DNA double-strand breaks (DSBs) are generated in four sequential steps ( Figure 2A ). In the first step, two noncoding RNAs, pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. In the second step, the tracrRNA is hybridized to the direct repeat of the pre-crRNA, which is then processed into a mature crRNA containing a separate spacer sequence. In the third step, the mature crRNA:tracrRNA complex guides Cas9 to a DNA target consisting of the protospacer and the corresponding PAM via het...

example 2

[0944] Example 2: CRISPR System Modifications and Alternatives

[0945] The ability to program sequence-specific DNA cleavage using RNA defines a new class of genome engineering tools for a variety of research and industrial applications. Several aspects of the CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting. Optimal Cas9 activity can be dependent on free Mg present in mammalian nuclei 2+ High levels of free Mg 2+Availability of NGG motifs (see e.g. Jinek et al., 2012, Science, 337:816), and a preference for NGG motifs located just downstream of protospacers limits targeting to the human genome average capacity per 12-bp in (Fig. 9, both positive and negative strands of human chromosomal sequences were evaluated). Some of these constraints can be overcome by exploring the diversity of CRISPR loci across microbial metagenomics (see e.g. Makarova et al., 2011, Nat Rev Microbiol, 9:467) . Other CRISPR loci can be grafted i...

example 3

[0946] Example 3: Sample target sequence selection algorithm

[0947] A software program is designed to identify candidate CRISPR target sequences on both strands of the input DNA sequence based on the desired guide sequence length and CRISPR motif sequence (PAM) for the specified CRISPR enzyme. For example, 5'-N can be searched on both the input sequence and the reverse complement of the x -NGG-3' to identify the target site of Cas9 from Streptococcus pyogenes with the PAM sequence NGG. Likewise, 5'-N can be searched both on the input sequence and the reverse x -NNAGAAW-3' to identify the target site of Cas9 of Streptococcus pyogenes CRISPR1 with the PAM sequence NNAGAAW. Likewise, 5'-N can be searched both on the input sequence and the reverse x -NGGNG-3' to identify the target site of Cas9 of Streptococcus pyogenes CRISPR3 with the PAM sequence NGGNG. Can be fixed by program or specified by userN x The value "x" in , such as 20.

[0948] Since multiple occurrences in ...

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Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are deliver)' systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex, as well as methods forthe design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicityand to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

Description

[0001] Related applications and cited references [0002] Claims from U.S. Provisional Patent Applications 61 / 836,123 filed June 17, 2013, 61 / 847,537 filed July 17, 2013, 61 / 862,355 filed August 5, 2013, August 28, 2013 61 / 871,301, 61 / 915,325 filed December 12, 2013, 61 / 979,733 filed April 15, 2014, and PCT / US 2013 / 074667 filed December 12, 2013 for purposes in the United States Priority, this application is also a continuation-in-part; and as may be permitted under US law, US equivalents or national phases hereto may further claim and claim priority to PCT / US 2013 / 074667 and PCT / US 2013 / 074667 Priority of the application from which priority is claimed. [0003] The aforementioned applications, and all documents cited therein or during their prosecution or ("Application Citations") and all documents cited or referenced in these Application Citations, and all documents cited or referenced herein ( "documents cited herein"), all documents cited or referenced in documents cited ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/10C40B40/08C12N9/22
CPCC12N9/22C12N15/1082C12N15/63A01K67/0275A61K48/00C12N15/86A01K2217/052A01K2227/105A01K2267/0312A01K2267/0331A01K2267/0362C12N2710/24144C12N2740/15043C12N2750/14143C12N15/907A61P1/16A61P43/00C12N2310/20C12N15/102A61K48/0058C12N7/00A61K48/0091C12N2750/14132C12N2750/14152C12Y301/00
Inventor 张锋丛乐然霏
Owner THE BROAD INST INC
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