Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders

Patent metadata

Field	Value
Publication number	US-2016354487-A1
Application number	US-201615179941-A
Country	US
Kind code	A1
Filing date	Jun 10, 2016
Priority date	Dec 12, 2013
Publication date	Dec 8, 2016
Grant date	—

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 delivery 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 SIN CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing SIN CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and 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.

First claim

1 . A non-naturally occurring or engineered self-inactivating CRISPR-Cas composition comprising:
I. a first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises:
(a) at least one first guide sequence capable of hybridizing to a target DNA,
(b) at least one tracr mate sequence, and
(c) at least one tracr sequence, and
wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,

II. a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme,
wherein parts I and II comprise a first CRISPR-Cas system, and wherein,

III. the composition further comprises
(a) at least one second guide sequence capable of hybridizing to a sequence in or of the CRISPR-Cas system,
(b) at least one tracr mate sequence, and
(c) at least one tracr sequence, and
wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, wherein parts I and III comprise a second CRISPR-Cas system,

and said composition when transcribed comprises
a first CRISPR complex comprising the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized or is hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
a second CRISPR complex comprising a CRISPR enzyme complexed with (1) the second guide sequence that is hybridized or hybridizable to a sequence of a polynucleotide comprising or encoding the CRISPR-Cas system, and (2) the tracr mate sequence that is hybridized to the tracr sequence,
wherein the first guide sequence directs sequence-specific binding of a first CRISPR complex to the target DNA, and
wherein the second guide sequence directs sequence-specific binding of a second CRISPR complex to a sequence comprising a polynucleotide comprising or encoding a component of the CRISPR-Cas system and whereby there is diminished activity of the first CRISPR-Cas system over a period of time, and the CRISPR-Cas composition is self-inactivating (“SIN CRISPR-Cas composition”).

2 . The composition of claim 1 , wherein the target DNA sequence is within a cell.

3 . The composition of claim 2 , wherein the cell is a eukaryotic cell.

4 . The composition of claim 3 , wherein the first CRISPR-Cas system and/or the second CRISPR-Cas system is codon optimized.

5 . The composition of claim 4 , wherein part II includes coding for one or more nuclear localization signals (NLSs).

6 . The composition of claim 5 , wherein the CRISPR enzyme comprises a C-terminal NLS and an N-terminal NLS.

7 . The composition of claim 1 , wherein the RNA of part I is chimeric RNA (chiRNA).

8 . The composition of claim 1 , wherein part I is encoded by a first viral vector and part II is encoded by a second viral vector.

9 . The composition of claim 8 , wherein the first and second viral vectors are lentiviral vectors or recombinant AAV.

10 . The composition of claim 9 , wherein the recombinant AAV genome comprises inverted terminal repeats (iTRs).

11 . The composition of claim 10 , wherein expression of the CRISPR enzyme is driven by the inverted terminal repeat (iTR) in the AAV genome.

12 . The composition of claim 1 , wherein the first regulatory element is a RNA polymerase type III promoter and the second regulatory element is a RNA polymerase type III promoter.

13 . The composition of claim 1 , wherein the first regulatory element is a U6 promoter or a H1 promoter.

14 . The composition of claim 1 , wherein the second regulatory element is a ubiquitous expression promoter or a cell-type specific promoter.

15 . The composition of claim 1 , wherein there is a selection marker comprising a FLAG-tag.

16 . The composition of claim 1 , which is delivered via injection.

17 . The composition of claim 1 , wherein the composition or a part thereof is delivered via a liposome, a nanoparticle, an exosome, or a microvesicle.

18 . The composition of claim 2 , wherein the first guide sequence directs sequence-specific binding of the first CRISPR complex to the target DNA sequence and alters expression of a genomic locus in the cell.

19 . The composition of claim 2 , wherein the first CRISPR complex mediates binding to or a double or single stranded DNA break, thereby editing a genomic locus in the cell.

20 . The composition of claim 1 , wherein the first and/or second CRISPR-Cas system is a multiplexed CRISPR enzyme system further comprising multiple chimeras and/or multiple multiguide sequences and a single tracr sequence.

21 . The composition of claim 1 , wherein the first CRISPR-Cas system is a multiplexed CRISPR enzyme system to minimize off-target activity.

22 . The composition of claim 1 , wherein the CRISPR enzyme is a nickase.

23 . The composition of claim 1 , wherein the CRISPR enzyme comprises one or more mutations.

24 . The composition of claim 23 , wherein the CRISPR enzyme comprises one or more mutations selected from D10A, E762A, H840A, N854A, N863A or D986A.

25 . The composition according to claim 23 , wherein the one or more mutations is in a RuvC1 domain of the CRISPR enzyme.

26 . The composition of claim 1 , wherein the CRISPR enzyme further comprises a functional domain.

27 . The composition of claim 1 , wherein the CRISPR enzyme is a Cas9.

28 . The composition of claim 1 , wherein the second complex binds to a sequence for CRISPR enzyme expression.

29 . The composition of claim 1 , wherein the second guide sequence is capable of hybridizing to (a) a sequence encoding the RNA or (b) a sequence encoding the CRISPR enzyme, or (c) a non-coding sequence comprising
i) a sequence within a regulatory element driving expression of non-coding RNA elements, ii) a sequence within a regulatory element driving expression of the CRISPR enzyme, iii) a sequence within 100 by of the ATG translational start codon of the CRISPR enzyme coding sequence, and iv) a sequence within an inverted terminal repeat of a viral vector.

30 . The composition of claim 1 , wherein the second guide sequence is expressed singularly to achieve inactivation of the first CRISPR-Cas system.

31 . The composition of claim 30 , wherein the second CRISPR complex induces a frame shift in CRISPR enzyme coding sequence causing a loss of protein expression.

32 . The composition of claim 30 , wherein the second guide sequence targets an iTR, wherein expression will result in the excision of an entire CRISPR-Cas cassette.

33 . The composition of claim 28 , wherein the second guide sequence is expressed in an array format to achieve inactivation of the first CRISPR-Cas9 system.

34 . The composition of claim 28 , wherein the second guide sequences is expressed in array format and targets both regulatory elements, thereby excising intervening nucleotides from within the first CRISPR-Cas system, effectively leading to its inactivation.

35 . The composition of claim 34 , wherein expression of the second guide sequences are driven by a U6 promoter.

36 . The composition of claim 1 , whereby the self-inactivation of the first CRISPR-Cas system limits duration of its activity and/or expression in targeted cells.

37 . T

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Univ Iowa Res Found

Inventors

• Zhang Feng
• Davidson Beverly
• Lin Chie-Yu
• Rodriguez Edgardo

Classifications

• A61P25/28

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• A61P21/04

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• A61P25/00

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