DELIVERY, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR TARGETING AND MODELING DISEASES AND DISORDERS OF POST MITOTIC CELLS
First Claim
1. A method of modifying an organism or a non-human organism by manipulation of a post-mitotic cell target sequence in a genomic locus of interest, to thereby invoke a phenotypic change in the cell, comprisingdelivering a non-naturally occurring or engineered composition comprising:
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I. a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises;
(a) a guide sequence capable of hybridizing to a post-mitotic cell target sequence in a eukaryotic cell,(b) a tracr mate sequence, and(c) a tracr sequence, andII. a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,wherein (a), (b) and (c) are arranged in a 5′
to 3′
orientation,wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the post-mitotic cell target sequence, andwherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the post-mitotic cell target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,wherein the polynucleotide sequence encoding the CRISPR enzyme is operably linked to regulatory sequence(s) expression of the CRISPR enzyme, whereby with expression of the CRISPR enzyme there is assembly of the CRISPR complex in the post-mitotic cell, and manipulation thereof.
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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 delivery systems and tissues or organ which include post mitotic cells 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 for the 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 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.
99 Citations
44 Claims
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1. A method of modifying an organism or a non-human organism by manipulation of a post-mitotic cell target sequence in a genomic locus of interest, to thereby invoke a phenotypic change in the cell, comprising
delivering a non-naturally occurring or engineered composition comprising: -
(A)—
I. a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises;(a) a guide sequence capable of hybridizing to a post-mitotic cell target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences, wherein (a), (b) and (c) are arranged in a 5′
to 3′
orientation,wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the post-mitotic cell target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the post-mitotic cell target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, wherein the polynucleotide sequence encoding the CRISPR enzyme is operably linked to regulatory sequence(s) expression of the CRISPR enzyme, whereby with expression of the CRISPR enzyme there is assembly of the CRISPR complex in the post-mitotic cell, and manipulation thereof. - View Dependent Claims (2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 25, 26, 27, 28, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises; (a) a first guide sequence capable of hybridizing to the first target sequence, (b) a first tracr mate sequence, (c) a first tracr sequence, (d) a second guide sequence capable of hybridizing to the second target sequence, (e) a second tracr mate sequence, and (f) a second tracr sequence, and optionally, wherein a linker sequence is present between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem; and II. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (a), (b), (c), (d), (e) and (f) are arranged in a 5′
to 3′
orientation, wherein the polynucleotide sequence comprises a linker sequence between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem, and wherein when transcribed, the first and the second tracr mate sequence hybridize to the first and second tracr sequence respectively and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,or II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and wherein components I and II are located on the same or different vectors of the system, and when transcribed, a first tracr mate sequence hybridizes to a first tracr sequence and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively; wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized to the first target sequence, and (2) the first tracr mate sequence that is hybridized to the first tracr sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized to the second target sequence, and (2) the second tracr mate sequence that is hybridized to the second tracr sequence, wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the non-human organism by minimizing off-target modifications.
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5. A method of modifying an organism or a non-human organism by manipulation of a post-mitotic cell target sequence in a genomic locus of interest comprising
delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors operably encoding a composition for expression thereof, wherein the composition comprises: -
a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises (A) a guide sequence capable of hybridizing to a kidney target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences, wherein (A), (b) and (c) are arranged in a 5′
to 3′
orientation,wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the kidney target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the kidney target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence. - View Dependent Claims (6)
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7. A method of treating or inhibiting a condition caused by a defect in a kidney target sequence in a genomic locus of interest in a subject or a non-human subject in need thereof comprising modifying the subject or a non-human subject by manipulation of the kidney target sequence and wherein the condition is susceptible to treatment or inhibition by manipulation of the kidney target sequence comprising providing treatment comprising:
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delivering a non-naturally occurring or engineered composition comprising an AAV or lentivirus vector system, comprising one or more AAV or lentivirus vectors operably encoding a composition for expression thereof, wherein the kidney target sequence is manipulated by the composition when expressed, wherein the composition comprises; (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises (A) a guide sequence capable of hybridizing to a kidney target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (A), (b) and (c) are arranged in a 5′
to 3′
orientation,wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the kidney target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the kidney target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, or (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to (A) a guide sequence capable of hybridizing to a kidney target sequence in a eukaryotic cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the kidney target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the kidney target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24)
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30. A composition comprising:
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(A)—
I. a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises;(A) a guide sequence capable of hybridizing to a kidney target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences, wherein (A), (b) and (c) are arranged in a 5′
to 3′
orientation,wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the kidney target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the kidney target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, or (B) I. polynucleotides comprising; (A) a guide sequence capable of hybridizing to a kidney target sequence in a eukaryotic cell, and (b) at least one or more tracr mate sequences, II. a polynucleotide sequence encoding a CRISPR enzyme, and III. a polynucleotide sequence comprising a tracr sequence, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the kidney target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the kidney target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA; for use in medicine or therapy;
or for use in a method of modifying an organism or a non-human organism by manipulation of a kidney target sequence in a genomic locus of interest;
or for use in a method of treating or inhibiting a condition caused by a defect in a kidney target sequence in a genomic locus of interest;
or for use in ex vivo gene or genome editing. - View Dependent Claims (31)
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Specification