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EP-3653704-B1 - COMPOSITIONS AND METHODS OF USE OF CRISPR-CAS SYSTEMS IN NUCLEOTIDE REPEAT DISORDERS

EP3653704B1EP 3653704 B1EP3653704 B1EP 3653704B1EP-3653704-B1

Inventors

  • ZHANG, FENG
  • DAVIDSON, BEVERLY
  • LIN, Chie-yu
  • RODRIGUEZ, EDGARDO

Dates

Publication Date
20260513
Application Date
20141212

Claims (18)

  1. A non-naturally occurring or engineered self-inactivating CRISPR-Cas composition comprising: I. a first promoter operably linked to a polynucleotide sequence encoding a first CRISPR-Cas system RNA that 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 promoter operably linked to a polynucleotide sequence encoding a Cas9, wherein parts I and II comprise a first CRISPR-Cas system, and, III. the composition further comprises (a) at least one second guide sequence capable of hybridizing to a sequence in or of the first 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 II and III comprise a second CRISPR-Cas system, wherein parts I, II, and III are encoded by one or more plasmid or viral vectors; wherein said Cas9 forms a first CRISPR complex comprising Cas9 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, and wherein said Cas9 forms a second CRISPR complex comprising Cas9 complexed with (1) the second guide sequence that is hybridized or hybridizable to a sequence of a polynucleotide comprising or encoding the first 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 the first CRISPR complex to the target DNA, and wherein the second guide sequence directs sequence-specific binding of the second CRISPR complex to a sequence comprising a polynucleotide comprising or encoding a component of the first 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.
  2. The composition of claim 1, wherein the target DNA sequence is within a cell; optionally, wherein the cell is a eukaryotic cell.
  3. The composition of claim 2, wherein the polynucleotide sequence encoding Cas9 is codon optimized for expression in a eukaryotic cell.
  4. The composition of claim 3, wherein the Cas9 comprises one or more nuclear localization signals (NLSs); optionally comprising a C-terminal NLS and an N-terminal NLS.
  5. The composition according to any one of the preceding claims, wherein part I is encoded by a first viral vector and part II is encoded by a second viral vector; optionally, wherein the first and second viral vectors are lentiviral vectors or recombinant AAV.
  6. The composition of claim 5, wherein the recombinant AAV genome comprises inverted terminal repeats (iTRs); optionally, wherein expression of Cas9 is driven by the inverted terminal repeat (iTR) in the AAV genome.
  7. The composition of any one of the preceding claims, - wherein the promoter of I is a U6 promoter or a H1 promoter; - wherein the promoter of II is a ubiquitous expression promoter or a cell-type specific promoter; or - wherein the promoter of I is a RNA polymerase type III promoter and the promoter of II is a RNA polymerase type III promoter.
  8. 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.
  9. The composition of claim 2, wherein the first CRISPR complex creates a double or single stranded DNA break, thereby editing a genomic locus in the cell.
  10. The composition of any of the preceding claims, wherein the first and/or second CRISPR-Cas system is a multiplexed CRISPR-Cas system further comprising multiple chimeras and/or multiple multiguide sequences and a single tracr sequence.
  11. The composition according any of the preceding claims, wherein Cas9 is a nickase.
  12. The composition of any one of the preceding claims, wherein the second CRISPR complex binds to a sequence for Cas9 expression.
  13. The composition of any of the preceding claims, wherein the second guide sequence is capable of hybridizing to (a) a sequence encoding the first CRISPR-Cas system RNA, (b) a sequence encoding Cas9, or (c) a non-coding sequence comprising i) a sequence within a regulatory element driving expression of the first CRISPR-Cas system RNA, ii) a sequence within a regulatory element driving expression of the Cas9, iii) a sequence within 100bp of the ATG translational start codon of the Cas9 coding sequence, and iv) a sequence within an inverted terminal repeat of a viral vector.
  14. The composition according to any of the preceding claims, further comprising a recombination template polynucleotide.
  15. A composition according to any one of the preceding claims for use in a method of treatment of a CTG, CAG, CGG, CCG, GAA, or TTC trinucleotide repeat disease or disorder; a CCTG tetranucleotide repeat disease or disorder; a ATTCT or AGAAT pentanucleotide repeat disease or disorder; a GGGGCC hexanucleotide repeat disease or disorder; or a CCCCGCCCCGCG or CGCGGGGCGGGG dodecanucleotide repeat disease or disorder, wherein the method is not a method of modifying the germ line genetic identity of human beings.
  16. The composition for use of claim 15, wherein part III is introduced into the cell sequentially or at a time point after the introduction of parts I and II.
  17. The composition for use of claim 15, the composition is provided by local delivery.
  18. The composition for use of claim 15, wherein the composition is provided as a single dose.

Description

RELATED APPLICATIONS This application claims priority from US provisional patent applications Serial Nos. 61/915,150, filed December 12, 2013; and 62/010,888 and 62/010,879, both filed June 11, 2014. The foregoing applications, and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein, may be employed in the practice of the invention. FIELD OF THE INVENTION The present invention generally relates to the delivery, engineering, optimization and therapeutic applications of systems, methods, and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. The invention relates to delivery, use, control and therapeutic applications of CRISPR-Cas systems and compositions, for brain and central nervous system (CNS) disorders and diseases. The invention relates to delivery, use, control and therapeutic applications of CRISPR-Cas systems and compositions, for nucleotide repeat elements (e.g., trinucleotide repeat, tetranucleotide repeat, nucleotide expansion elements) disorders and diseases. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with government support under the NIH Pioneer Award (1DP1MH100706) awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome. SUMMARY OF THE INVENTION The invention is set out in the appended set of claims. The references to methods of treatment in the present description are to be interpreted as references to the compositions of the present invention for use in a method of treatment of the human or animal body by therapy. The invention provides in an aspect, a non-naturally occurring or engineered self-inactivating CRISPR-Cas composition comprising: I. a first promoter operably linked to a polynucleotide sequence encoding a first CRISPR-Cas system RNAthat 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 promoter operably linked to a polynucleotide sequence encoding a Cas9, wherein parts I and II comprise a first CRISPR-Cas system, and,III. the composition further comprises (a) at least one second guide sequence capable of hybridizing to a sequence in or of the first 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 II and III comprise a second CRISPR-Cas system,wherein parts I, II, and III are encoded by one or more plasmid or viral vectors;wherein said Cas9 forms a first CRISPR complex comprising Cas9 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,and wherein said Cas9 forms a second CRISPR complex comprising Cas9 complexed with (1) the second guide sequence that is hybridized or hybridizable to a sequence of a polynucleotide comprising or encoding the first 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 the first CRISPR complex to the target DNA, andwherein the second guide sequence directs sequence-specific binding of the second CRISPR complex to a sequence comprising a polynucleotide comprising or encoding a component of the first CRISPR-Cas system and whereby there is diminished activity of the first CRISPR-Cas system over a period of time, and the C