METHODS FOR CORRECTING ALPHA-ANTITRYPSIN POINT MUTATIONS

• US 20150166984A1
• Filed: 07/08/2014
• Published: 06/18/2015
• Est. Priority Date: 12/12/2013
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Liu, David R., Komor, Alexis Christine
• Application Number:
  US14/326,290
• Publication Number:
  US 20150166984A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 38/50 acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/6472 Cysteine endopeptidases (3.4.22)
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• CPC Class Codes: C12P 19/34 : Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Y 301/00 : Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/22 : Endodeoxyribonucleases producing 3'-phosphomonoesters (3.1.22)
• CPC Class Codes: C12Y 304/22062 : Caspase-9 (3.4.22.62)
• CPC Class Codes: C12Y 305/04 : in cyclic amidines (3.5.4)
• CPC Class Codes: C12Y 305/04001 : Cytosine deaminase (3.5.4.1)
• CPC Class Codes: C12Y 305/04004 : Adenosine deaminase (3.5.4.4)
• CPC Class Codes: C12Y 305/04005 : Cytidine deaminase (3.5.4.5)
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a nucleic acid encoding a mutant α-antitrypsin protein to correct a point mutation associated with a disease or disorder, e.g., with chronic obstructive pulmonary disease (COPD) disease. The methods provided are useful for correcting an α-antitrypsin point mutation within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Claims

1. A method of editing a nucleic acid molecule encoding an α

-antitrypsin protein, the method comprising contacting the nucleic acid molecule with

(a) a fusion protein comprising a nuclease-inactive Cas9 domain and a deaminase domain; and

(b) an sgRNA targeting the fusion protein of (a) to the α

-antitrypsin-encoding nucleic acid molecule;

wherein the nucleic acid molecule comprises a T>

C and/or an A>

G point mutation in the α

-antitrypsin-encoding nucleic acid molecule as compared to a wild-type α

-antitrypsin-encoding nucleic acid molecule,

and wherein the α

-antitrypsin-encoding nucleic acid molecule is contacted with the fusion protein and the sgRNA in an amount effective and under conditions suitable for the deamination the mutant C or G nucleotide base.

2. The method of claim 1, wherein the deaminase is a cytidine deaminase.

3. The method of claim 1, wherein the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.

4. The method of claim 3, wherein the deaminase is an APOBEC1 family deaminase.

5. The method of claim 3, wherein the deaminase is an activation-induced cytidine deaminase (AID).

6. The method of claim 1, wherein the deaminase is an ACF1/ASE deaminase.

7. The method of claim 1, wherein the deaminase is an adenosine deaminase.

8. The method of claim 7, wherein the deaminase is an ADAT family deaminase.

9. The method of claim 1, wherein the deaminase domain is fused to the N-terminus of the Cas9 domain.

10. The method of claim 1, wherein the deaminase domain is fused to the C-terminus of the Cas9 domain.

11. The method of claim 1, wherein the Cas9 domain and the deaminase domain are fused via a linker.

12. The method of claim 1, wherein the linker comprises a (GGGGS)n (SEQ ID NO:

91), a (G)n, an (EAAAK)n (SEQ ID NO;

     5), or an (XP)n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30.

13. The method of claim 1, wherein the T>

C and/or an A>

G point mutation in the α

-antitrypsin-encoding nucleic acid molecule is associated with a disease or disorder, and wherein the deamination of the mutant C or G residue results in a sequence that is not associated with a disease or disorder.

14. The method of claim 1, wherein the deamination results in a correction of the T>

C and/or an A>

G point mutation to restore the wild-type sequence.

15. The method of claim 1, wherein the sequence associated with the disease or disorder encodes a protein, and wherein the deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein.

16. The method of claim 1, wherein the T>

C and/or an A>

G point mutation causes an amino acid sequence in the α

-antitrypsin protein as compared to the wild type α

-antitrypsin protein.

17. The method of claim 1, wherein the α

-antitrypsin protein comprises an L55P substitution caused by an T>

C point mutation in codon 55 of the α

-antitrypsin gene.

18. The method of claim 17, wherein the α

-antitrypsin point mutation is associated with chronic obstructive pulmonary disease (COPD).

19. The method of claim 17, wherein the contacting results in deamination of the mutant cytidine residue in codon 55 of the α

-antitrypsin gene, thus correcting the T>

C point mutation.

20. The method of claim 1, wherein the contacting is in vivo in a subject having or diagnosed with the disease or disorder.

21. The method of claim 1, wherein the method further comprises detecting the deamination the mutant C or G nucleotide base.

22. The method of claim 21, wherein the detecting is via PCR.