DNA sequences comprising dystrophin minigenes and methods of use thereof

• US 7,001,761 B2
• Filed: 04/30/2001
• Issued: 02/21/2006
• Est. Priority Date: 04/28/2000
• Status: Expired due to Term

• Current Assignee: Asklepios Biopharmaceutical Incorporated (Bayer AG)
• Original Assignee: Asklepios Biopharmaceutical Incorporated (Bayer AG)
• Inventors: Xiao, Xiao
• Primary Examiner(s): Priebe, Scott D.
• Assistant Examiner(s): WHITEMAN, BRIAN A
• Application Number:
  US09/845,416
• Publication Number:
  US 20030171312A1
• Time in Patent Office: 1,758 Days
• Field of Search: 435/320.1, 435/455, 536/23.1, 536/23.5, 514/74, 424/99.2
• US Class Current: 435/320.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/4708 Duchenne dystrophy
• CPC Class Codes: C12N 2799/025 where the vector is derived from a parvovirus

Abstract

The present invention provides a series of novel dystrophin minigenes that retain the essential biological functions. The expression of the dystrophin minigenes may be controlled by a regulatory element along with a small polyadenylation signal. The entire gene expression cassettes may be readily packaged into a viral vector, preferably an AAV vector. The present invention further defines the minimal functional domains of dystrophin and provides ways to optimize and create new versions of dystrophin minigenes. Finally, the present invention provides a method of treatment for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

Claims

1. An isolated nucleotide sequence comprising a dystrophin minigene encoding a protein or the complement of the dystrophin minigene, wherein the protein comprises:

(a) a N-terminal domain of a dystrophin protein or a modified N-terminal domain of the dystrophin protein;

(b) five rod repeats of the dystrophin protein;

(c) an H1 domain of a dystrophin gene and an H4 domain of the dystrophin protein; and

(d) a cysteine-rich domain of the dystrophin protein, wherein said nucleotide sequence has fewer than 5,000 nucleotides.

2. An isolated nucleotide sequence comprising a dystrophin mingene encoding a protein or the complement of the dystrophin minigene, wherein the protein comprises:

(a) a N-terminal domain of a dystrophin protein or a modified N-terminal domain of the dystrophin protein;

(b) six rod repeats of the dystrophin protein;

(c) an H1 domain of a dystrophin protein and an H4 domain of the dystrophin protein; and

(d) a cysteine-rich domain of the dystrophin protein, wherein said nucleotide sequence has fewer than 5,000 nuclotides.

3. An isolated nucleotide sequence consisting of SEQ ID NO:

2, or which is the complement of SEQ ID NO;

2.

9. A recombinant adeno-associated virus vector, comprising any one of the nucleotide sequences of claims 3, 4, 5, 6, 7 and 8, operably linked to an expression control element.

10. The recombinant adeno-associated virus vector of claim 9, wherein the control element is an MCK promoter or a CMV promoter.

4. An isolated nucleotide sequence consisting of SEQ ID NO:

6, or which is the complement of SEQ ID NO;

6.

11. A recombinant adeno-associated virus vector, comprising the nucleotide sequence of claim 4, operably linked to an expression control element.

5. An isolated nucleotide sequence consisting of SEQ ID NO:

9, or which is the complement of SEQ ID NO;

9.

12. A recombinant adeno-associated virus vector, comprising the nucleotide sequence of claim 5, operably linked to an expression control element.

6. An isolated nucleotide sequence consisting of SEQ ID NO:

10, or which is the complement of SEQ ID NO;

10.

13. A recombinant adeno-associated virus vector, comprising the nucleotide sequence of claim 6, operably linked to an expression control element.

7. An isolated nucleotide sequence consisting of SEQ ID NO:

12, or which is the complement of SEQ ID NO;

12.

14. A recombinant adeno-associated virus vector, comprising the nucleotide sequence of claim 7, operably linked to an expression control element.

8. An isolated nucleotide sequence consisting of SEQ ID NO:

14, or which is the complement of SEQ ID NO;

14.

15. A recombinant adeno-associated virus vector, comprising the nucleotide sequence of claim 8, operably linked to an expression control element.