Antisense compound for inducing immunological tolerance

• US 8,008,469 B2
• Filed: 11/15/2007
• Issued: 08/30/2011
• Est. Priority Date: 10/23/2003
• Status: Expired due to Fees

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Moulton, Hong M., Hinrichs, David J., Mourich, Dan V., Iversen, Patrick L.
• Primary Examiner(s): McGarry; Sean
• Application Number:
  US11/941,033
• Publication Number:
  US 20090111977A1
• Time in Patent Office: 1,384 Days
• Field of Search: None
• US Class Current: 536/24.5
• CPC Class Codes: A61K 31/675 having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 2319/10 containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• CPC Class Codes: C12N 15/1132 against retroviridae, e.g. HIV
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2810/6054 Retroviridae

Abstract

A method and conjugate for selectively killing antigen-activated T cells are disclosed. The conjugate is composed of a substantially uncharged antisense compound targeted against the human cFLIP protein, and a reverse TAT (rTAT) polypeptide coupled covalently to the antisense compound. The rTAT polypeptide is effective to produce selective uptake of the conjugate into antigen-activated T cells, relative to the uptake of the conjugate into non-activated T cells. The cFLIP antisense compound causes activation induced cell death (AICD) of activated lymphocytes. The method is useful in treating transplantation rejection and autoimmune conditions.

Claims

1. An antisense conjugate for use in inducing immunologic tolerance in a subject, comprising

(a) a substantially uncharged antisense oligonucleotide compound

(i) composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an, adjacent subunit, and containing 20-25 subunits,

(ii) comprising the base sequence identified by SEQ ID NO;

17,

(iii) effective, when hybridized to the processed human cFLIP transcript, to block expression of cFLIP in lymphocytes, and

(b) covalently coupled to the antisense oligonucleotide compound, an arginine-rich peptide effective to enhance the uptake of the antisense compound into lymphocytes.

2. The conjugate of claim 1, wherein the morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

3. The conjugate of claim 2, wherein X═

NR₂, where each R is independently hydrogen or methyl.

6. The conjugate of claim 1, wherein said arginine-rich peptide has the sequence identified by SEQ ID NO:

1 or 2.

7. The conjugate of claim 1, wherein the lymphocytes are naï

ve T-cells.

8. The conjugate of claim 1, wherein the lymphocytes are antigen-activated T-cells.

4. An antisense conjugate for use in inducing immunologic tolerance in a subject, comprising

(a) a substantially uncharged antisense oligonucleotide compound

(i) composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, and containing 12-40 subunits,

(ii) having a base sequence that is complementary to at least 12 contiguous bases in a region extending from −

30 to +30 bases with respect to the AUG start site in a processed human cFLIP transcript, corresponding to base positions 452 to 512 in SEQ ID 16, and

(iii) effective, when hybridized to the processed human cFLIP transcript, to block expression of cFLIP in lymphocytes, and

(b) covalently coupled to the antisense oligonucleotide compound, an arginine-rich peptide effective to enhance the uptake of the antisense compound into lymphocytes wherein said arginine-rich peptide has the sequence identified by SEQ ID NO;

2.

5. The conjugate of claim 4, wherein said antisense compound has a base sequence that is complementary to at least 12 contiguous bases extending from −

12 to +12 bases with respect to the AUG start site in a processed human cFLIP transcript, corresponding to SEQ ID NO;

4 in SEQ ID NO;

16.

9. The conjugate of claim 4, wherein the morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure:

10. The conjugate of claim 9, wherein X═

NR₂, where each R is independently hydrogen or methyl.