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Method and antisense composition for selective inhibition of HIV infection in hematopoietic cells

a technology of hematopoietic cells and antiviral conjugates, applied in the field of new drugs, can solve the problems of no curative anti-retroviral drugs against aids, emergence of viral resistance, etc., and achieve the effect of a greater intracellular uptak

Inactive Publication Date: 2005-10-06
AVI BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0040] In one aspect, the invention includes a method of achieving selective uptake of a substantially uncharged antisense compound into activated human hematopoietic cells, e.g., macrophage or T lymphocyte cells. The method includes exposing a population of human macrophage or T lymphocyte cells that include activated human macrophage or T lymphocyte cells to an rTAT-antisense conjugate composed of (i) the antisense compound and (ii) covalently coupled thereto, a reverse TAT (rTAT) polypeptide having the sequence identified as SEQ ID NO:1. This exposing is effective to achieve a greater level of intracellular uptake of the oligonucleotide analog into activated macrophage or T-lymphocyte cells than is achieved (i) by exposing non-activated macrophage or T-lymphocyte cells to the same antisense conjugate, or (ii) by exposing activated macrophage or T-lymphocyte cells to the oligonucleotide analog in the absence of the rTAT polypeptide.

Problems solved by technology

Although considerable effort is being put into the successful design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist.
Despite the success obtained with HAART, approximately 30-50% of patients ultimately fail resulting in the emergence of viral resistance.

Method used

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  • Method and antisense composition for selective inhibition of HIV infection in hematopoietic cells
  • Method and antisense composition for selective inhibition of HIV infection in hematopoietic cells
  • Method and antisense composition for selective inhibition of HIV infection in hematopoietic cells

Examples

Experimental program
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example 1

Preparation of rTAT-Antisense Conjugates

[0146] A. Production of PMO and Peptide Conjugated PMOs.

[0147] The PMOs were synthesized at AVI BioPharma (Corvallis, Oreg.) as previously described (Summerton and Weller, 1997). Purity of full length oligomers was >95% as determined by reverse-phase high-pressure liquid chromatography (HPLC) and MALDI TOF mass spectroscopy. Peptide conjugated forms of the PMO where produced by attaching the carboxy terminal cysteine of the peptide to the 5′ end of the PMO through a cross-linker N-[□-maleimidobutyryloxy] succinimide ester (GMBS) (Moulton and Moulton, 2003), as detailed below in section C. The peptides used in this study designated as P002 (RRRQRRKKRC, SEQ ID NO:1) (Moulton and Moulton, 2003) and P003 (RRRRRRRRRFFC, SEQ ID NO:2). The lyophilized PMO or peptide-conjugated PMO were dissolved in sterile H2O prior to use in cell cultures or dilution in PBS prior to injection in to mice.

[0148] B. 3′-Fluoresceination of a PMO (Phosphorodiamidate-L...

example 2

Uptake of rTAT-Antisense Conjugates Selectively into Activated T Cells

[0158] The DO11.10 transgenic mouse system (Murphy, Heimberger et al. 1990) was used as a source of splenocytes and T cells. This transgenic mouse contains the gene for the T cell receptor (TCR) from the T cell hybridoma, DO11.10, that recognizes chicken ovalbumin (OVA). Virtually all thymocytes and peripheral T cells in these mice express the OVA-TCR which is detected by the KJ26 monoclonal antibody.

[0159] A. Uptake in Naïve and Activated Murine Lymphocytes

[0160] Freshly isolated splenocytes from B6 mice were plated (1.5 million / well) into 96 well V-bottom plates and incubated with PMO-fl, P002-PMO-fl or P003-PMO-fl [10 μM, 10 μM and 5 μM in culture media, respectively]. Lymphocyte activating substances derived from bacterial (LPS), murine cytokine (Gamma IFN), mitogenic plant lectin (PHA), chemical activator (PMA+ION) or culture media control (naïve cell treatment) were added to individual cultures as follows...

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Abstract

A method and conjugate for selectively targeting activated hematopoietic cells, e.g., macrophage or T-lymphocyte cells, are disclosed. The conjugate is composed of a substantially uncharged antisense compound targeted against HIV, 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 activated, HIV-infected cells, e.g., activated, HIV-infected macrophage and T-lymphocyte cells. An exemplary embodiment of the invention provides an antisense compound directed to the HIV Vif gene, causing the production of defective HIV virions in an infected individual.

Description

[0001] This application claims priority to U.S. Provisional Ser. No. 60 / 514,064 filed on Oct. 23, 2003 which is incorporated herein in its entirety by reference.FIELD OF THE INVENTION [0002] The present invention is drawn to novel antiviral conjugates and their use in inhibiting HIV infection and replication in hematopoietic cells, in particular, macrophage and T lymphocyte cells. References [0003] Agrawal, S., S. H. Mayrand, et al. (1990). “Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides.”Proc Natl Acad Sci USA 87(4): 1401-5. [0004] Akhtar, S., S. Basu, et al. (1991). “Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes).”Nucleic Acids Res 19(20): 5551-9. [0005] Aldrovandi, G. M. and J. A. Zack (1996). “Replication and pathogenicity of human immunodeficiency virus type 1 accessory gene mutants in SCID-hu mice.”J Virol 70(3): 1505-11. [0006] Anderson, C. M., W. Xiong, et al. (1999). “Distribution ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61KA61K31/675A61K48/00C12N15/113
CPCA61K31/675C07K2319/10C12N15/1132C12N2310/11C12N2810/6054A61P31/18A61P37/06
Inventor MOURICH, DANIVERSEN, PATRICKBESTWICK, RICHAD
Owner AVI BIOPHARMA
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