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

a technology of hematopoietic cells and antisense, 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 effects of inhibiting the synthesis of hiv vif protein, inhibiting the reverse transcription of viral rna, and inhibiting the mrna transcript transcription

Inactive Publication Date: 2007-02-15
AVI BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The invention is an antiviral compound that targets the human immunodeficiency virus (HIV-1). The compound is made up of a sequence of 12 to 40 morpholino subunits that have a complementary sequence to a region of HIV-1 positive strand RNA. The compound can form a heteroduplex structure with the viral RNA and inhibit its synthesis, transcription, and reverse transcription. The compound can selectively inhibit the replication of HIV-1 in infected human hematopoietic cells."

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
Comparison scheme
Effect test

example 1

Preparation Antisense PMO And Peptide Conjugates

[0152] A. Production of PMO and Peptide Conjugated PMOs.

[0153] The PMOs were synthesized at AVI BioPharma (Corvallis, OR) 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.

[0154] A schematic of a synthetic pathway that can be u...

example 2

Uptake of rTAT-Antisense Conjugates Selectively into Activated T Cells

[0166] 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.

[0167] A. Uptake in Naïve and Activated Murine Lymphocytes

[0168] 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 (naive cell treatment) were added to individual cultures as follow...

example 3

Inhibition of HIV-1 Replication in Human H9 Cells by a Peptide-Conjugated Antisense PMO Targeted to the HIV-1 Vif AUG Start Codon

[0171] The human T-cell line H9 was grown and harvested using standard protocols. Cells were pelleted and resuspended in RPMI-1640 supplemented with 0.1% fetal bovine serum (FBS). From this cell suspension, 5×10ˆ6 cells were infected in bulk with HIV-1 (strain NL4-3) at a multiplicity of infection (MOI) equal to 0.001 in a T25 flask The cells were incubated in the presence of HIV-1 for 2 hours at 37 degrees C. The cells were pelleted by centrifugation and resuspended in 20 ml of RPMI-1640+10% FBS. The infected cell suspension was used to seed a 24 well plate at 1×10ˆ5 cells per well. The final volume per well was adjusted to one ml. Peptide conjugated Vif-AUG4 PMO (Vif4-P007; SEQ ID NO:5 conjugated to SEQ ID NO:3) was added to each well at concentrations ranging from 10 to 5000 nanomolar and incubated for 5-7 days at 37 degrees C. A P007 conjugated scramb...

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Abstract

The invention provides antisense antiviral compounds and methods of their use in inhibition of growth of human immunodeficiency virus-1 (HOV-1), as in treatment of a viral infection. The antisense antiviral compounds have morpholino subunits linked by uncharged phosphorodiamidate linkages interspersed with cationic phosphorodiamidate linkages. An exemplary embodiment of the invention provides an antisense compound directed to the HIV Vif gene, causing the production of defective HIV- 1 virions in an infected individual.

Description

[0001] This is a continuation-in-part of U.S. patent application Ser. No. 10 / 971,959, filed Oct. 21, 2004, now pending, which claims the benefit of priority to U.S. Provisional Application No. 60 / 514,064, filed Oct. 23, 2003. Both applications are incorporated herein by reference.FIELD OF THE INVENTION [0002] The present invention is drawn to novel antiviral antisense oligomers and their use in inhibiting HIV- 1 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 ty...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C07F9/6533A61KA61K31/675C12N15/113
CPCA61K31/675C07K2319/10C12N2810/6054C12N2310/11C12N15/1132A61P31/18A61P37/06
Inventor MOURICH, DAN V.IVERSEN, PATRICK L.BESTWICK, RICHARD K.WELLER, DWIGHT D.
Owner AVI BIOPHARMA
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