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Ribozymes targeting retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs

A sequence and ribozyme technology, applied in the direction of medical preparations containing active ingredients, medical raw materials derived from viruses/phages, antiviral agents, etc., can solve short serum half-life, rapid bile clearance rate, poor oral utilization, etc. question

Inactive Publication Date: 2006-08-02
GENE SHEARS PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most protease inhibitors have short serum half-lives, rapid bile clearance and poor oral availability (Debouck, 1992)

Method used

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  • Ribozymes targeting retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs
  • Ribozymes targeting retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs
  • Ribozymes targeting retroviral packaging sequence expression constructs and recombinant retroviruses containing such constructs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Cleavage of Mo-MLV Psi Packaging Sequence Catalyzed by Ribozyme in Vitro

[0142] To show that the target site is indeed cleavable, an in vitro cleavage reaction was performed in cell culture medium prior to detection of the ribozyme.

[0143]Four sites were selected in the Mo-MLV packaging region based on the presence of GUC bases and the potential accessibility of sites within the RNA secondary structure suggested from Zuker's FOLDRNA program (Zuker et al., 1981). These sites were named 243, 274, 366 and 553 based on the nucleotide distance from the 5' end of the viral transcript (see Figure 2). These nucleotide positions are described in RNA tumor viruses (Coffin, 1985). Two types of ribozymes were designed: three mononucleases targeting positions 243, 274, and 366, respectively, with an arm-length range of 12 nucleotides, and one multinuclease targeting all four sites Enzymes with indirect arms of sequence length between each target site. The sites and overall de...

Embodiment 2

[0149] Anti-Mo-MLV packaging site (Psi) construct

[0150] After exhibiting efficient in vitro cleavage, the engineered ribozyme along with a long antisense sequence complementary to the Psi packaging region was cloned into neo r 3' untranslated region of the gene (Figure 4). neo r is a prokaryotic gene encoding a phosphorylase and thereby inactivates neomycin or the neomycin analogue G418. The latter is toxic to mammalian cells, exogenous neo r The expression of the gene allows the cell to survive. with neo r The construct of the gene-associated SV40 promoter is within the mammalian expression vector pSV2neo and is shown schematically in FIG. 4 .

[0151] The ribozyme insert and antisense control were cloned into neo by blunt end ligation r Sma I site in the 3' untranslated region. The resulting vectors were designated pSV243, pSV274, pSV366, pSVM7 and pSVasPsi (antisense construct), respectively.

Embodiment 3

[0153] Transfection of the construct into the 3T3-Mo-MLV producing cell line

[0154] Various pSV2neo-based constructs were transfected into 3T3-Mo-MLV cells using the calcium phosphate transfection method (Chen et al., 1987). Positive colonies were those formed after 9-12 days of growth in the presence of 500 μg / ml G418. For each construct, 4-7 colonies were isolated using cloning cylinders. These colonies were grown, stored in liquid nitrogen, and used for further testing. After 10-14 days of selection in 500 μg / ml G418, several stable clonal cell lines were established for each construct. To determine the integration of the transfected DNA expression constructs, genomic DNA was prepared from specifically transfected cell lines and then subjected to Southern analysis. Genomic DNA was digested with restriction enzymes Hind III and Nru I to generate neo r Fragment with insert (ribozyme or antisense sequence). then use neo r Specific probes confirm the presence of the con...

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Abstract

This invention is directed to a synthetic non-naturally occurring oligonucleotide compound which comprises nucleotides whose sequence defines a conserved catalytic region and nucleotides whose sequence is capable of hybridizing with a predetermined target sequence within a packaging sequence of an RNA virus. Preferably, the viral packaging sequence is a retrovirus packaging sequence or the HIV-1 Psi packaging sequence. The RNA virus may be HIV-1, Feline Leukemia Virus, Feline Immunodeficiency Virus or one of the viruses listed in Table I. The conserved catalytic region may be derived from a hammerhead ribozyme, a hairpin ribozyme, a hepatitis delta ribozyme, an RNAase P ribozyme, a group I intron, a group II intron. The invention is also directed to multiple ribozymes, combinations of ribozymes, with or without antisense, and combinations of ribozymes, with antisense, and TAR decoys, polyTARs and RRE decoys targeted against the RNA virus. Vectors are also described. Further, methods of treatment and methods of use both in vivo and ex vivo are described.

Description

[0001] This application is a continuation-in-part of US Serial Application No. 08 / 178082 (filed January 5, 1994). In this application, the author and year of each document are in parentheses. All references are listed in alphabetical order after the experimental section. These documents are hereby incorporated by reference in their entirety in order to more fully describe the state of the art to which this invention pertains. Background of the invention: [0002] retrovirus [0003] Retroviruses are viruses with RNA as their genomic material. They stably integrate cDNA copies of their genomic RNA into the host genome, thus utilizing the host cell for their replication (Miller, 1992, and Brown, 1987). The viral genome consists of long terminal repeats (LTRs) at both ends (5' and 3') of the proviral cDNA form of the virus. From 5' to 3', the LTR consists of U3 and U5 linked by a short sequence called R. Transcription starts in the 5'LTR and terminates at the polyA site in t...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/48C12N15/49C12N15/74C12N15/79C12N15/85C12N5/10C07H21/02A61K31/70A61K48/00A61P31/18A01N63/00C12N15/09A61K35/26A61K35/28A61K35/76A61K35/761A61K38/00A61P1/16A61P31/12A61P33/02A61P35/02A61P37/04C07H21/04C12N15/00C12N15/113C12Q1/68C12R1/92
CPCC12N15/113A61K38/00C12N15/1131C12N15/1132C12N2310/111C12N2310/12C12N2310/121C12N2310/122A61P1/16A61P31/12A61P31/18A61P33/02A61P35/02A61P37/04
Inventor G·P·西蒙斯L·-Q·申
Owner GENE SHEARS PTY LTD
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