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Tailored recombinase for recombining asymmetric target sites in a plurality of retrovirus strains

a technology of retrovirus and target site, which is applied in the direction of transferases, peptide/protein ingredients, directed macromolecular evolution, etc., can solve the problems of restricted use of these enzymes and limited library screening in directed protein evolution

Inactive Publication Date: 2015-04-16
HEINRICH PETTE INST LEIBNIZ INST FUR EXPE VIROLOGIE - STIFTUNG BUGERLICHEN RECHTS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The tailored recombinase effectively excises proviral DNA from a wide range of retrovirus strains, including HIV-1, potentially eradicating both integrated and non-integrated viral genomes, offering a broader therapeutic application than existing strain-specific approaches.

Problems solved by technology

However, since the site-specific recombinases function through specific interactions of the recombinase enzyme subunits with their cognate DNA target sequences, the use of these enzymes is restricted by the requirement that the targeted DNA regions must contain appropriately positioned target sites (LEWANDOSKI, 2001).
Consequently, library screening in directed protein evolution has been limited to “one-by-one” approaches that maintain the compartments, and the advantages associated with screening pools of candidates have not been available.

Method used

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  • Tailored recombinase for recombining asymmetric target sites in a plurality of retrovirus strains
  • Tailored recombinase for recombining asymmetric target sites in a plurality of retrovirus strains
  • Tailored recombinase for recombining asymmetric target sites in a plurality of retrovirus strains

Examples

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example 1

Identifying Asymmetric Target Sequences Present in a Plurality of Strains

[0185]The asymmetric target sequences were identified by a method divided in 3 steps: generation of a position weight matrix based on known recognition sites of a recombinase, provision of genomic sequences that are to be searched for recognition sites, and a binary search for potential target sites in the provided sequences and scoring of the resulting hits based on the initial position weight matrix, wherein the nucleotides are transformed into binary space.

[0186]SEQ ID NO:1 was found to be present in 348 of 379 HIV-1 strains of subtype B. Furthermore, SEQ ID NO:1 was found to be present in 32 of 40 HIV-1 strains of subtype A. It is located in the R region of the LTR.

[0187]SEQ ID NO:2 was found to be present in 288 of 379 HIV-1 strains of subtype B. SEQ ID NO:2 was not found to be present in any of the searched 40 HIV-1 strains of subtype A. It is located in the U3 region of the LTR.

example 2

Generation of a Tailored Recombinase Recognising and Recombining an Asymmetric Target Sequence within the LTR of HIV-1

[0188]To start the evolution process, HIV-1 LTR sequences were selected which are highly conserved among HIV-1 strains. SEQ ID NO:1 and SEQ ID NO:2 were found to comply with these criteria and to represent asymmetric target sequences for which a tailored recombinase can be selected. In the following, the generation of a tailored recombinase recognizing SEQ ID NO:1 is described in detail.

[0189]A simplified outline of the evolution strategy and the progress made to date is outlined in FIG. 2. Overall, the evolution of a new Tre recombinase specifically recognizing the desired target sequence was conducted as disclosed in WO 2008 / 083931 and described by Sarkar et al. (Sarkar et al., Science 2007).

[0190]The start library of recombinases was generated by pooling and family shuffling Cre and several known Cre-like recombinases, namely a library of Cre mutants (Cre / Fre) (Bu...

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Abstract

The present invention relates to a method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences within the long terminal repeat (LTR) of proviral DNA of a plurality of retrovirus strains inserted into the genome of a host cell, as well as to the obtained expression vector, cells transfected with this, expressed recombinase and pharmaceutical compositions comprising the expression vector, cells and / or recombinase. Pharmaceutical compositions are useful, e.g., in treatment and / or prevention of retrovirus infection. In particular, asymmetric target sequences present in a plurality of HIV strains are disclosed, as well as tailored recombinases capable of combining these sequences (Tre 3.0 and 4.0) and expression vectors encoding them.

Description

STATEMENT REGARDING SEQUENCE LISTING[0001]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing 440098—402C1_SEQUENCE_LISTING.txt. The text file is 119 KB, was created on Dec. 2, 2014, and is being submitted electronically via EFS-Web.[0002]The present invention relates to a method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences within the long terminal repeat (LTR) of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell, as well as to the obtained expression vector, cells transfected with these, expressed recombinase and pharmaceutical compositions comprising the expression vector, cells and / or recombinase. Pharmaceutical compositions are useful, e.g., in treatme...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/12C12N15/10
CPCC12N9/1241C12N15/1058A61K38/45A61P31/14A61P31/18C12N9/22C12N2740/16022
Inventor HAUBER, JOACHIMCHEMNITZ, JANBUCHHOLZ, FRANKCHUSAINOW, JANET
Owner HEINRICH PETTE INST LEIBNIZ INST FUR EXPE VIROLOGIE - STIFTUNG BUGERLICHEN RECHTS
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