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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

[0048]However, if sequences are found in the LTR that have only homology to either the left or the right half-site sequence of a known target site, these sequences could nevertheless be used in the practice of the present invention. The size of the target site belonging to the recombinase, whose native target sequence shows homology to sequences within the LTR, is known to the skilled person. For example, if homology is found within the LTR sequence to a target sequence recognised by the Cre recombinase, an asymmetric target site to be recognised by Cre recombinase should consist of 34 nucleotides with two half-site sequences of 13 nucleotides each separated by a spacer of 8 nucleotides. Accordingly, the homologous sequence within the LTR is defined as either the left or the right half-site or the spacer of the asymmetric target site depending on the homology to the sequence of the known target site. Thus, sequences with homology to the left half-site of a known target sequence are defined as left half-site, sequences with homology to the right half-site of a known target sequence are defined as right half-site. Starting from this definition, the other parts of the asymmetric target sites are defined under consideration of the structure of the known target site. Thus, having defined for example a right half-site sequence within the LTR over homology to a loxP site (recognised by Cre recombinase), the other sequences corresponding to the spacer and the left half-site of the asymmetric target sequence can easily be defined. The spacer sequence is for example defined by counting 8 nucleotides upstream of the 5′ end of the sequence defined as right half-site sequence, whereas the left half-site sequence is similarly defined by counting 13 nucleotides upstream of the 5′ end of the previously defined spacer sequence.
[0177]However, global immune activation therapy or specific activation of provirus gene expression or similar therapy strategies greatly benefits from the concurrent removal of proviral DNA, thereby reducing in the patient the pool of infected cells.

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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