Tailored recombinase for good tolerance and high specificity of asymmetric target sites in recombination of multiple retroviral strains

Abstract

The present invention relates to methods for preparing expression vectors encoding well-tolerated and highly specific tailored recombinases that are capable of recombining asymmetric target sequences within the long terminal repeats (LTRs) of proviral DNA of a variety of retroviral strains that can be inserted into the genome of a host cell, as well as the expression vectors obtained, the cells transfected with these, the recombinases expressed and pharmaceutical compositions comprising said expression vectors, cells and / or recombinases. The pharmaceutical compositions can be used, for example, for the treatment and / or prophylaxis of retroviral infections, in particular HIV infections. In particular, the present invention relates to well-tolerated and highly specific tailored recombinases that are capable of combining asymmetric target sequences in more than 90% of HIV-1 strains, thereby excising HIV-1 sequences and expression vectors encoding them.

Description

[0001] This invention relates to a method for preparing expression vectors encoding well-tolerated and highly specific modified recombinases, and to the obtained expression vectors, cells transfected with these vectors, expressed recombinases, and pharmaceutical compositions comprising said expression vectors, cells, and / or recombinases, wherein the modified recombinases are capable of recombining asymmetric target sequences within the long terminal repeats (LTRs) of proviral DNA from various retroviral strains that can be inserted into the genome of host cells. The pharmaceutical compositions can be used, for example, to treat and / or prevent retroviral infections, particularly HIV infection. Specifically, this invention relates to well-tolerated and highly specific modified recombinases capable of combining asymmetric target sequences in more than 90% of HIV-1 strains, thereby excising HIV-1 sequences and expression vectors encoding them.

[0002] Retroviral infections, such as those caused by human immunodeficiency virus (HIV), remain one of the most important and widespread human diseases.

[0003] One approach to treating retroviruses such as HIV is to target the provirus that has inserted into the host cell's genome. Removing the proviral DNA from the host's genome, for example, would prevent further HIV replication, and unlike current methods, it has the potential to eradicate even dormant viruses present in the host's genome.

[0004] One class of proteins considered for this alternative approach is site-specific recombinases (FLOWERS et al., 1997). Site-specific recombinases mediate many functions in the properties of gene rearrangement to genome segregation, such as, for example, the excision, induction, or integration of defined DNA units (reviewed in STARK et al., 1992).

[0005] One of the simplest and most easily understood recombinases is the Cre recombinase from bacteriophage P1, which breaks down genomic dimers into monomers through recombination between two identical (i.e., symmetrical) double-stranded DNA sites of a specific sequence (HOESS & ABREMSI, 1985). Cre recombinase has been found to be widely used in mouse genetics (NAGY, 2000). Cre is a 38 kDa protein named for its function, as it induces recombination (STERNBERG & HAMILTON, 1981). The prerequisite for this recombination is that two recombination sites recognized by Cre are aligned in an antiparallel orientation and then bound by four identical Cre subunits that form a loop, with each subunit contacting two adjacent subunits and half of a recombination site (HOESS & ABREMSI, 1985). The recombination site recognized by Cre is called loxP (from the cross-( x ) of Gene locus, P 1( locus o fcrossing over( x ), P1); STERNBERG & HAMILTON, 1981) which is palindromic, except for its eight innermost base pairs (called the spacer), which confer directionality to the site.

[0006] Some site-specific recombination systems, including the Cre / loxP-system, function without the aid of accessory proteins or cofactors and function under a wide variety of cellular conditions. However, since site-specific recombinases function through the specific interaction of a recombinase subunit with its cognate DNA target sequence, the use of these enzymes is limited by the requirement that the DNA region being targeted must contain an appropriately positioned target site (LEWANDOSKI, 2001). To date, no wild-type recombinase has been identified that recognizes natural retroviral sequences as its DNA target sequence.

[0007] Extensive mutational and structural analyses of site-specific recombinases have been performed in recent years to alter their properties and to achieve a better understanding of the complex mechanisms of these enzymes (for review, see VAN DUYNE, 2001; and COATES et al., 2005). Many studies have focused on the Cre recombinase to explore its evolution. Several studies have shown that it is possible to alter its target specificity when several nucleotides in the loxP recognition site of Cre are changed (BUCHHOLZ & STEWART, 2001; SANTORO & SCHULTZ, 2002; RUFER & SAUER, 2002). Further studies addressed the engineering of mutated loxP target sites containing sequences from the LTR of HIV-1 to develop possible target sites for using Cre as an antiviral strategy (LEE & PARK, 1998; LEE et al., 2000).

[0008] The method of directed evolution is an efficient method to select enzymes with altered specificity (reviewed in Yuan et al., 2005; and JOHANNES & ZHAO, 2006). At the beginning, this method was used to isolate improved enzymes by selecting RNA molecules with altered substrate sites based on RNA. The use of PCR-based methods allowed the screening of very large libraries and the recovery of successful coding regions from a pool of candidates. In contrast, in directed evolution of proteins, the screening and recovery of improved mutants identified by changes in protein properties requires a method to recover the nucleic acid sequence encoding the protein. The link between protein and its encoding sequence has often been maintained by compartmentalization. Therefore, library screening in directed protein evolution was limited to the "one-by-one" approach of maintaining compartments and did not yet benefit from the advantages associated with screening a pool of candidates.

[0009] This limitation has been overcome by the development of a method allowing the cross-linking of proteins with their respective messenger RNA (mRNA) using mRNA-protein fusions and ribosome display. Therefore, functional screening for improved protein properties is linked to the direct recovery of the corresponding encoding molecule and large pools have been screened in vitro (see e.g. BUCHHOLZ et al., 1998). A further improvement of directed protein evolution was achieved by so-called substrate-linked protein evolution (SLiPE; BUCHHOLZ & STEWART, 2001), in which the substrate of a recombinant enzyme is located on the same DNA molecule as the protein coding region. In this way, when the recombinant enzyme is expressed within a compartment, it acts on the DNA substrate next to its own coding region. Therefore, a library can be screened by PCR as a pool to amplify only the candidate coding region next to the altered substrate. This allows the convenient screening of large libraries for the rapid recovery of successful coding regions. This method was used to alter the DNA specificity of the Cre recombinase and adapt it to a novel recognition target site (BUCHHOLZ & STEWART, 2001).

[0010] In view of the potential of site-specific recombinases and the need for an AIDS therapy that eliminates the HIV-1 provirus from the genome of a host cell, WO 2008 / 083931 discloses the generation of tailored recombinases (TREs) that are able to recombine into an asymmetric target site within the LTR of a proviral DNA of a retrovirus inserted into the genome of a host cell, thus eliminating the provirus from the genome of the host cell. The engineered recombinase, Tre, disclosed in the examples recognizes a specific asymmetric site present in a specific HIV-1 strain. The asymmetric target site has some homology to the symmetric loxP site recognized by Cre. WO 2008 / 083931 recognizes that due to the high sequence variability of retroviruses, in particular HIV, different tailored recombinases can have to be adjusted for the treatment of patients with different HIV strains, or a collection of recombinases containing tailored recombinases specific for various target sequences has to be made.

[0011] In contrast, WO 201 1 / 147590 A2 provides tailored recombinases that are able to excise a variety of retroviruses, e.g. HIV strains. Thus, the generated recombinases can be used for a variety of HIV infections without the need to generate a new recombinase for each strain. The inventors found that, despite the high sequence variability of retroviruses, using an innovative approach, an asymmetric target sequence could be identified that is present in a high percentage of viruses of a specific subtype. Surprisingly, a target sequence (SEQ ID NO: 1) could be identified that is present in 96% of HIV-1 subtype B strains, i.e. the prevalent strains in Europe and the Americas. Another target sequence (SEQ ID NO: 2) was identified that is present in a lower percentage of HIV-1 strains. Using Cre (SEQ ID NO: 6) as a basis for molecular directed evolution, they also identified several tailored recombinases that are able to recombine the asymmetric target sequence and provided a consensus sequence of these tailored recombinases, e.g. SEQ ID NO: 7 or Tre 3.0 (SEQ ID NO: 8, able to recombine SEQ ID NO: 1).

[0012] In view of this, the inventors solved the problem of providing an improved tailored recombinase able to recombine asymmetric target sequences present in a variety of HIV-1 strains. The inventors have surprisingly found that a tailored recombinase having a sequence different from the consensus sequence SEQ ID NO: 8 as taught in WO 201 1 / 147590 A2 is also highly active on the asymmetric target sequence SEQ ID NO: 1 present in 96% of all HIV-1 subtype B strains, i.e. the epidemic strains in Europe and America, and has improved characteristics. The tailored recombinase according to the present invention preferably comprises the consensus amino acid sequence of SEQ ID NO: 9, more preferably a more specific consensus sequence of SEQ ID NO: 10 or any one of SEQ ID NOs: 1 1-13. The tailored recombinase of the present invention has improved specificity compared to the tailored recombinases according to the prior art and is therefore better tolerated by humans, in particular in human T cells. Said recombinases are preferably highly specific in that they do not have any detectable residual activity on the known target sequences for which they were developed, e.g. on loxP (SEQ ID NO: 4), loxH (SEQ ID NO: 5) or also on loxLTR Tre 1.0 (SEQ ID NO: 3).

[0013] The present invention provides for the first time a method for generating expression vectors encoding well-tolerated and highly specific tailored recombinases able to recombine asymmetric target sequences within the LTRs of proviral DNA of a variety of retroviral strains of one species inserted into the genome of a host cell. The recombinases have been tailored by dividing the substrate into a plurality of new subsets having smaller differences from the original target and progressively tailoring the recombinases to recognize these subsets to recognize asymmetric target sites different from their natural symmetric target sites that can be present in a variety of retroviral strains (WO 2008 / 083931 and WO 201 1 / 147590). The combinatorial approach allows the selection of functional molecules recognizing asymmetric target sites within a given sequence. It has thus been possible to generate enzymes with remote novel asymmetric target specificity during directed molecular evolution through substrates intermediates. This method is also used in the present invention. The present invention complements the methods taught in WO 2008 / 083931 and WO 201 1 / 147590 as it introduces the step of selecting a tailored recombinase well-tolerated by human cells, in particular human T cells.

[0014] In particular, the present application provides a method for making an expression vector encoding a well-tolerated tailored recombinase that is capable of recombining an asymmetric target sequence within the LTR of proviral DNA of a plurality of retroviral strains that can be inserted into the genome of a host cell, comprising the steps of: identifying a sequence in the LTR sequence of proviral DNA of a plurality of retroviral strains that has at least 30% homology to the left half-site sequence and the right half-site sequence of at least one known recombinase target site, wherein the homologous sequences are separated by a spacer of 5-12 nucleotides, and wherein the asymmetric target sequence is found to exist in a plurality of retroviral strains; and generating by repeating the following steps:

[0015] i) molecularly directed evolution of at least one recombinase recognizing a known homologous target site using as a substrate a modified target sequence based on the asymmetric target sequence but modified to contain only a limited number of variations from the known target sequence; wherein in each round the target sequence can differ from the target sequence on which the known recombinase acts by one, two or three nucleotides; and

[0016] ii) shuffling of the recombinase library to obtain a recombinase library that is capable of recombining target sequences that are more homologous to the asymmetric target sequence;

[0017] until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of retroviral DNA;

[0018] the repeating steps of molecularly directed evolution and shuffling of the library to negative selection for recombination on a known target site;

[0019] selecting one or more of the tailored recombinases by expressing the library in human cells, in particular human T cells, and culturing the human cells expressing the tailored recombinases for at least 1 week, preferably at least 2 weeks, and isolating the nucleic acid of the recombinases from the cultured cells expressing a selectable marker;

[0020] and, optionally, cloning the nucleic acid encoding the recombinases into a suitable expression vector.

[0021] The present application in particular provides a method for making a nucleic acid or an expression vector encoding a well-tolerated and highly specific tailored recombinase that is capable of recombining an asymmetric target sequence within the LTR of proviral DNA of a plurality of retroviral strains, comprising the steps of:

[0022] (a) identifying sequences having at least 30% homology to at least one known recombinase target site in the sequence of the left half-site and the right half-site of the LTR of proviral DNA of a plurality of retroviral strains, wherein the homologous sequences are separated by a spacer of 5-12 nucleotides, and wherein the asymmetric target sequence is found in a plurality of retroviral strains;

[0023] (b) identifying two sequences, wherein the first sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the left half-site of the known target site, and is referred to as "half-site sequence 1", and wherein the second sequence corresponds to the sequence of the asymmetric target sequence of step (a) homologous to the right half-site, and is referred to as "half-site sequence 2";

[0024] (c) determining the nucleotides within the sequences of step (b) that differ from the corresponding homologous left and right half-site sequences of the at least one known homologous target site of step (a);

[0025] (d) generating a first subset of two target nucleic acids comprising target sequences, wherein the first target sequence is referred to as sub-site 1 and comprises, in 5' to 3' order, the half-site sequence 1 of step (b), the spacer sequence of the asymmetric target sequence, and the reverse repeat of the half-site sequence 1 adjacent to each other, and wherein the second target sequence is referred to as sub-site 2 and comprises, in 5' to 3' order, the reverse repeat of the half-site sequence 2, the spacer sequence of the asymmetric target sequence, and the half-site sequence 2 of step (b) adjacent to each other;

[0026] (e) generating a second subset of target nucleic acids comprising modified target sequences based on the target sequences in the first subset of step (d),

[0027] wherein, in the sequence based on sub-site 1, in the left half-site sequence, the portion of nucleotides that differ from the corresponding homologous half-site sequence of the at least one known target site of step (a) are replaced by the natural nucleotides found in the known target site, up to one, two or three nucleotides that differ from the known target site, wherein the right half-site of the modified target sequence is formed by the reverse repeat of the modified left half-site sequence, which is separated from the modified left half-site sequence by the spacer sequence of the asymmetric target sequence, and

[0028] wherein in the sequence based on sub-site 2, in the right half-site sequence, a part of the nucleotides which differs from the respective homologous half-site sequence of the at least one known target site of step (a) is replaced by the natural nucleotides found in said known target site, up to the point where the half-site sequence contains one, two or three nucleotides which differ from said known target site, wherein the left half-site of the modified target sequence is formed by the inverted repeat of the modified right half-site sequence, the inverted repeat of the modified left half-site sequence is separated from the modified right half-site sequence by the spacer sequence of the asymmetric target sequence,

[0029] such that in all modified half-site sequences of one target sequence derived from the first subset of step (d) put together, all deviating nucleotides can be found, whereas none of the modified half-site sequences contains all deviating nucleotides alone,

[0030] (f) applying molecular directed evolution to the library of recombinases evolved in step (e) using each nucleic acid of the second subset obtained in step (e) as substrate, respectively, for the recognition of the known homologous target site according to step (a);

[0031] (g) shuffling the library of recombinases evolved in step (f), wherein all libraries of recombinases evolved on the sequence based on sub-site 1 are combined and shuffled, and wherein all libraries of recombinases evolved on the sequence based on sub-site 2 are combined and shuffled;

[0032] (h) applying molecular directed evolution, preferably substrate tethered protein evolution, to the shuffled library obtained in step (g) using each nucleic acid of the subset according to step (d) as substrate;

[0033] (i) shuffling the library of recombinases evolved in step (h);

[0034] (j) applying molecular directed evolution, preferably substrate tethered protein evolution, to the shuffled library obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as substrate, until at least one recombinase is obtained which has activity on the asymmetric target sequence within the LTR of the retroviral DNA of step (a);

[0035] (k) isolating the nucleic acid encoding the at least one recombinase obtained in step (j) from the library and cloning it into an evolution vector which allows for the negative selection of a tailored recombinase which recombines the known target site according to step (a), thereby obtaining a library;

[0036] (l) applying molecular directed evolution, preferably substrate tethered protein evolution, to the library obtained in step (k);

[0037] (m) shuffling the library obtained in step (1);

[0038] (n) isolating the nucleic acid encoding at least one tailored recombinase obtained in step (m) and cloning it into a vector for expressing the encoded recombinase and a selectable marker in a human cell, thereby obtaining a library of vectors,

[0039] (o) transforming a human cell, preferably a human T cell, with the library of vectors obtained in step (n);

[0040] (p) culturing the cells expressing the selectable marker for at least 1 week and selecting for high expression of the selectable marker;

[0041] (q) isolating the nucleic acid encoding a recombinase from the cells expressing the selectable marker obtained in step (p);

[0042] (r) selecting the nucleic acid encoding a recombinase that is able to recombine the asymmetric target sequence of step (a);

[0043] (s) isolating the nucleic acid encoding at least one recombinase obtained in step (f) from the library; and,

[0044] (t) optionally, cloning the nucleic acid obtained in step (s) into a suitable expression vector.

[0045] In step (a) of the method of the application, the sequence of the LTR of the proviral DNA can be determined, such as for example by DNA sequencing using chain termination inhibitors (SANGER et al., 1977). However, if the sequence of the LTR of the retroviral DNA inserted into the genome of a host has been determined, this sequence can be determined by reference to a database. Based on the sequence information, a computer-based analysis of the sequence information is performed to identify sequences therein having at least 30% homology to the left half-site sequence and the right half-site sequence, respectively, of a known target site of a known recombinase, the left half-site sequence and the right half-site sequence of the known target site of the known recombinase being separated by a suitable spacer of 5-12 nucleotides, wherein the asymmetric target sequence is found to be present in a plurality of retroviral strains. Preferably, the homology to the left half-site sequence and the right half-site sequence of the known target site is at least 40% or at least 50%. Preferably, the plurality of strains comprises more than 10 strains, more preferably more than 100 strains, more than 130 strains, more than 200 strains or more than 300 strains, such as HIV strains. The strains can be from a subtype of a virus, such as HIV-1, HIV-1 subtypes A, B and C, preferably HIV-1 subtype B. Thus, the recombinase obtained or the expression vector encoding the same can be used for treating infections with a plurality of strains (e.g., more than 50%, more than 70%, more than 80%, more than 90% or all known strains of a retrovirus or a subtype thereof).

[0046] The term "recombinase" as used herein refers to a protein involved in recombination. Thus, a recombinase recognizes and binds to two specific DNA sequences called "recombination sites" or "target sites" and mediates recombination between the two target sites. The term "recombinase" thus means any protein component of any recombination system that mediates DNA rearrangement in specific DNA loci. Naturally occurring recombinases recognize symmetric target sites composed of two identical sequences called "half sites" of approximately 9-20 bp forming an inverted repeat, wherein the half site sequences are separated by a spacer sequence of 5-12 bp. Recombinases from the tyrosine integrase family are characterized by having a tyrosine as active site nucleophile for DNA cleavage, while recombinases from the serine integrase family use a serine, not a tyrosine.

[0047] In one embodiment of the application, the at least one known recombinase whose target sequence is used in step (a) and to which molecular directed evolution is applied in steps (h) and (j) belongs to the family of serine integrases. Preferred recombinases belonging to the family of serine integrases are selected from the group consisting of phiC31 integrase (COMBES et al., 2002), any component of the Gin or Hin recombination system, Tn3 resolvase (KRASNOW & COZZARELLI, 1983) or any other member of the large serine recombinase family, Ragl, Rag2 or any other component of the VDJ recombination system or a variant thereof.

[0048] In another embodiment, the recombinase belongs to the family of tyrosine integrases. Preferred recombinases belonging to the family of tyrosine integrases are selected from the group consisting of Cre from bacteriophage PI (ABREMSKI et al., 1983, 1984), FLP recombinase from yeast (VOLERT & BROACH, 1986), Dre from bacteriophage D6 (SAUER & MCDERMOTT, 2004), R recombinase from Zygosaccharomyces rouxii plasmid pSRl, A recombinase from Kluveromyces drosophilarium plasmid pKDl, A recombinase from Kluveromyces waltii plasmid pKWl, TnpI from Bacillus transposon Tn4430, any component of the lambda Int recombination system or a variant thereof. Preferably, the recombinase is a Cre recombinase or a variant thereof.

[0049] The term variant in this context refers to a protein derived by deletion, substitution and / or addition of amino acids from the above-mentioned proteins and retaining some or all of the functions inherent in the proteins from which they are derived.

[0050] In a preferred embodiment, the recombinase known is a chimeric recombinase obtained by "family shuffling", e.g. as described by Cramer et al. (1998). A prerequisite for the application of family shuffling is significant homology between the recombinases used to generate the chimeric recombinase. An example of a chimeric recombinase that can be used in the present application is a chimeric recombinase consisting of the sequence of the recombinase Cre and the sequence of the recombinase Dre, respectively.

[0051] In a more preferred embodiment, the recombinase is the Cre recombinase recognizing the 34 bp symmetric target site called loxP. The loxP site (and also other recombination sites of wild-type recombinases) is palindromic, with two 13 bp repeats separated by eight innermost base pairs, which represent the so-called spacer, which confers directionality to the site. Recombination occurs by cleavage within the spacer sequence. Depending on the relative position and orientation of the two loxP sites involved, Cre catalyzes DNA integration, excision or rearrangement (Hoess & Abremski, 1985).

[0052] A useful recombinase is Zre isolated from Salmonella enterica or variants, fragments and homologues thereof, e.g. having at least about 70%, at least about 80%, at least about 90% or at least about 95% homology to the wild-type sequence and having recombinase function. Zre recombinases recombine DNA at zox sites. They can be used alone or in the context of a library to start the method of the present application.

[0053] In a most preferred embodiment, the recombinase library is used as a starting point for molecular evolution, e.g. a recombinase library comprising different wild-type and / or adapted / shuffled recombinases, e.g. as described in e.g. Example 2 of WO 2011 / 147590 A2. Such libraries are preferably used as a starting point for generating tailored recombinases capable of recognizing SEQ ID NO: 1 or, alternatively, SEQ ID NO: 2.

[0054] The tailored recombinases obtained by the method of the present application are capable of recombining the asymmetric target sequence within the LTR of proviral DNA of a plurality of retroviral strains. The proviral DNA targeted by the recombinase can be inserted into the genome of a host cell. Alternatively, the tailored recombinases of the present application can recombine the asymmetric target sequence within the LTR of proviral DNA of a plurality of retroviral strains that have not (yet) integrated into the genome of a host cell, i.e. exist as a pre-integration complex (PIC) that is not integrated. Thus, HIV that has not integrated into the genome of a host cell as well as HIV that has already integrated can be inactivated by the tailored recombinases of the present application.

[0055] It should be noted that the terms "target sequence", "target site" and "recombination site" are used interchangeably in the present application and also in the art.

[0056] In contrast to naturally occurring recombinases that recognize symmetrical target sites, the method of the present application provides tailored recombinases that recognize target sites that are not composed of palindromic sequences separated by a spacer. Rather, in asymmetrical target sites, the sequences do not form a symmetrical inverted repeat. Thus, a tailored recombinase that is able to recognize an asymmetrical target site should recognize and recombine a target site that is composed of half sites of different sequences.

[0057] Within the asymmetrical target site, the sequences referred to as "left half site" and "right half site", respectively, are defined by their homology to the left and right half sites of known target sites. The sequence located between the sequences homologous to the left and right half sites of known target sites is referred to as the spacer.

[0058] However, if sequences are found within the LTR that only have homology to the sequence of the left or right half site of a known target site, these sequences can still be used in the practice of the present application. The size of the target site belonging to a recombinase whose natural target sequence shows homology to sequences within the LTR is known to the person skilled in the art. For example, if homology to a target sequence recognized by the Cre recombinase is found within the LTR sequence, the asymmetrical target site recognized by the Cre recombinase should consist of 34 nucleotides with two half site sequences of 13 nucleotides each separated by a spacer of 8 nucleotides. Thus, the homologous sequence within the LTR is defined as the left or right half site or the spacer of the asymmetrical target site depending on the homology to the sequence of the known target site. Thus, the sequence having homology to the left half site of the known target sequence is defined as the left half site and the sequence having homology to the right half site of the known target sequence is defined as the right half site. Starting from this definition, the other parts of the asymmetrical target site are defined taking into account the structure of the known target site. Thus, having defined the right half site sequence within the LTR, for example, with respect to the homology to the loxP site (recognized by the Cre recombinase), the other sequences corresponding to the spacer and the left half site of the asymmetrical target sequence can easily be defined. The spacer sequence is defined, for example, by counting 8 nucleotides upstream of the 5' end of the sequence of the right half site sequence and the left half site sequence is defined analogously by counting 13 nucleotides upstream of the 5' end of the previously defined spacer sequence.

[0059] In this context and throughout the application homology means sequence identity. Preferred comparison for homology purposes is the comparison of at least two sequences using standard techniques known in the art including, but not limited to, the local homology algorithm of SMITH & WATERMAN (1981), the homology alignment algorithm of NEEDLEMAN & WUNSCH (1970) or the similarity search method of PEARSON & LIPMAN (1988). For the purposes of the present application, sequence homology is preferably determined using the ClustalW computer program available from the European Bioinformatics Institute (EBI), unless otherwise specified.

[0060] In view of the requirement that two identical target sites must be present in the genome of the provirus to allow the recombinase to excise the sequence between the two target sites, the sequence of the proviral DNA is looked for in step (a) of the method of the present application at least twice in the genome. Such sequences are, for example, the LTR sequences of the proviral DNA. Thus, preferably the sequence of the LTR is looked for, since the 5'-LTR and the 3'-LTR of the proviral DNA are identical. The asymmetric target site present in the 5'-LTR is also present in the 3'-LTR, thus allowing excision of the proviral DNA located between the LTRs.

[0061] Among the sequences identified within the LTR sequences that have sufficient homology to known target sites, the sequence having the highest homology to the sequence of the target site of the known recombinase is preferably selected. However, sequences other than the one having the highest homology can also be selected, for example, those sequences that are present in the highest number of retroviral strains or that are present in the target retroviral strain, for example, if the patient is infected with a particular strain.

[0062] It should be noted that the potential of the method of the present application even allows to tailor a recombinase recognizing asymmetric target sites having less than 30% homology, e.g. at least 11% or at least 20% homology to known target sites of the recombinase. However, in order to ensure the presence of residual recombination activity of the respective asymmetric target site or sub-site, it is preferred to look at sequences having at least 30% homology to the left half-site and right half-site sequences of known target sites of the known recombinase. In a further preferred embodiment, asymmetric target sequences are selected having at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, more preferably 85%, particularly preferably 90%, and most preferably 95% homology to the left half-site and right half-site sequences of known target sites of the known recombinase.

[0063] In one embodiment of the present application, the sequences selected within the LTRs have homology to the symmetric loxP target site recognized by the site-specific recombinase Cre.

[0064] In a preferred embodiment, the library of recombinases is used as a starting point for molecular evolution, e.g. a library of recombinases comprising different wild-type and / or adapted / reshaped recombinases, such as the library described in Example 2 of WO 2011 / 147590 A2. Exemplary libraries comprise Cre and recombinases derived therefrom. It can further comprise Tre, Dre, recombinases from Salmonella and Shewanella and / or recombinases derived therefrom. The library can comprise, e.g., Cre, Dre, Dre"Cre-ed", Shewanella recombinase (Shew), Shew"Cre-ed", and / or Zre, as disclosed in WO 2011 / 147590 A2. Tre is a tailored recombinase as disclosed in WO 2008 / 083931, which is further referred to as Tre 1.0.

[0065] In one embodiment, all recombinases of the library recognize target sequences having the same length of spacer. The total length of the half-site sequences 1 and 2 including the spacer is preferably 34 nucleotides.

[0066] If the at least one recombinase is a recombinase library, the homology is to a collection of known recombinase target sites (i.e. the homology at a given position to at least one target sequence is defined as homology). Thus, in step (c) only those nucleotides which do not correspond to a nucleotide in at least one known target sequence are defined as deviating nucleotides. In case of a recombinase library, the "natural nucleotide" in step (e) can be the nucleotide present at this position in any known target sequence, preferably it is the nucleotide present at this position in several or most known target sequences.

[0067] In order to identify target sequences present in a variety of retroviral strains, the known specific sites of recombinases which have been described in the literature can be used as queries to search a stretch of the genome for conserved asymmetric target sequences. However, given the repetitive nature of the region, the use of standard sequence similarity search tools is excluded. Sarkar et al., 2007, used BLAST (ALTSCHUL et al., 1997) to find lox-like binding sites throughout HIV strains. When performed across HIV-1 LTR sequences, BLAST search for lox-like sites resulted in finding only one site present in a single strain. BLAST does not perform well for such short redundant sequences, and alternative programs such as HMMER (EDDY et al., 1998), RepeatMasker or the palindrome program from the Embosssuite software package proved to be inadequate as well. WO 201 1 / 147590 A2 identified asymmetric target sequences found in a variety of retroviral strains with a specific program using a position weight matrix based on the flanking regions of known specific sites of recombinases and binary operations on the sequences after they were converted into bit strings.

[0068] For HIV-1, a suitable asymmetric target sequence was determined which has the sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. This makes it possible to generate recombinases which can actually be used as therapeutic agents against the retroviral genome in a significant number of patients, because these recombinases target recognition sites present in as many retroviral strains as possible.

[0069] The left half-site and right half-site sequences of SEQ ID NO: 1 and 2 are underlined and the spacer is in bold print:

[0070]

[0071] SEQ ID NO: 1 is identical in 96% of the searched HIV-1 subtype B strains (1024 / 1067), in 92% of the searched HIV-1 subtype C strains (624 / 679) and in 82% of the searched HIV-1 subtype A strains (71 / 87). SEQ ID NO: 2 is identical in a lower percentage of B and C subtype strains.

[0072] SEQ ID NO: 1 has 54% homology to a collection of known recombinase target sites and SEQ ID NO: 2 has 42% homology to a collection of these sequences (for the left half-site and the right half-site, respectively). The homology to individual known target sites is lower, e.g. at least 30% for SEQ ID NO: 1 and at least 11% for SEQ ID NO: 2. In particular, where the homology to known target sites is low individually, it can be advantageous to use a library of recombinases as starting material, e.g. for generating tailored recombinases capable of recombining SEQ ID NO: 1 or SEQ ID NO: 2, said library being a library comprising Cre, Fre, Dre, Zre and Tre.

[0073] In step (b) of the method of the application, the sequence of the asymmetric target site within the LTR of the provirus which is homologous to the left half-site of a known target site is defined as "half-site sequence 1". The sequence of the asymmetric target site within the LTR of the provirus which is homologous to the right half-site of a known target site is defined as half-site sequence 2. The sequence between the sequences representing the left half-site and the right half-site is referred to as the spacer.

[0074] In step (c), the nucleotides within "half-site sequence 1" and "half-site sequence 2", respectively, of the sequence of step (b) which differ from the sequence of the respective homologous left half-site and right half-site sequence of a known target are determined by sequence alignment and sequence comparison. In this context, the sequence of "half-site sequence 1" is compared to the respective natural half-site, which is preferably the left half-site sequence, while the sequence of "half-site sequence 2" is compared to the other half-site forming the palindromic natural target site, which is preferably the right half-site sequence.

[0075] WO 2011 / 147590 A2 Figure 1 The results of this comparison of such SEQ ID NO: 1 and 2 to a library of recombinases are shown. Deviating nucleotides are shown in front of a dark background.

[0076] This comparison does not necessarily have to be performed after step (b) and before step (d) of the method of the application, but can also be performed in different stages of the method after step (a) and before step (e).

[0077] In step (d), a first subset of two target nucleic acids comprising target sequences is generated, wherein the first target sequence is referred to as sub-site 1 and comprises in 5' to 3' order the half-site sequence 1 of step (b), the spacer sequence of the asymmetric target site and the reverse repeat of the half-site sequence 1 adjacent to each other, and wherein the second target sequence is referred to as sub-site 2 and comprises in 5' to 3' order the reverse repeat of the half-site sequence 2, the spacer sequence of the asymmetric target site and the half-site sequence 2 of step (b) adjacent to each other. The target sequences of the first subset are palindromic oligonucleotide sequences having the structure of a symmetric target site. These artificial symmetric target sites are synthesized based on the half-site sequences of step (b) by being the reverse repeat complementary to the missing half-site sequence in each oligonucleotide sequence, wherein the sequence of "half-site sequence 1" and "half-site sequence 2" are used to be complementary to the second half-site sequence at the opposite end of the spacer sequence, respectively. Thus, the first target sequence in the first subset (referred to as "sub-site 1") comprises a reverse repeat consisting of "half-site sequence 1" and the reverse repeat of "half-site sequence" separated by the spacer sequence, while the second target sequence in the first subset (referred to as "sub-site 2") comprises a reverse repeat consisting of the reverse repeat of "half-site sequence 2" and "half-site sequence 2" separated by the spacer sequence. In "sub-site 1", the sequence is arranged as follows: 5'- "half-site sequence 1" - spacer - "reverse repeat of half-site sequence 1" - 3', and in "sub-site 2", the sequence is arranged as follows: 5'- "reverse repeat of half-site sequence 2" - spacer - "half-site sequence 2" - 3'.

[0078] The spacer sequence within each two synthetic target sequences of the first subset is preferably identical and corresponds to the sequence of the LTR representing or defining the spacer sequence of the asymmetric target site. However, in another embodiment, the spacer sequence can comprise one or two sequence deviations derived from nucleotide substitutions.

[0079] Generally, this step represents a first partitioning of the sequence of the asymmetric target site selected for tailoring the specific recombinase (see WO 2008 / 083931, which is incorporated herein by reference in its entirety, and WO 2011 / 147590 A2, which is also incorporated herein by reference in its entirety). In this step, sequences are generated which carry symmetric target sites derived from the half-sites of the asymmetric target site selected for tailoring the specific recombinase. As a result, each mutation present in one of the half-sites of the asymmetric target site (i.e. the difference to the target site recognized by the original (wild-type) recombinase) has now been distributed between the symmetric target sequences in the first subset. Figure 1 Figure 2 which is also incorporated herein by reference in its entirety). In this step, sequences are generated which carry symmetric target sites derived from the half-sites of the asymmetric target site selected for tailoring the specific recombinase. As a result, each mutation present in one of the half-sites of the asymmetric target site (i.e. the difference to the target site recognized by the original (wild-type) recombinase) has now been distributed between the symmetric target sequences in the first subset.

[0080] ​In step (e) of the method of the application, a second subset of target nucleic acids comprising modified target sequences is generated based on the target sequences of the first subset of step (d). In a sequence based on sub-site 1, in the left half-site sequence, a part of the nucleotides which differs from the respective cognate half-site sequence of the at least one known target site of step (a) is replaced by the natural nucleotides found in said known target site, up to the point where said half-site sequence contains one, two or three (preferably two) nucleotides which differ from said known target site, wherein the right half-site of said modified target sequence is formed by the reverse repeat of the modified left half-site sequence, which is separated from said modified left half-site sequence by the spacer sequence of the asymmetric target sequence.

[0081] In a sequence based on sub-site 2, in the right half-site sequence, a part of the nucleotides which differs from the respective cognate half-site sequence of the at least one known target site of step (a) is replaced by the natural nucleotides found in said known target site, up to the point where said half-site sequence contains one, two or three (preferably two) nucleotides which differ from said known target site, wherein the left half-site of said modified target sequence is formed by the reverse repeat of the modified right half-site sequence, which is separated from said modified right half-site sequence by the spacer sequence of the asymmetric target sequence.

[0082] For example, if one sub-site comprises six deviating nucleotides, such as the library of recombinases shown in Figure 1 Figure 1 of WO 201 1 / 147590 A2, based on two sub-sites of SEQ ID NO: 1 or sub-site 2 of SEQ ID NO: 2, three modified target sequences can be generated based on said sub-site, each containing two (different) deviating nucleotides in the left half-site (if based on sub-site 1 ) or the right half-site (if based on sub-site 2). Thus, in each modified target sequence, the sequence of the respective sub-site is modified to correspond to the sequence of four nucleotides of the known target sequence (or at least one known target sequence) (Figure 1 of WO 201 1 / 147590 A2). Of course, also six modified target sequences can be generated (each containing one of the deviating nucleotides) or two target sequences (each containing three of the deviating nucleotides). Figure 2

[0083] In another example, if one sub-site comprises nine deviating nucleotides, such as the library of recombinases shown in Figure 1 Figure 1 of WO 201 1 / 147590 A2, sub-site 1 of SEQ ID NO: 2, three modified target sequences can be generated based on said sub-site, each containing three (different) deviating nucleotides in the half-site.

[0084] ​The result is that in all modified half-site sequences of one target sequence derived from the first subset of step (d) put together, all deviating nucleotides can be found, while none of the modified half-site sequences alone comprises all deviating nucleotides.

[0085] Again, the reverse repeat is generated based on the modified half-site sequences such that a spacer sequence separates the two sequences of the reverse repeat (see WO 201 1 / 147590 A2, paragraph to ). The spacer sequence within each modified target sequence derived from the new subset of the higher subset is preferably identical and corresponds to the sequence of the LTR representing or defining the spacer sequence of the asymmetric target site. However, in another embodiment, the spacer sequence can comprise one or two sequence deviations derived from the nucleotide substitutions. Using this approach, the number of mutations representing the differences to the target site recognized by the wild-type recombinase in the target sequences of each subset is less than in the starting asymmetric target sequence, but all mutations are still represented in one of the target sequences (see WO 2008 / 083931, paragraph to , WO 201 1 / 147590 A2, paragraph to ). Figure 2 Figure 1 Figure 2 ) of WO 2008 / 083931.

[0086] The term "deviating nucleotide" as used herein refers to a nucleotide within the asymmetric target sequence identified or defined within the LTR or a nucleotide within the target sequences of a subset generated according to the present application which deviates from (i.e. is different from) the nucleotide present in the corresponding position of the respective homologous sequence of the known homologous symmetric target sequence of the known recombinase selected in step (a) of the method of the present application. In this context, the terms "deviating nucleotide" and "mutation" are used interchangeably.

[0087] WO 2008 / 083931 teaches that if the target sequence used as a substrate differs from the natural target sequence by no more than 3 nucleotides, the molecular directed evolution using the target sequence as a substrate can be used to fine-tune the recombinase. Therefore, the generation of subsets in the above-described different orders is used to reduce the number of deviating nucleotides of each target sequence to 3 or less (see WO 2008 / 083931, paragraph to ). The stepwise reduction of the number of deviating nucleotides finally results in a number of subsets of target sequences of different orders with a reduced number of deviating nucleotides until a final subset is produced which can be used as a substrate for molecular directed evolution. When different subsets are generated and thereby the number of deviating nucleotides is reduced, the differences to the target site recognized by the wild-type recombinase are distributed among several target sequences each not comprising more than 3 of these deviating nucleotides, while the final order of target sequences as a whole still represents all deviating nucleotides. Figure 1

[0088] ​​​Optionally, in the method of the application, further subsets of target sequences can be generated from the second subset of target sequences by stepwise repeating the procedure of step (e), i.e. by splitting the target sequences into respective half-site sequences and generating new palindromes based on these half-site sequences after changing the sequence of the half-sites derived from the second subset of target sequences, each time generating a new subset of target sequences, wherein the half-site sequences used for generating the inverted repeats contain fewer nucleotides different from the respective cognate half-site sequences of the at least one known cognate target site. These additional target sequences can be used in additional steps of directed molecular evolution and shuffling of the recombinase library. Of course, this additional step can also be performed only for some of the sequences, e.g. for those sequences where recombinases with low recombination efficiency were obtained. If additional subsets are generated and recombinases evolved on these, the evolved library of recombinases is used in step (f) of the method of the application.

[0089] From the second subset of target sequences obtained in step (e), a third subset can be generated, and subsequently, if necessary, a fourth, fifth, sixth subset etc. However, the generation of a third subset is usually only necessary if the target sequences of the second subset still contain more than three deviating nucleotides. However, if the target sequences of a previous subset still contain more than three deviating nucleotides, it is only necessary that they are suitable for generating the next subset. It should be noted that in one embodiment, subsets of target sequences will be generated until the target sequences of the final subset contain only one deviating nucleotide. Thus, the number of subsets generated for each half-site sequence of an asymmetric target site can differ depending on the number of deviating nucleotides in each half-site sequence. For example, it can be necessary to generate only two subsets for the left half-site sequence, whereas for the right half-site three or four subsets have to be generated in order to distribute the deviating nucleotides among several target sequences such that no single target sequence contains more than 3 of these deviating nucleotides.

[0090] The principle of generating further subsets of target sequences to reduce the number of deviating nucleotides to a number lower than 3 is exemplified in WO 2008 / 083931, page 6, lines 1-8. Figure 1 The principle of generating further subsets of target sequences to reduce the number of deviating nucleotides to a number lower than 3 is exemplified in WO 2008 / 083931, page 6, lines 1-8. Figure 2 Specific examples of modified target sequences are provided.

[0091] In step (f), the method of molecular directed evolution is applied to at least one recombinase recognizing the known cognate target site using as substrate the final or second subset of target sequences obtained in step (e) containing one, two or three nucleotides different from the respective cognate half-site sequences of the known cognate target site of step (a).

[0092] The term "final subset" as used herein refers to the last subset generated in step (e), i.e. if no additional subset is generated for the second subset. Depending on the number of biasing nucleotides in the asymmetric target site and the number of subsets that have to be generated in order to reduce the number of biasing nucleotides of each target sequence to below 3, the "final subset" can correspond to any subset, e.g. the second, third, fourth or later subset, and can be different for the half-site sequences of asymmetric target sequences within an LTR. If a recombinase has been evolved on an additional subset of modified target sequences with fewer nucleotides different from the respective cognate half-site sequence of the known cognate target site before, the recombinase obtained in this step is used.

[0093] Of course, the method of the application can be started with a specific recombinase for a specific modified target sequence and another recombinase (or library) for another specific modified target sequence. Methods of molecular directed evolution, also known as laboratory evolution or in vitro evolution, are known in the art (for a review see YUAN et al., 2005 and references therein; JOHANNES & ZHAO, 2006).

[0094] In a first step of molecular directed evolution, a library of randomly mutated recombinase sequences is generated by methods known in the art, e.g. by using error-prone PCR and DNA shuffling (reviewed in e.g. YUAN et al., 2005) or the methods disclosed in international patent application WO 2002 / 44409. The plasmid comprising each library of mutated recombinases also contains one of the target sequences of the final subset obtained in step (f). After transfection of the generated plasmid library into appropriate cells, expression of the recombinases can be performed and molecular directed evolution is carried out as known to the skilled person.

[0095] In a preferred embodiment, the molecular directed evolution employed in step (f) of the method of the application is Substrate-linked protein evolution (SLiPE; Buchholz & Stewart, 2001 ; International Patent Application WO 02 / 44409). Substrate-linked protein evolution can be performed as described in detail in the examples of WO 2008 / 083931 or WO 201 1 / 147590 A2. Briefly, the target sequence obtained in step (e) is cloned into a plasmid (so-called evolution vector) together with a coding sequence for a recombinase that is randomly mutated. Random mutagenesis is performed by error-prone PCR (see BUCHHOLZ & STEWART, 2001 ). The generated library of plasmids is then transfected into E. coli cells to allow expression of the recombinase. By using an inducible promoter to drive expression of the recombinase, the level of expression can be regulated. After overnight incubation, plasmid DNA is isolated from the cells and digested with Nde\ to cleave non-recombined plasmids, and subsequently only the recombined plasmids are amplified with primers. The PCR product of the recombined plasmids results in a 1.7 Kb band. The PCR product is digested with BsrGI and Xbal, and subcloned back into a similarly digested evolution vector for the next round of evolution.

[0096] In step (g), the libraries of recombinases evolved in step (f) are combined and shuffled. Techniques for DNA shuffling are known in the art (for a review, see MINSHULL & STEMMER, 1999; STEMMER, 1994). The libraries of recombinases evolved based on sub-site 1 on the modification of the target sequence are combined and shuffled, and separately, the libraries of recombinases evolved based on sub-site 2 on the modification of the target sequence are combined and shuffled. The combined and shuffled libraries are then cloned into a new generation of vectors comprising the next higher subset of target sequences, i.e. the next higher subset is the subset generated in step (d) if two subsets are generated. For example, the vectors of the library evolved based on sub-site 1 on the sequence comprise the sequence of sub-site 1 as target sequence, and the vectors of the library evolved based on sub-site 2 on the sequence comprise the sequence of sub-site 2 as target sequence.

[0097] In step (h), the method of molecular directed evolution is applied to the shuffled library obtained in step (g) using the next higher subset of target sequences, which can be according to the subset of step (d) as discussed. In this step, the same method of molecular directed evolution as previously applied in step (f) can be used, but also a different method of molecular directed evolution can be used in this step of the method of the application. Examples of different methods of molecular directed evolution are described in e.g. YUAN et al. (2005). Preferably, the method of substrate-linked protein evolution is also applied to the combined and shuffled library.

[0098] This step results in a recombinase recognizing and recombining a mutated combination (and thus an increased number) of target sequences carrying different target sequences from the lower subset. The mutated combination of different libraries from the lower subset of target sequences results in a synergistic effect and leads to the production of a recombinase which now recombines a higher subset of target sequences, demonstrating that the evolutionary strategy through intermediates can be used to achieve the desired activity.

[0099] In step (i), step (g), i.e. combination and shuffling of the recombinase library, and (j), i.e. applying molecular directed evolution to the combined and shuffled library, is repeated until at least one recombinase is obtained which is active on the asymmetric target sequence present in the LTR of the pro viral DNA.

[0100] In a method wherein two subsets of target sequences have to be generated to generate target sequences having only one, two or three nucleotide deviations, the recombinase library evolved, e.g. for the second subset of target sequences, is combined and shuffled and molecular directed evolution is applied to this shuffled library using the target sequences of the first subset. In the next step, the recombinase library comprising recombinases recognizing the target sequences of the first subset is evolved by molecular directed evolution using the asymmetric target sequence of step (a) within the LTR of the pro viral DNA to obtain at least one recombinase which is active on the asymmetric target sequence within the LTR of the retroviral DNA. In this step, the method of molecular directed evolution is preferably a method of substrate tethered protein evolution.

[0101] "at least one recombinase" refers to the fact that the method of the present invention can result in one or more (single) recombinases each of which is active in recombining the asymmetric target sequence by itself. It is not intended to encompass several different recombinases which are only able to recombine the asymmetric target sequence together. In fact, the method of the present invention does not result in selecting a recombinase which needs to be combined with other, different recombinases to recombine the asymmetric target sequence, since each single cell only expresses one recombinase.

[0102] The method steps (a) - (j) are known per se in the art, in particular from WO 2011 / 147590.

[0103] After step (j), the library of recombinases is negatively selected for recombination of known symmetric target sites according to step (a), e.g. for recombination of loxP and / or loxH.

[0104] This selection can be achieved by at least one cycle comprising one or more steps of targeted molecular evolution and shuffling of the vector library.

[0105] To this end, the nucleic acid encoding the at least one tailored recombinase evolved in the previous step can be isolated from the vector used therein and cloned into a suitable evolution vector. The vector allows for negative selection recombination of known target sites according to step (a), e.g. for recombination of loxP (SEQ ID NO: 4) and / or loxH (SEQ ID NO: 5) by the tailored recombinase. Thereby a library of vectors is obtained. Then directed molecular evolution, preferably substrate-linked protein evolution (SLiPE) as known to the skilled person and according to the principles outlined above is utilized.

[0106] For example, the evolution vector can be constructed such that it comprises the final asymmetric target sequence (e.g. SEQ ID NO: 1) and the known target sites (e.g. loxP and / or loxH), each twice to allow recombination. Recombination at the known target sites and subsequent restriction digestion leads to a linear product which does not comprise two specific primer sites in the order to allow PCR amplification of the product. If no recombination occurs at all, the vector is linearized by restriction digestion and no amplification by PCR occurs. In contrast, recombination at the final asymmetric target site (e.g. SEQ ID NO: 1) excises the restriction sites, i.e. the vector is not linearized by restriction digestion, and the tailored recombinase can be amplified by PCR. Error-prone PCR is used to generate variability. The evolution can be carried out in E. coli.

[0107] The library obtained after one or more, preferably about ten cycles of directed molecular evolution can be shuffled. One or more further cycles of directed molecular evolution and / or shuffling can be carried out.

[0108] Optionally, the negative selection for recombination of several known target sites, e.g. for recombination of loxP and loxH, can be carried out alternately, e.g. one cycle of evolution of negative selection for loxP can alternate with one cycle of evolution of negative selection for loxH. For example, about 10 to about 30 or about 15-20 cycles of negative selection can be carried out in combination with about two rounds of DNA shuffling. Between these evolution cycles, the amount of transcriptional activator L-arabinose can be varied, e.g. from 100 pg / mL to 1 pg / mL. Preferred vectors and methods are shown in Figure 2 and the examples.

[0109] After a plurality of evolution cycles have been carried out, the specificity of the tailored recombinase for the final asymmetric target sequence and the potential residual activity for the known target sequence can be checked for one or more clones.

[0110] If the specificity is not yet satisfactory, further evolution cycles should be carried out.

[0111] As Figure 3The negative selection removes the residual activity of the tailored recombinase (such as those taught by WO 2011 / 147590 A2 (TRE3)) on known target sequences of loxP and loxH as shown in Figure 1. The residual activity of the generated recombinase on known target sites (e.g. loxP and / or loxH) is not detectable even in the presence of high amounts (50 or 100 pg / ml) of the transcriptional activator L-arabinose, i.e. in the presence of high amounts of the recombinase. This shows that the obtained tailored recombinase is highly specific for its target sequence (in this case for SEQ ID NO: 1) as the specific asymmetric target sequence is recombined but the known symmetric target sequence is not.

[0112] This has the advantage that when the tailored recombinase of the present application is applied for human therapy applications, the risk of cross-reactions with human sequences and recombination of human sequences in host cells is minimal. This is one factor contributing to the tolerability of the tailored recombinase in human cells. However, not only the short-term results of the expression of the recombinase are at issue here, but also, even at low potency, safety aspects such as the possible carcinogenic effect of non-specific recombination are at issue. The elimination of even residual activity of the tailored recombinase thus contributes to the safety and reliability of the resulting tailored recombinase in a therapeutic environment.

[0113] The selection against recombination of known symmetric target sequences is followed by the selection of tailored recombinases which are well tolerated in human cells.

[0114] In step (n) of the method of the present application, the nucleic acid encoding the at least one tailored recombinase obtained in step (m) is isolated and cloned into a vector for expression of the encoded recombinase and a selectable marker in eukaryotic cells, preferably human cells, thereby obtaining a library of vectors. The nucleic acid can be isolated from the respective plasmids within the library using appropriate restriction enzymes. Methods of restriction enzyme digestion are known to the skilled person. The nucleic acid encoding the recombinase can then be recovered by known methods such as gel electrophoresis. It can be cloned into an appropriate expression vector for expression in eukaryotic cells, e.g. human cells, as known in the art or as described below. For example, retroviruses, e.g. lentiviral vectors, can be used, e.g. as described in WO 2011 / 147590 A2 (TRE3), the disclosure of which is incorporated herein by reference. Figure 4A The expression of the encoded tailored recombinase and the selectable marker is preferably constitutive, or it can be induced by a suitable agent.

[0115] The selectable marker can confer resistance to an antibiotic, or it can be a fluorescent protein such as green fluorescent protein (GFP) or a derivative thereof (e.g. EBFB, ECFP, YFP). Fluorescent proteins such as GFP allow easy sorting of cells according to the intensity of expression.

[0116] In step (1), the eukaryotic cells, preferably human cells, preferably human T cells, are transformed with the library of vectors obtained in step (k). Methods known in the art can be used. The transformed cells are typically human cells, however, if a non-human patient is intended to be treated, it is advisable to test the tolerance in cells of that patient species. The human cells are preferably hematopoietic cells, such as preferably T cells, in particular CD4+ T cells, but stem cells, such as CD34+ stem cells, can also be used. Primary cells, such as primary T cells, preferably primary CD4+ T cells, can be used, but cell lines, such as Jurkat T cells, can also be employed.

[0117] In step (p), the cells expressing the selectable marker are cultured for a period of time sufficient to select for TRE recombinases well tolerated by human cells. The selection is based on the assumption that the expression of the marker is correlated with the expression of the tailored recombinase. Cells are selected for expression of the marker, such as GFP positive cells, preferably cells with strong expression of GFP, are selected. Since the expression of the selectable marker is correlated with the expression of the tailored recombinase, these cells will also express the tailored recombinase. Thus, cells expressing the tailored recombinase in amounts that are detrimental to their survival or their reproductive capacity are eliminated or reduced. Preferably, the cells expressing the marker are cultured for at least 1 week, at least two weeks, at least 3 weeks or at least 4 weeks. Thus, the tailored recombinase expressed in T cells will be well tolerated by human cells, such as human T cells, i.e. it is not toxic to the cells, or preferably also not detrimental to the survival and reproduction of the cells. Preferably, during the culturing, the cells are selected at least once, preferably 2, 3 or 4 times, for high expression of the selectable marker. With fluorescent proteins, selection can be performed by fluorescence-activated cell sorting. With antibiotic resistance genes, increasing amounts of antibiotic can be added to the culture medium.

[0118] While the expression of wild-type Cre recombinase in human cells has been established for a long time and has been shown without problems at reasonable expression levels, overexpression of Cre can be toxic (LOONSTRA et al., 2001). The present inventors found that a significant number of the mutated tailored recombinases according to the present application are detrimental to the survival and / or reproduction of human T cells when strongly overexpressed. Interestingly, even if one can expect low specificity of the tailored recombinases and residual activity on target sites such as loxP and loxH (sequences present in the human genome) leading to low tolerance in human cells, the selection for tolerance in human T cells alone, without prior selection for high specificity, is not sufficient to eliminate residual cross-reactivity on loxP or loxH. Thus, the combination of only two selection steps with the previously known methods of the present application leads to tailored recombinases that are well tolerated and highly specific.

[0119] In step (q), after the culturing and selection steps, the nucleic acids encoding the at least one recombinase are isolated from the cells expressing the selectable marker obtained in step (p).

[0120] Step (r) is optional and adds a further option of nucleic acid encoding a recombinase which is able to recombine the asymmetric target sequence of step (a), preferably with high activity of recombination. The recombination activity is preferably tested in human cells, in particular human CD4+ T cells, but it can also be tested in E. coli.

[0121] In step (s), nucleic acids of recombinases active on the asymmetric target sequence of step (a) within the LTR of the retroviral DNA are isolated from the library. The nucleic acids can be isolated from the respective plasmids within the library using appropriate restriction enzymes. Methods of restriction enzyme digestion are known to the skilled person. The nucleic acids encoding the recombinases can then be recovered by known methods such as gel electrophoresis.

[0122] The nucleic acids can be stored (preferably at temperatures below -80°C) or can optionally be cloned into expression vectors in step (t) for further analysis, for protein expression methods, or for administration to a subject for the treatment and / or prevention of a retroviral infection, in particular an HIV infection and / or AIDS. Suitable expression vectors are known in the art or disclosed below.

[0123] The development of tailored recombinases specifically targeting the asymmetric sequences within the HIV-l LTR such as SEQ ID NO: 1 allows for excision of the respective provirus from the proviral chromosomal integration in most subjects infected with HIV-l. Expression vectors encoding such recombinases, cells transfected therewith and / or recombinase proteins derived therefrom have medical uses, e.g. the treatment and / or prevention of HIV-l infection. Preferred methods of making such tailored recombinases or expression vectors encoding the same are taught in WO 2011 / 147590. The inventors add to the methods described in WO 2011 / 147590 the following steps: active selection for high specificity, i.e. no detectable cross-reactivity to the known target sequence of step (a) (or to e.g. loxP and loxH), and active selection for tolerance of the tailored recombinase in human cells such as human T cells. As described, this significantly improves the medical use of the tailored recombinases for excision of HIV proviral genomes from human T cells.

[0124] The proviral DNA that can be inserted into the genome of a host cell or that can not yet be inserted is preferably DNA of a retrovirus. Retroviruses comprise a large and diverse family of enveloped RNA viruses. The hallmark feature of the family is its replication strategy, which includes reverse transcription of the viral RNA into linear double-stranded DNA and subsequent integration of this DNA (proviral DNA) into the genome of the host cell as an essential step. Retroviruses are subdivided into seven groups defined by evolutionary relatedness. Five of these groups (a-, b-, d-, e-, and g-retroviruses) represent retroviruses with oncogenic potential, and the other two groups are lentiviruses and spumaviruses. The human pathogenic human T-cell leukemia viruses type I and II (HTLV-I and HTLV-II) belong to the d-retrovirus group, while the AIDS viruses human immunodeficiency viruses type 1 and 2 (HIV-I and HIV-2) belong to the lentivirus group (for review, see the standard textbook "Retroviruses", COFFIN JM, HUGHES SH, VARUS HE (eds), 1997, Cold Spring Harbor Laboratory Press, New York).

[0125] In one embodiment, the proviral DNA that can be inserted into the genome of a host cell is DNA of a retrovirus selected from the group consisting of mouse mammary tumor virus (MMTV), Mason Pfizer monkey virus (MPMV), human T-cell leukemia virus type I (HTLV-I), human T-cell leukemia virus type II (HTLV-II), simian T-cell leukemia virus type I (STLV-I), simian T-cell leukemia virus type II (STLV-II), bovine leukemia virus (BLV), feline leukemia virus (FeLV), and Moloney murine leukemia virus (MoMLV).

[0126] In another embodiment, the retrovirus is a lentivirus selected from the group consisting of human immunodeficiency virus type 1 (HIV-I), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Maedi-visna virus (MVV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV). As mentioned above, HIV, in particular HIV-I, is preferred.

[0127] The asymmetric target sequence identified in step (a) of the method of the present application is located in the 5'-LTR and the 3'-LTR of the HIV provirus. Preferably, the asymmetric target sequence located in the 5'-LTR and the 3'-LTR of the HIV provirus has the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0128] In a preferred embodiment, the method of molecular directed evolution applied in the inventive method is the method of substrate-linked protein evolution (SLiPE; BUCHHOLZ & STEWART, 2001 ; see also WO 02 / 44409).

[0129] By performing the inventive method as described herein, the inventors generated several nucleic acids encoding well-tolerated tailored recombinases and the tailored recombinases themselves. Thus, the present application provides a well-tolerated tailored recombinase, or a nucleic acid encoding the same, the tailored recombinase comprising or consisting of a sequence according to any one of SEQ ID NOs: 9-13.

[0130] Surprisingly, it was found that these tailored recombinases differ from the consensus sequences SEQ ID NOs: 7 and 8 of tailored recombinases capable of recombining asymmetric target sequences as taught in WO 201 1 / 147590, and all other previously known recombinases.

[0131] In particular, the newly analyzed well-tolerated tailored recombinases capable of recombining the asymmetric target sequence of SEQ ID NO: 1 with high specificity surprisingly comprise a Q89L mutation.

[0132] In one embodiment, the tailored recombinase of the present application comprises a sequence according to SEQ ID NO: 9 (Tre 3.1 consensus sequence 85%). SEQ ID NO: 9 represents a consensus sequence, wherein each mutation (compared to the Cre amino acid sequence) is present with a probability of 85%. For this assay, 100 individual clones generated by the inventive method were analyzed by Sanger sequencing, and the entire generated Tre 3.1 library (33,000 reads of unique 200bp-sequences) was analyzed by next generation sequencing. The variable amino acids are represented by X, which can represent any naturally occurring amino acid (see Figure 1 ). About one third of the amino acids of SEQ ID NO: 9 are highly variable, i.e. these positions do not need to be conserved in order for the recombinase to be capable of recombining the asymmetric target sequence of SEQ ID NO: 1 and to be well-tolerated by humans. On the other hand, about two thirds of the amino acid positions seem to be important for recombining the asymmetric target sequence of SEQ ID NO: 1 with high specificity and / or to be well-tolerated by humans.

[0133] In a preferred embodiment, the tailored recombinase of the application comprises a sequence according to any one of SEQ ID NOs: 11-13, most preferably SEQ ID NO: 11. These tailored recombinases were selected for their specificity, i.e. while other recombinases generated according to the application still have a low, but detectable recombination activity on loxP, loxH or lox LTR 1.0, with the recombinases of SEQ ID NOs: 11-13, this recombination activity is not detectable, as shown in the examples. Thus, the application provides a well-tolerated and highly specific tailored recombinase (i.e. a functional tailored recombinase) capable of recombining asymmetric target sequences within the LTRs of proviral DNA of a variety of retroviral strains that can be inserted into the genome of a host cell, which preferably comprises SEQ ID NOs: 11-13, preferably SEQ ID NO: 11. Such a tailored recombinase is, for example, obtainable according to the method of the application.

[0134] In one embodiment, the tailored recombinase can comprise a sequence according to SEQ ID NO: 10. This sequence, Tre 3.1 consensus sequence 100% (3 clones), is the consensus sequence of the three preferred recombinases of SEQ ID NOs: 11-13.

[0135] The tailored recombinase can also have at least 95% amino acid identity, preferably at least 99% amino acid identity or 100% amino acid identity to SEQ ID NO: 10 or it can differ from SEQ ID NO: 10 by only one or two amino acids and it comprises the following defined amino acid exchanges compared to the Cre sequence (SEQ ID NO: 6): V7L, P12S, P15L, M30V, H40R, M44V, S51T, Y77H, K86N, Q89L, G93A, S108G, C155G, A175S, A249V, R259D, E262R, T268A, D278G, P307A, N317T, I320S. It can also comprise the following exchanges: N160T, R241Q, K244I, N319E. Preferably, it comprises the following amino acid exchanges compared to Cre: N3I, V7L, N10S, P12S, P15L, V23A, M30V, F31L, H40R, M44V, S51T, Y77H, K86N, Q89L, G93A, S102F, S108G, N111D, K122R, A131T, S147A, D153E, C155G, N160T, F163L, I166V, I174V, A175S, V182I, G198S, D232S, R241Q, K244I, A249V, Q255R, R259D, A260V, E262R, G263K, T268A, D278G, P307A, N317T, N319E, I320S.

[0136] These specific exchanges make the enzyme particularly suitable for recombination at the target sequence of SEQ ID NO: 1 or at a target sequence having high sequence identity to SEQ ID NO: 1, e.g. at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO: 1. The present inventors could surprisingly show that a single amino acid variation, namely Q89L, ensures that the tailored recombinase is well tolerated in human cells, e.g. human hematopoietic cells or human T cells, and has high specificity as it has no detectable activity in recombining the original target sequences loxP or loxH and preferably also no detectable activity on loxLTR Tre 1. Activity can be detected by gel electrophoresis of samples comprising loxP (SEQ ID NO: 4), loxH (SEQ ID NO: 5), or, for comparison, loxLTR Tre 3 comprising SEQ ID NO: 1 (each in contact with the tailored recombinase), e.g. by inducing its expression from a suitable vector, as exemplified in the Examples and Figure 3As shown in the figure, the Tre 3.0 recombinase (produced according to WO2011 / 147590), although quite specific compared to other recombinases, exhibits residual activity for loxP and loxH under the conditions shown, while for uTre (the recombinase of the present invention), the recombinant product is only visible for loxLTR containing SEQ ID NO:1. This high specificity minimizes the risk of unwanted recombination in the human genome.

[0137] The sequence of the modified recombinase of the present invention is not disclosed in WO 2008 / 083931 or WO 2011 / 147590. Specifically, the prior art does not teach or imply that a modified recombinase capable of recombining the asymmetric target sequence of SEQ ID NO:1 has an amino acid exchange Q89L. In contrast, WO 2011 / 147590 explicitly teaches that this position should remain Q (see all specific or common sequences disclosed therein). The sequence of the modified recombinase of the present invention also differs from naturally occurring recombinases such as Cre, Dre, Fre, or Zre, which is evident from its ability to recombine the asymmetric target sequence within the LTR of the proviral DNA of various retroviral strains that can be inserted into the genome of host cells, preferably SEQ ID NO:1.

[0138] If a recombinase capable of recombining the asymmetric target sequence within the LTR of the proviral DNA of multiple retroviral strains inserted into the genome of a host cell will recombinate the target sequence of SEQ ID NO:1, then it preferably contains 85% of the common sequence Tre3.1, SEQ ID NO:9, or 100% of the common sequence Tre 3.1, SEQ ID NO:10, or one of the specific sequences SEQ ID NO:11-13.

[0139] Functional recombinases capable of recombining asymmetric target sequences within the LTR of proviral DNA from various retroviral strains that can be inserted into the genome of host cells (which can be obtained, for example, by the method of the present invention) may differ from the sequences described herein, but the sequences described herein provide valuable guidance to those skilled in the art to generate recombinases capable of recombining asymmetric target sequences such as SEQ ID NO:1, even without implementing the method of the present invention.

[0140] Preferably, the amino acid exchange relative to the reference sequence is a conservative substitution, which is well known to the person skilled in the art (e.g. Creighton (1984), Proteins. W. H. Freeman and Company (Eds)). For example, a conservative substitution replaces one amino acid from the group of negatively charged amino acids with another. Most preferably, the exchange results in one of the amino acids present in any one of SEQ ID NOs: 11-13 at the relevant position.

[0141] The tailored recombinase capable of recombining asymmetric target sequences within the LTR of proviral DNA of a variety of retroviral strains that can be inserted into the genome of a host cell can also comprise a combination of 2 or more sequences selected from SEQ ID NOs: 11-13, for example a C-terminal portion of any one of these sequences, for example SEQ ID NO: 11, and a N-terminal portion of any other of these sequences, for example SEQ ID NO: 12. The C-terminal portion can have a length of 1-342 amino acids. In a combination of two sequences, the N-terminal portion can have a length of 1-342 amino acids. The tailored recombinase can also be a combination of three or more portions derived from these sequences. The combination comprises the TRE 3.1 consensus motif, for example SEQ ID NO: 10 or preferably SEQ ID NO: 9.

[0142] The present application also provides a nucleic acid encoding a tailored recombinase capable of recombining asymmetric target sequences such as SEQ ID NO: 1 within the LTR of proviral DNA of a variety of retroviral strains that can be inserted into the genome of a host cell, the tailored recombinase comprising an amino acid sequence as defined above.

[0143] In the context of the present application, a nucleic acid or protein comprising a sequence can consist of said sequence.

[0144] It can alternatively comprise other sequences, for example a signal sequence providing expression / localization in a particular cellular compartment, such as a nuclear localization signal, as in SEQ ID NO: 14 (the nuclear localization signal is at positions 2-9 of SEQ ID NO: 14). If the protein is to be used in a pharmaceutical composition, it is particularly preferred to express it as a fusion protein with a protein transduction domain, such as a tat protein transduction domain, which allows protein transduction of target cells. Preferably, the tailored recombinase of the present application to be used in a pharmaceutical composition is prepared as a fusion protein with a nuclear localization sequence and with a protein transduction domain (e.g. from tat), and a nucleic acid encoding a tailored recombinase of the present application can encode this fusion protein. For example, the following protein transduction domains can be used in a fusion protein with a tailored recombinase of the present application, which preferably further comprises a nuclear localization signal:

[0145] - the basic domain of the HIV-1 Tat transactivator (Fawell S, Seery J, Daikh Y, Moore C, Chen LL, Pepinsky B, Barsoum J. Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):664-8.)

[0146] - the homeodomain of the Drosophila Antennapedia (Derossi D, Joliot AH, Chassaing G, Prochiantz A. The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem. 1994 Apr 8;269(14): 10444-50.)

[0147] - the HSV VP22 transcription factor (Elliott G, O'Hare P. Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell. 1997 Jan 24;88(2):223-33.) - the cell permeable translocation motif (TLM) of the Pre S2 surface antigen of the hepatitis B virus (HBV) (Oess S, Hildt E. Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens. Gene Ther. 2000 May;7(9):750-8.).

[0148] In case the protein is to be purified, a tag facilitating the purification of the protein can also be added, such as a His-tag.

[0149] The codon usage of the nucleic acid of the application encoding a Tre recombinase as defined above can be chosen by the skilled person. For example, codon usage can be chosen which is suitable for expression in human cells, in particular if expression in human cells is intended, for example for therapeutic purposes. Codon usage can also be based on the codon usage of, for example, Cre recombinase.

[0150] The tailored recombinase or the nucleic acid encoding the tailored recombinase can be obtained by or obtainable by the inventive method as described herein. It can also be obtained based on the sequences disclosed herein, optionally by combining and / or further altering these sequences, optionally testing the activity of the recombinogenic asymmetric target site such as SEQ ID NO: 1.

[0151] The application further provides a composition, for example a library, comprising two or more nucleic acids encoding a tailored recombinase as defined above, for example encoding two or more tailored recombinases comprising different sequences according to SEQ ID NO: 9, preferably according to any one of SEQ ID NO: 10 or 11-13. In one embodiment, the composition comprises nucleic acids encoding a tailored recombinase comprising two or more, three or more, four or more, five or more, ten or more, 20 or more or 25 or more recombinases comprising a sequence according to any one of SEQ ID NO: 9-13 or a combination of these sequences. Such a composition, in particular where the nucleic acids are expression vectors, can be particularly suitable as a pharmaceutical composition as described below.

[0152] In the methods of the application, the nucleic acid encoding a tailored recombinase having activity on asymmetric target sequences within the LTR of retroviral DNA is preferably cloned into an expression vector. An expression vector is a genetic construct used to express a protein encoded by a nucleic acid within the vector. Such expression vectors can be self-replicating extrachromosomal vectors or vectors that integrate into the host genome. Typically, these expression vectors include transcriptional and translational regulatory nucleic acids operably linked to the nucleic acid encoding a tailored recombinase of the application.

[0153] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0154] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. The linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Transcriptional and translational control nucleic acids are generally appropriate for the host cell in which the tailored recombinase is to be expressed. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

[0155] The expression vector used in the present application can be a retroviral vector, a lentiviral vector, a spumavirus vector, an adenoviral vector or an adeno-associated viral vector. However, in a preferred embodiment, the expression vector is a lentiviral vector selected from HIV-1 -, SIV-, FIV- or EIAV-derived lentiviral vectors. Lentiviral vectors are for example described in SCHAMBACH et al. (2006) or in European Patent Application No. 1 100 0751.5.

[0156] In a preferred embodiment of the present application, the expression vector comprises a cellular promoter, a bacterial promoter, a viral promoter or a hybrid promoter.

[0157] Generally, for the purposes of the present application, the promoter can be a constitutive or an inducible promoter. Furthermore, the promoter can be a naturally occurring promoter, such as a bacterial promoter, a cellular promoter or a viral promoter, or a hybrid promoter. Hybrid promoters combining elements of more than one promoter are known in the art and can be used in the present application. Furthermore, the promoters used in the present application can also be derivatives of naturally occurring promoters. A "derivative" of a naturally occurring promoter as used herein can be a combination of promoters or of cis-active elements of sequences obtained from different sources, or alternatively, can be obtained by deletion or mutation of cis-active elements in a particular naturally occurring promoter (EDELMAN et al., 2000; ALPER et al., 2006; HARTENBACH & FUSSENEGGER, 2006).

[0158] In one embodiment of the present application, the constitutive promoter or derivative thereof is selected from or derived from the promoter of cytomegalovirus, Rous sarcoma virus, murine leukemia virus-related retroviruses, phosphoglycerokinase gene, mouse spleen focus-forming virus or human elongation factor 1 alpha.

[0159] In another embodiment of the present application, the inducible promoter or derivative thereof is selected from or derived from the LTR or derivative thereof derived from lentiviruses, spumaviruses and delta retroviruses.

[0160] In this context, the term "LTR" refers to the 5' and 3' long terminal repeats of the provirus having promoter function (for review see the standard textbook "Retroviruses" (COFFIN JM, HUGHES SH, VARMUS HE (eds) 1997, Cold Spring Harbor Laboratory Press, New York).

[0161] Preferably, the inducible promoter or derivative thereof is selected from or derived from an LTR or derivative thereof derived from HIV-1, HIV-2, MVV, EIAV, CAEV, SIV, FIV, BIV, HTLV-I and HTLV-II.

[0162] The present application further provides a method for preparing a tailored recombinase, wherein the method comprises the aforementioned method for preparing an expression vector encoding a tailored recombinase and the further step of expressing the tailored recombinase (or a fusion polypeptide comprising the amino acid sequence of the tailored recombinase) from the expression vector in a suitable host cell.

[0163] Preferably, the finally obtained recombinases are tested in mammalian cells to ensure that they function in the mammalian cell environment. Furthermore, in order to obtain good expression in mammalian cells, the recombinases can be optimized for expression in these cells (e.g. codon usage optimization using methods well known in the art. See e.g. SHIMSHÉ et al., 2002) or a signal sequence required for the guidance of the protein to the nucleus of the mammalian cell, such as an NLS sequence (MACARA, 2001) can be added to the nucleic acid of the tailored recombinase. Expression of the nucleic acid encoding the tailored recombinase cloned into an expression vector can be carried out using, for example, a bacterial, insect or mammalian expression system, e.g. according to step (1) of the method for preparing an expression vector encoding a tailored recombinase. However, other expression systems known in the art can also be employed. Methods for introducing foreign nucleic acids into mammalian, insect or bacterial hosts, as well as other hosts, are also well known in the art and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into the nucleus.

[0164] The fusion protein is prepared by methods well known in the art. For example, the expression vector in which the nucleic acid encoding the tailored recombinase has been cloned already comprises a nucleic acid sequence encoding a second polypeptide or protein. By cloning the nucleic acid encoding the tailored recombinase in frame with the sequence of the second polypeptide or protein, both sequences will be expressed as a fusion protein.

[0165] Host cells for expressing the tailored recombinase from an expression vector include prokaryotic cells, such as, for example, bacterial cells or yeast cells, or preferably eukaryotic cells, such as, for example, insect cells or mammalian cells, most preferably human cells. The host cells can be hematopoietic cells, for example adult hematopoietic stem cells or T-cells, for example CD4+ cells. The cells can be derived from a subject infected with a retrovirus, and the cells can be administered back to the subject after transformation, and optionally culturing and / or propagating.

[0166] The present application further provides a method for preparing a transformed adult stem cell, wherein the method comprises the aforementioned method for preparing an expression vector encoding a tailored recombinase, and the further step of introducing the expression vector obtained in the aforementioned method for preparing an expression vector encoding a tailored recombinase in vitro into a suitable adult stem cell.

[0167] In another aspect, the present application relates to a nucleic acid as disclosed herein and / or obtainable from the aforementioned methods of the present application. It is also provided herein a nucleic acid encoding a tailored recombinase defined by a sequence.

[0168] As used herein, a "nucleic acid" is a polymeric compound composed of covalently linked subunits called nucleotides. Nucleic acids include polyribonucleic acids (RNAs), such as mRNA, and polydeoxyribonucleic acids (DNAs), both of which can be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.

[0169] In another aspect, the present application also relates to an expression vector obtainable from the aforementioned methods of the present application, and to an expression vector comprising a nucleic acid encoding a tailored recombinase as defined herein.

[0170] As used herein, the term "protein" includes proteins, polypeptides, and peptides. As will be appreciated by the skilled person, the nucleic acid sequences of the present application can be used to generate protein sequences. Another aspect of the present application is a tailored recombinase protein as obtainable, for example, from the aforementioned methods of the present application, which recombinase can optionally be a fusion protein comprising a functional recombinase. In one embodiment, the tailored recombinase protein can be prepared as a fusion polypeptide using techniques well known in the art. In a preferred embodiment, the tailored recombinase protein is linked to a second polypeptide. Preferably, the fusion polypeptide is obtained from the aforementioned methods of the present application, wherein the tailored recombinase is linked to a second polypeptide.

[0171] In one embodiment, the tailored recombinase protein is prepared as a fusion polypeptide to increase expression. In another embodiment, the tailored recombinase protein is prepared as a fusion polypeptide to allow the polypeptide to be introduced into a living cell. Typically, purified proteins cannot enter cells because they cannot pass through the cell membrane due to their size. However, fusion of specific peptide sequences to proteins can result in the uptake of these fusion proteins into cells. In the cell, the protein can exert its function. Site-specific recombinases, including Cre recombinase, have been successfully delivered into cells using this approach (Peitz et al., 2002). Cell permeable recombinases have been further described by Nolden et al. (2006) and Lin et al. (2004). Thus, this strategy can be used to deliver the tailored recombinase into a cell to remove the provirus from the infected cell. Thus, the second polypeptide in the fusion polypeptide can comprise a signal peptide. The signal peptide can be a protein transduction domain such as a TAT peptide or a peptide from the third helix of the Antennapedia homeodomain (Derossi et al., 1994, 1996; Vives et al., 1997; Vives, 2003; Richard et al., 2005) or a NLS (nuclear localization sequence) for delivery of the fusion polypeptide into the nucleus of a eukaryotic cell (Macara, 2001).

[0172] Another aspect of the present application relates to an adult stem cell obtainable from the aforementioned method for preparing the transformed adult stem cell of the present application. The stem cell is preferably infected or transfected with an expression vector according to the present application. In a preferred embodiment, the adult stem cell is a stem cell from the hematopoietic lineage expressing the tailored recombinase, the aforementioned fusion polypeptide or comprising the aforementioned expression vector. Hematopoietic stem cells (HSCs) are CD34+ cells of bone marrow origin, which can be purified from the G-CSF-mobilized peripheral blood of a donor (e.g. an HIV-infected patient) by conventional leukapheresis (Scherr & Eder, 2002). The in vitro genetically modified cells can then be formulated for reinfusion into the patient.

[0173] In the prior art, the term "stem cell" denotes a cell which (a) has the capacity for self-renewal and (b) the capacity to give rise to at least one and often many specialized cell types due to its asymmetric division capacity (Donovan & Gearhart, 2001). Adult stem cells can be isolated from different tissues of an adult, i.e. a differentiated individual. Such stem cells are referred to in the prior art as "multipotent adult stem cells". The essential difference between embryonic pluripotent stem cells and adult multipotent stem cells lies in the number of differentiated tissues that can be obtained from the respective cells.

[0174] In another embodiment, the expression vector of the present application is used to transform T-cells, such as CD4+ primary cells (blood cells) of a patient infected with a retrovirus, such as HIV.

[0175] Alternatively, the tailored recombinase of the present application can be formulated for delivery by a virus-like particle (VLP). The VLP can be used to package a Tre mRNA, a Tre protein, such as a fusion protein, or a DNA, such as a DNA plasmid expressing Tre, or a construct comprising a promoter-Tre cDNA-polyA site. Thus, the nucleic acid of the present application can further contain a packaging signal.

[0176] In another step of the method of the present application, the nucleic acid of the present application, the expression vector comprising a nucleic acid sequence encoding the tailored recombinase of the present application, the recombinase protein, the fusion protein or the somatic stem cell obtained by the method of the present application is formulated as a pharmaceutical composition for the prevention and / or treatment of a retroviral infection and / or for reducing the viral load in a subject infected with a retrovirus, such as HIV, in particular HIV-1. Another object of the present application is a pharmaceutical composition obtained by the aforementioned method. The pharmaceutical composition is preferably present in the form of a solution suitable for intravenous application (infusion).

[0177] The pharmaceutical preparation can further comprise one or more pharmaceutically acceptable carriers, excipients and / or adjuvants. Suitable carriers, excipients and adjuvants for use in pharmaceutical compositions are known in the art.

[0178] When administered to a subject, the pharmaceutical composition of the present application will preferably reduce the viral load in a subject infected with a retrovirus to less than 5000 genome equivalents per ml of plasma, preferably less than 500 genome equivalents per ml of plasma, and more preferably less than 50 genome equivalents per ml of plasma. Thus, the pharmaceutical composition of the present application comprising an expression vector encoding a tailored recombinase (or a tailored recombinase as a protein or fusion polypeptide or a stem cell comprising said expression vector) is capable of reducing the viral load in a subject infected with a retrovirus by eradicating the genetic reservoir of the retrovirus within the host cell, thereby preventing further life cycles of the virus.

[0179] The term "viral load" as used herein refers to the HIV RNA equivalent (i.e. genome) associated with 1 ml of patient plasma (DYBUL et al., 2002), for example. Thus, the viral load is determined by measuring the amount of viral DNA in a sample obtained from a patient. Currently, there are three major types of viral load assays available:

[0180] 1) HIV RNA reverse-transcriptase-polymerase chain reaction (RT-PCR): Amplicor(TM) HIV-1 Monitor Test; Roche Diagnostics;

[0181] 2) branched DNA (bDNA): Versant(TM) HIV RNA Assay; Bayer Diagnostics; and

[0182] 3) nucleic acid sequence based amplification (NASBA): NucliSens(TM) Assay; bioMerieux.

[0183] In a preferred embodiment, the pharmaceutical composition of the present application is capable of reducing the viral load in a subject infected with a retrovirus to less than 5000 genome equivalents per ml of plasma, preferably less than 500 genome equivalents per ml of plasma, and more preferably less than 50 genome equivalents per ml of plasma. Patients with a viral load of less than 5000 genome equivalents per ml of plasma are considered to be relatively well-adapted to drug therapy. However, the current goal of AIDS therapy is to reduce the viral load to below the detection limit of the viral load assay, which is currently below about 50 genome equivalents per ml of plasma. The pharmaceutical composition preferably reduces the viral load of a retrovirus selected from the group consisting of mouse mammary tumour virus (MMTV), Mason Pfizer monkey virus (MPMV), human T-cell leukaemia virus type I (HTLV-I), human T-cell leukaemia virus type II (HTLV-II), simian T-cell leukaemia virus type I (STLV-I), simian T-cell leukaemia virus type II (STLV-II), bovine leukaemia virus (BLV), feline leukaemia virus (FeLV) and Moloney murine leukaemia virus (MoMLV). In yet another preferred embodiment, the retrovirus to be treated with the pharmaceutical of the present application is a lentivirus. The lentivirus is preferably selected from the group consisting of human immunodeficiency virus type 1 (HIV-I), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Maedi-visna virus (MVV), equine infectious anaemia virus (EIAV) and caprine arthritis encephalitis virus (CAEV). Most preferably, the retrovirus is HIV, in particular HIV-I. However, it will be apparent to the person skilled in the art that the present application is also applicable to retroviral infections with other retroviruses than those mentioned above.

[0184] The subject infected with a retrovirus for which the pharmaceutical composition is administered is selected from the group consisting of humans, primates, monkeys, cattle, horses, goats, sheep and domestic cats. However, the subject is preferably a human.

[0185] Generally, an effective amount of the expression vector, tailored recombinase or transformed cell of the application should be administered to the subject. Administration can be, for example, intravenous or intramuscular administration.

[0186] In one embodiment, the pharmaceutical composition is formulated for concomitant administration with other active agents of highly active antiretroviral therapy (HAART). The highly active antiretroviral therapy HAART is a combination therapy targeting viral reverse transcriptase, protease and fusion (GULIC et al., 1997; LALEZARI et al., 2003).

[0187] In another embodiment, the pharmaceutical composition is formulated for administration simultaneously or after systemic immune activation therapy or specific activation of proviral gene expression. The premise of immune activation therapy is based on the hypothesis that intentional activation of latently HIV-infected cells can accelerate the eradication of persistent viral reservoirs. Elimination occurs via immune clearance through programmed death of those cells actively expressing HIV-1 (pro-apoptotic) products (KULKOSKY & BRAY, 2006). Systemic immune activation (activation of immune cells, including resting cells) is generally achieved by, for example, administration of an immunotoxin, a cytokine (e.g., IL-2) or a T cell activating antibody (e.g., OKT3).

[0188] In view of the fact that systemic T cell activation apparently also induces viral replication and increases the number of potential HIV-1 target cells beyond the level that can be contained by HAART (FRASER et al., 2000), immune activation to intentionally activate the HAART-resistant latent reservoirs unfortunately fails to permanently eliminate HIV-1 and viral rebound (for review, see KULKOSKY & BRAY 2006; MARCELLO, 2006; SHEHU-XHILAGA et al., 2005), it is necessary to further treat HIV with specific therapy. One approach is to activate transcription of otherwise quiescent viral genomes. Specific activation of latent proviral gene expression can be achieved by administration of prostratin or the human cytokine IL-7, both of which appear to reactivate latent HIV-1 in the absence of cell proliferation (MARCELLO, 2006). Furthermore, selective transcriptional activation of HIV-1 can also be achieved by histone-deacetylase (HDAC1) inhibitors such as, for example, valproic acid, which ultimately induces a growth halo of HIV-1 from resting cells in the absence of cell activation (MARCELLO, 2006; LEHRMAN et al., 2005).

[0189] However, systemic immune activation therapy or specific activation of proviral gene expression or similar therapeutic strategies greatly benefit from the simultaneous removal of proviral DNA, thereby reducing the pool of infected cells in the patient.

[0190] The present application also provides a method of treating and / or preventing a retroviral infection, in particular an HIV infection, in a subject. In one embodiment, the sequence of the retrovirus infecting a subject is analyzed in a sample obtained from the subject, and if the proviral DNA from the subject comprises the asymmetric target sequence identified in step (a) for which the recombinase has been selected, at least one expression vector encoding a tailored recombinase, at least one tailored recombinase or at least one cell, e.g. one adult stem cell, transformed with said expression vector, is administered to said subject. The sample obtained from the subject can be a blood sample, e.g. comprising infected CD4+ cells. SUMMARY

[0192] Figure 1 : Figure 1 An alignment of the following protein sequences is provided:

[0193] (a) Cre recombinase SEQ ID NO: 6;

[0194] (b) Tre consensus sequence according to WO 2011 / 147590, SEQ ID NO: 7, consensus sequence for Tre recombinase specific for asymmetric target sites within the HIV-1 LTR;

[0195] (c) Tre recombinase 3.0 consensus sequence according to WO 2011 / 147590, SEQ ID NO: 8, Tre recombinase specific for SEQ ID NO: 1 ;

[0196] (d) Tre recombinase 3.1 consensus sequence 85%, SEQ ID NO: 9, individual mutations in the consensus sequence compared to the Cre sequence are present in 85% of all clones generated by the method of the present application and by high-throughput sequencing (33,000 reads of unique 200bp-sequences);

[0197] (e) Tre recombinase 3.1 consensus sequence 100%, SEQ ID NO: 10, consensus sequence for three Tre recombinases 3.1 selected for high specificity for SEQ ID NO: 1 (no activity on loxP, loxH or loxLTR Tre 1.0 (SEQ ID NO: 3-5)) and human tolerance;

[0198] (f) Exemplary Tre 3.1 recombinase uTre, SEQ ID NO: 11, is highly specific for SEQ ID NO: 1 and well tolerated by humans. Modified recombinases according to this sequence are termed uTre, for "universal Tre".

[0199] Bold letter designations indicate conserved amino acids, variable and non- specified positions are indicated by X. Positions of mutated Cre in SEQ ID NO: 9, 10, and / or 11 are underlined in the Cre sequence, and the position of the mutation is provided above the sequence. The only Q89L exchange found in Tre3.1 is italicized.

[0200] Figure 2 : Figure 2 Exemplary evolution vectors for evolutionary selection for recombination on loxP are shown. Corresponding vectors for selection for recombination on loxH can be readily constructed. loxLTR comprises SEQ ID NO: 1. Tre3 evolution round 51-69 with pEVOloxLTR-loxP and pEVOloxLTR-loxH, or 100-1 μg / mL transcriptional activator L-ara, includes two rounds of DNA shuffling.

[0201] Figure 3 : Figure 3 High specificity of uTre compared to Tre3 is shown. Tre3: clone isolated from Tre3.0 library round 43. uTre: clone isolated from Tre3.1 library round 71. Recombination products are marked with one triangle, non-recombination products are marked with two triangles. uTre recombines loxLTR comprising SEQ ID NO: 1 in E. coli under conditions where the modified recombinase is expressed (induced by L-arabinose). In contrast, Tre3 recombines loxLTR and loxP as well as loxH, i.e., it has a relatively loose specificity.

[0202] Figure 4: Figure 4A Exemplary lentiviral expression vectors for constitutive expression of selectable marker EGFP and tre library in human cells are shown. Figure 4B Flowchart of cell screening for well-tolerated, highly specific Tres is shown. Final screening for highly active Tres confirms activity in human cells. Individual clones of recombinases are selected and subjected to further analysis.

[0203] Figure 5: Figure 5 shows anti-viral uTre activity in tissue culture in two representative cultures. PM1 T cells were transduced with vectors encoding GFP alone (control, open circles) or uTre and GFP (uTre, filled squares). Subsequently, cultures were infected with HIV-1 and viral load was monitored over time using a p24 antigen ELISA. This experiment shows that the tailored recombinase is effective in reducing viral load. After several weeks (8 or 9 weeks) viral load was no longer detectable by p24 antigen ELISA.

[0204] Figure 6 Significant anti-viral uTre activity in primary human CD4+ cells derived from HIV infected patients is shown. Cells were transduced with vectors expressing GFP alone (control experiment; left panel) or uTre and GFP (uTre; right panel). Viral replication was monitored by HIV-1 p24 antigen release (open circles) and the percentage of transduced (GFP+) human CD4+ cells was monitored by FACS (filled squares). Expression of uTre resulted in a significant anti-viral effect and protection of CD4+ cells. It is noteworthy that the drop in viral load between day 15 and day 20 in the control experiment reflects cell death due to un-inhibited viral replication.

[0205] Figure 7 Figure 7 Anti-viral uTre activity in HIV infected humanized mice is shown. Immunodeficient mice were transplanted with human CD34+ hematopoietic stem cells / HSCs (control) or uTre expressing CD34+ HSCs (animals #1 and #2). Subsequently, animals were infected with HIV-1 and viral load (determined as HIV-1 RNA copies / ml; open circles) and the percentage of human CD45+ CD4+ cells in all lymphocytes (filled squares) were monitored over time. Example

[0206] Example 1 :

[0207] Materials and methods as described in WO 2008 / 083931, WO 2011 / 147590 and BUCHHOLZ & STEWART, 2001 were used if not otherwise specified. Tailored recombinases capable of recombining asymmetric target sequences in multiple different HIV-1 strains were prepared as described in WO 2011 / 147590. The resulting tre libraries were used in further experiments.

[0208] Example 2:

[0209] To enhance uTre specificity, additional rounds of evolution were performed selecting for recombination activity on loxP and loxH. For this purpose, the evolved Tre library obtained from evolution round 50 was cloned into an evolution vector containing two loxLTR sites (SEQ ID NO: 1) flanked by two loxP sites or two loxH sites, respectively. An exemplary vector is shown in Figure 2 After induction of recombinase expression, recombination on loxLTR results in removal of the only existing Ndel site, while recombination on loxP or loxH does not. Plasmid DNA isolated after each evolution round was digested with Ndel, and recombinase-encoding sequences that have successfully recombined loxLTR, but not loxP or loxH, were amplified by PCR and subcloned back into the evolution vector for the next evolution round. A total of 19 additional Tre3 evolution rounds were performed, including two rounds of DNA shuffling, alternating selection for recombination on loxP and loxH.

[0210] Example 3:

[0211] To screen for uTre-recombinases with significantly reduced cytotoxicity (i.e. cytopathogenicity), the tre library was linked to a lentiviral vector constitutively expressing EGFP from an internal SFFV LTR promoter and the tre library from a constitutive EF1 alpha promoter Figure 4A ). Transduction of Jurkat T cells allowed for sequential sorting (by FACS) of high GFP expressing cells, followed by isolation of non-toxic uTre clones Figure 4B ). To this end, transduced T cell cultures were cell sorted with increasing stringency for EGFP expression at day 3, day 10 and day 24 post transduction. After an additional week of incubation, the remaining tre library was isolated and selected clones were analyzed for enhanced Tre activity.

[0212] Example 4:

[0213] To analyze uTre activity in a cell line, cultures of PM-1 T cells were transduced with ASLV-derived retroviral vectors expressing uTre and GFP or GFP alone (negative control vector). Notably, GFP expression allowed for tracking of transduced cells. At day 10 post transduction, cells were infected with HIV-1 Bal . The impact of uTre expression on HIV-1 replication was monitored by weekly ELISA measurement of the amount of viral p24 antigen in culture supernatant. As shown (Figure 5), p24 release was significantly reduced in uTre transduced cultures, while it remained stable or even increased in control cultures (expressing GFP only).

[0214] Example 5:

[0215] Analysis of uTre activity in primary CD4+ cells derived from HIV-1 infected patients. CD4+ cells were stimulated with CD3 / CD28 magnetic beads for 48 hours. Subsequently, cells were transduced with lentiviral vectors expressing either GFP alone (serving as a negative control) or uTre together with GFP. Cells were cultured in the presence of 100 IU of IL2 for 20 days. Viral load (measured by p24 antigen ELISA) and human transduced CD4+ cell counts (by FACS analysis) were monitored at the indicated days post-transduction. As shown in Figure 6 Figure 2, uTre expression resulted in a significant anti-viral effect (indicated by open circles) and protection of CD4+ cells (indicated by solid squares). In contrast, the decrease in viral load between day 15 and day 20 in the control experiment reflects cell death due to un-inhibited viral replication.

[0216] Example 6:

[0217] Analysis of uTre activity in vivo. Immunodeficient NOG mice (NOD.Cg-Prkdc scid IL2rg tm1Wjl / SzJ) were transplanted with either human CD34+ hematopoietic stem cells / HSCs (control) or uTre expressing CD34+ HSCs. Subsequently, animals were infected with HIV-1 Bal and viral load (detected by ultrasensitive PCR based assay) and percentage of human CD45+ CD4+ cells (by FACS analysis) were monitored over time. As shown in Figure 7 Figure 3, uTre expression resulted in a significant anti-viral activity in vivo.

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[0290] WO 2002 / 44409

[0291] WO 2008 / 083931

[0292] WO 2011 / 147590.

Claims

1. A nucleic acid encoding a tailored recombinase that is capable of recombining asymmetric target sequences SEQ ID NO: 1 within the long terminal repeat of proviral DNA of multiple HIV-1 strains, wherein the amino acid sequence of the tailored recombinase consists of the amino acid sequence according to any one of SEQ ID NOs: 11-13, wherein the recombination of SEQ ID NO: 1 by the tailored recombinase is highly specific, because the tailored recombinase does not recombine loxP or loxH with detectable activity, wherein the loxP sequence is SEQ ID NO: 4 and the loxH sequence is SEQ ID NO:

5.

2. The nucleic acid of claim 1, wherein the tailored recombinase consists of the amino acid sequence according to SEQ ID NO:

11.

3. A tailored recombinase encoded by the nucleic acid of any one of claims 1 to 2.

4. A fusion protein comprising the tailored recombinase of claim 3.

5. A transformed cell comprising the nucleic acid of any one of claims 1 to 2.

6. The transformed cell of claim 5, wherein the cell is a stem cell from the hematopoietic lineage.

7. A pharmaceutical composition comprising the nucleic acid of any one of claims 1 to 2, the tailored recombinase of claim 3, the fusion protein of claim 4 and / or the transformed cell according to any one of claims 5 or 6.

8. Use of the pharmaceutical composition of claim 7 for the manufacture of a medicament for the treatment or prevention of a retroviral infection in a subject, wherein the retrovirus is HIV-1.

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