TAILORED RECOMBINASE FOR RECOMBINING ASYMMETRIC TARGET SITES IN A PLURALITY OF RETROVIRUS STRAINS
The present invention relates to a method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences within the long terminal repeat (LTR) of proviral DNA of a plurality of retrovirus strains inserted into the genome of a host cell, as well as to the obtained expression vector, cells transfected with this, expressed recombinase and pharmaceutical compositions comprising the expression vector, cells and/or recombinase. Pharmaceutical compositions are useful, e.g., in treatment and/or prevention of retrovirus infection. In particular, asymmetric target sequences present in a plurality of HIV strains are disclosed, as well as tailored recombinases capable of combining these sequences (Tre 3.0 and 4.0) and expression vectors encoding them.
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing 440098—402C1_SEQUENCE_LISTING.txt. The text file is 119 KB, was created on Dec. 2, 2014, and is being submitted electronically via EFS-Web.
The present invention relates to a method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences within the long terminal repeat (LTR) of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell, as well as to the obtained expression vector, cells transfected with these, expressed recombinase and pharmaceutical compositions comprising the expression vector, cells and/or recombinase. Pharmaceutical compositions are useful, e.g., in treatment and/or prevention of retrovirus infection. In particular, asymmetric target sequences present in a plurality of HIV-1 strains are disclosed, as well as tailored recombinases capable of combining these sequences (Tre 3.0 and 4.0) and expression vectors encoding them.
TECHNICAL BACKGROUNDRetroviral infections such as for example infections by the human immunodeficiency virus (HIV) are still one of the most important and most widespread human diseases.
One approach to treatment of retrovirus, e.g., HIV, is to target the provirus inserted into the genome of the host cell. Excision of the proviral DNA from the host's genome for example would prevent further HIV replication and differs from current methodologies in that it has the potential to eradicate even dormant virus present in the genome of the host.
One class of proteins that were considered for use in this alternative approach are site-specific recombinases (F
One of the simplest and best understood recombinases is the Cre recombinase from bacteriophage P1 that resolves genome dimers into monomers by recombination between two identical double-stranded DNA sites of a particular sequence (H
Some site-specific recombination systems, including the Cre/loxP-system function without accessory proteins or cofactors and function under a wide variety of cellular conditions. However, since the site-specific recombinases function through specific interactions of the recombinase enzyme subunits with their cognate DNA target sequences, the use of these enzymes is restricted by the requirement that the targeted DNA regions must contain appropriately positioned target sites (L
Extensive mutational and structural analyses of site-specific recombinases have been carried out in recent years to alter their properties and to achieve a better understanding of the intricate mechanisms of these enzymes (for a review see
The method of directed evolution is a powerful method to select enzymes with altered specificities (reviewed in Yuan et al., 2005; and J
This limitation has been overcome by the development of methods that allow the crosslinking of proteins to their respective messenger RNAs (mRNAs) using mRNA-protein fusions and ribosome display. Functional screens for improved protein properties were thus coupled to direct retrieval of corresponding coding molecules, and large pools have been screened in vitro (see for example B
In view of the potential of site-specific recombinases and the need of finding an AIDS therapy eradicating HIV-1 provirus from the genome of host cell, WO 2008/083931 disclosed generation of a tailored recombinase (TRE) that is capable of recombining asymmetric target sites within the LTR of proviral DNA of a retrovirus inserted into the genome of a host cell, thus excising the provirus from the genome of the host cell. The engineered recombinase disclosed in the examples, Tre, recognizes a specific loxP-like site present in a particular HIV-1 strain. WO 2008/083931 appreciated that, due to the high sequence variability of retroviruses, in particular, HIV, for treatment of a patient with a different HIV strain, a different tailored recombinase might have to be adapted, or a collection of recombinases prepared containing tailored recombinases specific for a variety or target sequences.
In light of this, the inventors now addressed the problem of providing a tailored recombinase capable of excising a plurality of retrovirus, e.g., HIV strains. Thus, the generated recombinase can be employed for a plurality of HIV infections, without generation of a new recombinase for every strain. This problem is solved by the present invention.
DESCRIPTION OF THE INVENTIONThe present inventors for the first time provide a method for generating an expression vector encoding a tailored recombinase capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains of one species inserted into the genome of a host cell. Recombinases have been tailored to recognise asymmetric target sites different from their native symmetric target sites by splitting up the substrate into a number of new subsets with smaller differences from the original target and stepwise tailoring recombinases to recognise these subsets (WO 2008/083931). A combinatorial approach allows selection of functional molecules recognising the asymmetric target site within a given sequence. Thus, using the approach of traversing through substrate intermediates during directed molecular evolution, it is possible to produce enzymes with remote novel asymmetric target specificities. The present invention employs this approach to the problem of providing tailored recombinases able to recombine a plurality of retrovirus strains, preferably, strains from one species. They found that in spite of the high sequence variability of retroviruses, it is possible to identify asymmetric target sequences present in a high proportion of the viruses of a particular subtype.
The invention provides a method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell, comprising steps of identifying in the sequence of the LTR of proviral DNA of a plurality of retrovirus strains sequences with a homology of at least 30% 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 in a plurality of retrovirus strains; and generating, through repeated steps of
i) molecular directed evolution on at least one recombinase recognising the known homologous target site using as substrate modified target sequences based on the sequence of 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 may vary from the target sequence on which the recombinase is known to act in one, two or three nucleotides; and
ii) shuffling the recombinase libraries to obtain recombinase libraries able to recombine target sequences more homologous to the asymmetric target sequence;
until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of the retrovirus DNA; isolating the nucleic acid of the least one recombinase obtained; and cloning the nucleic acid encoding the recombinase into a suitable expression vector.
The method of generating a tailored recombinase disclosed in WO 2008/083931 can be used for generating a tailored recombinase capable of recombining an asymmetric target sequence.
The invention also provides a method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell, comprising the steps of
- (a) identifying in the sequence of the LTR of proviral DNA of a plurality of retrovirus strains sequences with a homology of at least 30% 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 in a plurality of retrovirus strains;
- (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 said 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”;
- (c) determining the nucleotides within the sequences of step (b) deviating from the corresponding homologous left half-site and right half-site sequences of the known homologous target site of step (a);
- (d) generating a first subset of two target nucleic acids comprising target sequences, wherein the first target sequence is designated subsite 1 and comprises, adjacent to each other and in 5′ to 3′ order, half-site sequence 1 of step (b), the spacer sequence of the asymmetric target sequence and an inverted repeat of half-site sequence 1, and wherein the second target sequence is designated subsite 2 and comprises, adjacent to each other and in 5′ to 3′ order, an inverted repeat of half-site sequence 2, the spacer sequence of the asymmetric target sequence and half-site sequence 2 of step (b);
- (e) generating a second subset of target nucleic acids comprising modified target sequences on the basis of the target sequences in the first subset of step (d),
- wherein, in sequences based on subsite 1, in the left half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the right half-site of said modified target sequence is formed by an inverted repeat of said modified left half-site sequence, which is separated from said modified left half-site sequence by the spacer sequence of the asymmetric target sequence, and
- wherein, in sequences based on subsite 2, in the right half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the left half-site of said modified target sequence is formed by an inverted repeat of said modified right half-site sequence, which is separated from said modified right half-site sequence by the spacer sequence of the asymmetric target sequence,
- such that in all modified half-site sequences originating from one target sequence of the first subset of step (d) taken together, all deviating nucleotides can be found, whereas none of said modified half-site sequences alone comprises all deviating nucleotides,
- (f) separately applying molecular directed evolution on at least one recombinase recognising a known homologous target site according to step (a) using each nucleic acid of the second subset obtained in step (e) as a substrate;
- (g) shuffling the recombinase libraries evolved in step (f), wherein all recombinase libraries evolved on sequences based on subsite 1 are combined and shuffled, and wherein all recombinase libraries evolved on sequences based on subsite 2 are combined and shuffled;
- (h) applying molecular directed evolution on the shuffled libraries obtained in step (g) using each nucleic acid of the subset according to step (d) as a substrate;
- (i) shuffling the recombinase libraries evolved in step (h);
- (j) applying molecular directed evolution on the shuffled library obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as a substrate, until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of the retrovirus DNA of step (a);
- (k) isolating the nucleic acid of the least one recombinase obtained in step (j) from the library; and
- (l) cloning the nucleic acid obtained in step (k) into a suitable expression vector.
In step (a) of the method of the present invention, the sequence of the LTR of the proviral DNA may be determined, such as for example by DNA sequencing using chain-terminating inhibitors (S
The term “recombinase” as used herein refers to a protein involved in recombination. As such recombinases recognise and bind two specific DNA sequences termed “recombination sites” or “target sites” and mediate recombination between these two target sites. Accordingly, the term “recombinase” is meant to refer to any protein component of any recombinant system that mediates DNA rearrangements in a specific DNA locus. Naturally occurring recombinases recognise symmetric target sites consisting of two identical sequences termed “half-site” 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 characterised by having a tyrosine as the active site nucleophile that is utilised for DNA cleavage, whereas recombinases from the serine integrase family use a serine instead of a tyrosine.
In one embodiment of the present invention, the at least one known recombinase whose target sequence is used in step (a) and upon 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 (C
In another embodiment, said 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 Phage P1 (A
The term variant in this context refers to proteins which are derived from the above proteins by deletion, substitution and/or addition of amino acids and which retain some or all of the function inherent in the protein from which they are derived.
In a preferred embodiment, the known recombinase is a chimeric recombinase obtained by for example “family shuffling” as described by C
In a more preferred embodiment the recombinase is the Cre recombinase recognising a symmetric target site of 34 bp known as loxP (SEQ ID NO:3). The loxP site (and also other recombination sites of wild-type recombinases) is palindromic with two 13 bp repeats separated by the eight innermost base pairs, which represent the so-called spacer, which imparts directionality to the site. Recombination takes place by cleavage within the spacer sequence. Depending on the relative location and orientation of the two participating loxP sites, Cre catalyses DNA integration, excision or rearrangement (H
One useful recombinase is Zre (SEQ ID NO:26) isolated from Salmonella enterica, or variants, fragments and homologues thereof, e.g., having a homology of at least about 70%, at least about 80%, at least about 90% or at least about 95% to the wildtype sequence, and having recombinase function. Zre recombinases recombine DNA at zox sites (
In one embodiment, a recombinase library is used as a starting point for molecular evolution, e.g., a recombinase library comprising different wildtype and/or adapted/shuffled recombinases, e.g., as described below or in Example 2. Such a library is preferably used as a starting point used for generation of the tailored recombinases able to recognize SEQ ID NO:1 and SEQ ID NO:2.
The tailored recombinase is capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains. The proviral DNA targeted by the recombinase may be inserted into the genome of a host cell. Alternatively, the tailored recombinase of the invention may recombine asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains which are not (yet) integrated into the genome of a host cell, i.e. which is present as a non-integrated pre-integration complex (PIC). Thus HIV which has not yet integrated into the genome of the host cell as well as HIV which already has integrated can be inactivated by the tailored recombinase of the invention.
It is to be noted that in the present invention and also in the art the terms “target sequence”, “target site” and “recombination site” are used interchangeably.
Contrary to the naturally occurring recombinases recognising symmetric target site, the method of the present invention provides tailored recombinases recognising target sites, which do not consist of palindromic sequences separated by a spacer. Instead, in the asymmetric target sites the sequences do not form a symmetric inverted repeat. Accordingly, a tailored recombinase able to recognise an asymmetrical target site should recognise and recombine target sites consisting of half-sites of varying sequence.
Within an asymmetric 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-site of a known target site. The sequence located between the sequences homologous to the left and right half-site of a known target site is referred to as spacer.
However, if sequences are found in the LTR that have only homology to either the left or the right half-site sequence of a known target site, these sequences could nevertheless be used in the practice of the present invention. The size of the target site belonging to the recombinase, whose native target sequence shows homology to sequences within the LTR, is known to the skilled person. For example, if homology is found within the LTR sequence to a target sequence recognised by the Cre recombinase, an asymmetric target site to be recognised by Cre recombinase should consist of 34 nucleotides with two half-site sequences of 13 nucleotides each separated by a spacer of 8 nucleotides. Accordingly, the homologous sequence within the LTR is defined as either the left or the right half-site or the spacer of the asymmetric target site depending on the homology to the sequence of the known target site. Thus, sequences with homology to the left half-site of a known target sequence are defined as left half-site, sequences with homology to the right half-site of a known target sequence are defined as right half-site. Starting from this definition, the other parts of the asymmetric target sites are defined under consideration of the structure of the known target site. Thus, having defined for example a right half-site sequence within the LTR over homology to a loxP site (recognised by Cre recombinase), the other sequences corresponding to the spacer and the left half-site of the asymmetric target sequence can easily be defined. The spacer sequence is for example defined by counting 8 nucleotides upstream of the 5′ end of the sequence defined as right half-site sequence, whereas the left half-site sequence is similarly defined by counting 13 nucleotides upstream of the 5′ end of the previously defined spacer sequence.
Homology in this context as well as in the whole application means sequence identity. A preferred comparison for homology purposes is to compare at least two sequences using standard techniques known in the art, including, but not limited to, the local homology algorithm of S
In view of the requirement of two identical target sites that must be present in the genome of the provirus to allow the recombinase to excise the sequence between these two target sites, sequences of the proviral DNA are scanned in step (a) of the method of the present invention that are present at least twice in the genome. Such sequences are for example the LTR sequences of the proviral DNA. Accordingly, the sequence of the LTR is preferably scanned, since the 5′-LTR and the 3′-LTR of the proviral DNA are identical. An asymmetrical target site present in the 5′-LTR is also present in the 3′-LTR and thus allows for excision of the proviral DNA located between the LTRs.
Out of the sequences identified within the LTR sequence having sufficient homology to known target sites, sequences are preferably chosen that have the highest homology to the sequence of the target site of known recombinases. However, it is also possible to select sequences other than those having the highest homology, e.g., those that are present in the highest number of retrovirus strains, or in the retrovirus strains of interest, e.g., if a patient is infected with a particular strain.
It is to be noted that the potential of the method of the present invention even allows tailoring recombinases that recognise asymmetric target sites with less than 30% homology to known target sites, e.g., at least 11% or at least 20% homology. However, to ensure the presence of residual recombination activity for the respective asymmetric target site, it is preferably scanned for sequences having a homology of at least 30% to the left half-site and the right half-site sequences of known target sites of known recombinases. In further preferred embodiments asymmetric target sequences having a homology of 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% to the left half-site and the right half-site sequences of known target sites of known recombinases are selected.
In one embodiment of the present invention, the sequence selected within the LTR has homology to the symmetric loxP target sites recognised by the site specific recombinase Cre.
In one embodiment, a recombinase library is used as a starting point for molecular evolution, e.g., a recombinase library comprising different wildtype and/or adapted/shuffled recombinases such as the library described in Example 2. An exemplary library comprises Cre and recombinases derived therefrom. It may also comprise Tre, Dre, recombinases from Salmonella and Shewanella and/or recombinases derived therefrom. The library may comprise, e.g., Cre, Dre, Dre “Cre-ed” (SEQ ID NO: 24), Shewanella recombinase (Shew), Shew “Cre-ed” (SEQ ID NO:25), and/or Zre (SEQ ID NO:26). One library is described in
In one embodiment, all recombinases in the library recognize a target sequence with the same length of spacer. The total length of the half-site sequences 1 and 2 including spacer preferably is 34 nucleotides.
If the at least one recombinase is a recombinase library, the homology is homology to the pool of known recombinase target sites (i.e., homology in a given position to at least one of a target sequences is defined as homology). Consequently, in step (c), only those nucleotides which do not correspond to a nucleotide in at least one of the known target sequences are defined as deviating nucleotides. In the case of a recombinase library, a “native nucleotide” in step (e) can be a nucleotide present in that position in any of the known target sequences, preferably, it is a nucleotide present in that position in several or most of the known target sequences.
To identify target sequences present in a plurality of retrovirus strains, the known recognition sites of recombinases, which have been described in literature, can be used as a query for a search for conserved asymmetric target sequences against a genomic stretch. Given the repetitive nature of regions, the use of standard sequence similarity search tools however is precluded. Sarkar et al., 2007, used BLAST (ALTSCHUL et al., 1997) to find a lox-like binding site across HIV strains. The BLAST search for the lox-like site when performed across HIV-1 LTR sequences resulted in the discovery of only one site present in a single strain. If recombinases, however, are to be used as therapeutic agents against retroviral genomes, it is critical to engineer recombinases to target recognition sites present across as many strains of the retrovirus as possible.
As BLAST does not perform well with such short redundant sequences, it was considered to use HMMER (EDDY et al, 1998), RepeatMasker or the palindrome program from the Emboss suite of packages. HMMER was developed to find remote homologs based on a probabilistic model of a sequence pattern being looked for, which is not the nature of the search intended to perform. HMMER could be forced to address this search question, but the amount of pre- and postprocessing of data and parameters would make it highly error-prone and inefficient. RepeatMasker only searches for repeats and low complexity regions that are already well characterized, which is again not the nature of this search. The Emboss palindrome program comes closest to addressing the search problem, but does not allow for a search to be defined, but rather results in a list of possibilities of lox-like sites. These would then have to be matched to the lox-site signature of interest. Clearly, such a strategy would only make the search convoluted and cumbersome. To address the lack of a program and a method to find lox-like sites, a program able to search degenerate lox-like sites in genomic sequences had to be developed.
An asymmetric target sequence found in a plurality of retrovirus strains may be identified according to this program, using an algorithm based on a position weight matrix for the flanking regions based on a known recognition site of a recombinase. Preferably, to make the search computationally efficient, binary operations are used on the sequences after they are transformed into bit strings.
In one preferred embodiment of the invention, the retrovirus is HIV, in particular HIV-1. For HIV-1, suitable asymmetric target sequences have been determined, having sequence set forth as SEQ ID NO:1 or SEQ ID NO:2 below.
The left half-site and right half site sequences are underlined and the spacer is printed in bold:
SEQ ID NO:1 is identical in 92% of the HIV-1 subtype B strains searched (348/379) and in 80% of the HIV-1 subtype A strains searched (32/40). SEQ ID NO:2 is identical in 76% of the subtype B strains searched (288/379). 82% of subtype C strains also comprise this sequence. SEQ ID NO:2 is not present in any of the subtype A strains searched.
As shown in
In step (b) of the method of the invention, the sequence of the asymmetric target site within the LTR of the proviruses which is homologous to the left half-site of the known target site is defined as “half-site sequence 1”. The sequence of the asymmetric target site within the LTR of the proviruses which is homologous to the right half-site of the known target site is defined as half-site sequence 2. The sequence between the sequences representing the left and the right half-site is referred to as the spacer.
In step (c), the nucleotides within “half-site sequence 1” and “half-site sequence 2”, respectively, of the sequences of step (b) deviating from the sequences of the corresponding homologous left half-site and right half-site sequences of the known target are determined by sequence alignment and sequence comparison. In this context, the sequence of “half-site sequence 1” is compared to the corresponding native half-site, which is preferably the left half-site sequence, whereas the sequence of “half-site sequence 2” is compared to other half-site forming the palindromic native target site, which is preferably the right half-site sequence.
This comparison must not necessarily performed after step (b) and prior to step (d) of the method of the invention, but can also be performed in a different phase of the method after step (a) and prior to step (e).
In step (d), a first subset of two target nucleic acids comprising target sequences is generated, wherein the first target sequence is designated subsite 1 and comprises, adjacent to each other and in 5′ to 3′ order, half-site sequence 1 of step (b), the spacer sequence of the asymmetric target sequence and an inverted repeat of half-site sequence 1, and wherein the second target sequence is designated subsite 2 and comprises, adjacent to each other and in 5′ to 3′ order, an inverted repeat of half-site sequence 2, the spacer sequence of the asymmetric target sequence and half-site sequence 2 of step (b). 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 synthesised on the basis of the half-site sequences of step (b) by complementing the missing half-site sequence in each oligonucleotide sequence as inverted repeat, wherein the sequence of “half-site sequence 1” and “half-site sequence 2”, respectively, is used to complement the second half-site sequence at the opposite end of the spacer sequence. Accordingly, the first target sequence in the first subset (referred to as “subsite 1”) comprises an inverted repeat consisting of the “half-site sequence 1” and the inversely repeated “half-site sequence 1′” separated by the spacer sequence, whereas the second target sequence in the first subset (referred to as “subsite 2”) comprises an inverted repeat consisting of the inversely repeated “half-site sequence 2′” and “half-site sequence 2” separated by the spacer sequence. In “subsite 1” the sequence are arranged as follows: 5′-“half-site sequence 1”-spacer-“inverted repeat of half-site sequence 1′”-3′, in “subsite 2” the sequence are arranged as follows: 5′-“inverted repeat of half-site sequence 2′”-spacer-“half-site sequence 2”-3′.
The spacer sequences within each two synthetic target sequences of the first subset are preferably identical and correspond to the sequence of the LTR representing or defined as the spacer sequence of the asymmetric target site. However, in a further embodiment, the spacer sequences may comprise one or two sequence deviations originating from nucleotide substitutions.
Generally, this step represents a first split up of the sequences of the asymmetric target site selected for tailoring a specific recombinase (see FIG. 1 of WO 2008/083931, which is fully incorporated herein by reference, and
In step (e) of the method of the invention, a second subset of target nucleic acids comprising modified target sequences is generated on the basis of the target sequences in the first subset of step (d). In sequences based on subsite 1, in the left half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three (preferably, two) nucleotides deviating from said known target site, wherein the right half-site of said modified target sequence is formed by an inverted repeat of said modified left half-site sequence, which is separated from said modified left half-site sequence by the spacer sequence of the asymmetric target sequence.
In sequences based on subsite 2, in the right half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three (preferably, two) nucleotides deviating from said known target site, wherein the left half-site of said modified target sequence is formed by an inverted repeat of said modified right half-site sequence, which is separated from said modified right half-site sequence by the spacer sequence of the asymmetric target sequence.
For example, if one subsite comprises six deviating nucleotides, such as both subsites based on SEQ ID NO:1 or subsite 2 of SEQ ID NO:2 with regard to the library of recombinases shown in
In another example, if one subsite comprises nine deviating nucleotides, such as subsite 1 of SEQ ID NO:2 with regard to the library of recombinases shown in
As a consequence, in all modified half-site sequences originating from one target sequence of the first subset of step (d) taken together, all deviating nucleotides can be found, whereas none of said modified half-site sequences alone comprises all deviating nucleotides,
Again, an inverted repeat is generated on the basis of the modified half-site sequence, such that the spacer sequence separates both sequences forming the inverted repeat (see
The term “deviating nucleotide” as used herein refers to a nucleotide within the asymmetric target sequence identified or defined within the LTR or within a target sequence of a subset generated according to the present invention that deviates (i.e. is different) from the nucleotide present at the same position in the corresponding homologous sequence of the known homologous symmetric target sequence of a known recombinase chosen in step (a) of the method of the present invention. In this context, the terms “deviating nucleotides” and “mutations” are used interchangeably.
WO 2008/083931 teaches that recombinases can be tailored using molecular directed evolution using target sequences as a substrate, if the target sequence used as a substrate differs in not more than 3 nucleotides from the native target sequence. Thus, the generation of subsets of different orders described above serves to reduce the number of deviating nucleotides per target sequence to 3 or less (see FIG. 1 of WO 2008/083931). The stepwise reduction of the number of deviating nucleotides finally yields a number of subsets of target sequences of different orders with decreasing numbers of deviating nucleotides until a final subset is created that can be used as a substrate for molecular directed evolution. While creating the different subsets and thereby reducing the number of deviating nucleotides, the differences to the target site recognised by the wild-type recombinase are spread between several target sequences that do not comprise more than 3 of these deviating nucleotides each, while the target sequences of the final order as a whole still represent all deviating nucleotides.
Optionally, in the method of the invention, further subsets of target sequences can be generated starting from the target sequences of the second subset by stepwise repeating the process of step (e), i.e. splitting up the target sequences into the respective half-site sequences and generating new palindromic structures on the basis of these half-site sequences after altering the sequence of the half-site derived from a target sequence of the second subset, each time generating a new subset of target sequences, wherein the half-site sequences used for generating the inverted repeats contain less nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target site. These additional target sequences can be used for additional steps of directed molecular evolution and shuffling of recombinase libraries. Of course, such an additional step can also only be performed for some of the sequences, e.g., for sequences wherein recombinases with a low efficiency of recombination are 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 invention.
Starting from the second subset of target sequences obtained in step (e), a third subset may be generated, followed by a fourth, fifth, sixth etc. subset if necessary. However, the generation of the third subset is generally only necessary, if the target sequences of the second subset still contain more than three deviating nucleotides. The same applies to the generation of the next subsets, which are only necessary, if the target sequences of the prior subset still contain more than three deviating nucleotides. It should be noted that in one embodiment, subsets of target sequences will be generated until the target sequences of the final subset only comprise one deviating nucleotide. Accordingly, depending on the number of deviating nucleotides in each half-site sequence, the number of subsets generated for each half-site sequence of the asymmetric target site may differ. It may for example be necessary to generate only two subsets for the left half-site sequence, whereas three or four subsets must be generated for the right half-site in order spread the deviating nucleotides between several target sequences such that a single target sequence does not comprise more than 3 of these deviating nucleotides.
The principle of generating further subsets of the target sequences for reducing the number of deviating nucleotides to numbers below three is illustrated in FIG. 1 of WO 2008/083931, and
In step (f), a method of molecular directed evolution is applied on the at least one recombinase recognising a known homologous target site of step (a), using a target sequence of the final or second subset obtained in step (e) containing one, two or three nucleotides deviating from the corresponding homologous half-site sequence of said known homologous target site as a substrate.
The term “final subset” as used herein refers to the last subset generated in step (e), i.e., if no additional subsets are generate, on the second subset. Depending on the number of deviating nucleotides in the asymmetric target site and number of subsets that had to be generated to reduce the number of deviating nucleotide per target sequence below 3, the “final subset” may correspond to any subset, for example the second, third, fourth or a later subset, and may be different for the half-site sequences of the asymmetric target sequence within the LTR. If recombinases have previously been evolved on additional subsets of modified target sequences having less nucleotides deviating from the corresponding homologous half-site sequence of said known homologous target site, the recombinase obtained in that step is used.
Of course, it is possible to start the process of the invention with a specific recombinase for a specific modified target sequence, and with another recombinase (or a library) for another specific modified target sequence.
Methods of molecular directed evolution, also referred to as laboratory evolution or in vitroevolution, are known in the art (for a review see Y
In a first step of molecular directed evolution, libraries of randomly mutated recombinase sequences are generated by methods known in the art, e.g. by using error prone PCR and DNA shuffling (reviewed in e.g. Y
In a preferred embodiment, the molecular directed evolution employed in step (f) of the method of the present invention is substrate-linked protein evolution (SLiPE; Buchholz & Stewart, 2001; International Patent application WO 02/44409). The substrate-linked protein evolution may be carried out as described in detail in the examples of WO 2008/083931. Briefly, the target sequences obtained in step (e) are cloned into a plasmid (the so-called evolution vector) together with a randomly mutated coding sequence for the recombinase. The random mutation is carried out by error-prone PCR (see B
In step (g), the recombinase libraries evolved in step (f) are combined and shuffled. The technology of DNA shuffling is known in the art (for a reviewed see M
The combined and shuffled libraries are then cloned into a new generation of vectors comprising the target sequences of the next higher subset, i.e., if two subsets are generated, the subset generated in step (d). For example, the vector for the library evolved on the sequences based on subsite 1 comprises the sequence of subsite 1 as a target sequence, and the vector for the library evolved on the sequences based on subsite 2 comprises the sequence of subsite 2 as a target sequence.
In step (h), the method of molecular directed evolution is applied on the shuffled libraries obtained in step (g) using the target sequence of the next higher subset, which, as discussed may be the subset according to step (d). In this step, the same method of molecular directed evolution as those applied before in step (f) can be used, but it is also possible to use a different method of molecular directed evolution in this step of the method of the present invention. Examples of different methods of molecular directed evolution were described for example by Y
This step yields recombinases recognising and recombining target sequences harbouring the combination (and thus increasing numbers) of mutations from the different target sequences of the lower subset. The combination of mutations from the different libraries of a lower subset of target sequences results in synergistic effects and leads to the generation of recombinases, which now recombine target sequences of a higher subset, demonstrating that an evolution strategy traversing through intermediates can be used to achieve a desired activity.
In step (i), the steps (g), i.e. combining and shuffling of recombinase libraries, and (j), i.e. the application of molecular directed evolution on the combined and shuffled libraries, are repeated until at least one recombinase is achieved that is active on the asymmetric target sequence present in the LTR of the proviral DNA.
For example, in a method wherein the generation of two subsets of target sequences was necessary to generate target sequences with only one, two or three nucleotide deviations, the recombinase libraries evolved for example for the second subset of target sequences are combined and shuffled and molecular directed evolution is applied on this shuffled library using the target sequences of the first subset. In the next (and final) step the asymmetric target sequence of step (a) within the LTR of the proviral DNA is used to evolve the recombinase library comprising recombinases recognising the target sequences of the first subset by molecular directed evolution to obtain at least one recombinase that is active on the asymmetric target sequence within the LTR of the retroviral DNA. In this step, the method of molecular directed evolution preferably is the method of substrate-linked protein evolution.
In step (k), the nucleic acid of a recombinase having activity on the asymmetric target sequence of step (a) within the LTR of the retroviral DNA is isolated from the library. The nucleic acid is isolated from the respective plasmid within the library using appropriate restriction enzymes. Method of restriction endonuclease digestion are known to skilled person. The nucleic acid encoding the recombinase can than be recovered by known methods such as for example gel electrophoresis.
The nucleic acid may be stored (preferably at temperatures below −80° C.) or may optionally be cloned in step (l) into an expression vector for use in further analysis, in protein expression methods, or for the administration to a subject for treating and/or preventing retrovirus infection, in particular, HIV infection and/or AIDS. Suitable expression vectors are known in the state of the art or disclosed below.
Preferably, the asymmetric target sequence identified in step (a) is localised in both the 5′-LTR and the 3′-LTR of the provirus to allow excision of the proviral DNA from the genome of the host cell.
Using substrate linked directed evolution and the sequences identified in SEQ ID NO:1 and SEQ ID NO:2 as a substrate, the present inventors are producing a tailored recombinase that recombines this asymmetric DNA target sequence present in the HIV-1 long terminal repeats of a plurality of HIV-1 strains. The development of such tailored recombinases that specifically targets asymmetric sequences within a plurality of HIV-1 LTRs allows the excision of the respective provirus from its chromosomal integration for the majority of subjects infected with HIV-1. Thus, in one embodiment, the tailored recombinase is derived from a library of recombinases comprising Cre recombinase, e.g., a library as described in
In one embodiment, the invention provides a method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of HIV-1 strains which may be inserted into the genome of a host cell, comprising the steps of
- (a) identifying SEQ ID NO:1 or SEQ ID NO:2 having homology to the left half-site sequence and the right half-site sequence of the recombinase target sites of a recombinase library comprising the recombinases specified in
FIG. 1 ; - (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 said 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”;
- (c) determining the nucleotides within the sequences of step (b) deviating from the corresponding homologous left half-site and right half-site sequences of the known homologous target site of step (a), as shown in
FIG. 1 ; - (d) generating a first subset of two target nucleic acids comprising target sequences, wherein the first target sequence is designated subsite 1 and comprises SEQ ID NO:8 (for SEQ ID NO:1), or SEQ ID NO:16 (for SEQ ID NO:2), and wherein the second target sequence is designated subsite 2 and comprises SEQ ID NO:12 (for SEQ ID NO:1), or SEQ ID NO:20 (for SEQ ID NO:2);
- (e) generating a second subset of target nucleic acids comprising modified target sequences on the basis of the target sequences in the first subset of step (d), comprising SEQ ID NO:9-11 and 13-15 (for SEQ ID NO:1), or SEQ ID NO:17-19 and 21-23 (for SEQ ID NO:2), respectively,
- (f) separately applying molecular directed evolution on said recombinase library using each nucleic acid of the second subset obtained in step (e) as a substrate;
- (g) shuffling the recombinase libraries evolved in step (f), wherein all recombinase libraries evolved on sequences based on subsite 1 are combined and shuffled, and wherein all recombinase libraries evolved on sequences based on subsite 2 are combined and shuffled;
- (h) applying molecular directed evolution on the shuffled libraries obtained in step (g) using each nucleic acid of the subset according to step (d) as a substrate;
- (i) shuffling the recombinase libraries evolved in step (h);
- (j) applying molecular directed evolution on the shuffled library obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as a substrate, until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of the retrovirus DNA of step (a);
- (k) isolating the nucleic acid of the least one recombinase obtained in step (j) from the library; and
- (l) cloning the nucleic acid obtained in step (k) into a suitable expression vector wherein the method of molecular directed evolution preferably is substrate-linked protein evolution.
The TRE recombinase tailored to recognize SEQ ID NO:1 is designated TRE 3.0. The TRE recombinase tailored to recognize SEQ ID NO:2 is designated TRE 4.0.
However, it is obvious for the person skilled in the art that other tailored site specific recombinases can be generated that recombine divergent target sites found in the genome of a plurality of retroviral provirus inserted into the genome of the host cell. Other candidate target sequences can for example be determined which are present in the genome of a plurality of retrovirus strains.
The proviral DNA which may be inserted into the genome of a host cell, or which may not yet be inserted, preferably is the DNA of a retrovirus. Retroviruses comprise a large and diverse family of enveloped RNA viruses. The hallmark feature of the family is its replicative strategy which includes as essential steps the reverse transcription of the viral RNA into linear double-stranded DNA and the subsequent integration of this DNA (proviral DNA) into the genome of the host cell. Retroviruses are subdivided into seven groups, defined by evolutionary relatedness. Five of these groups (alpha-, beta-, delta-, epsilon-, and gamma-retrovirus) represent retroviruses with oncogenic potential, and the other two groups are the lentiviruses and the spumaviruses. The human pathogenic human T cell leukemia viruses type I and type II (HTLV-I and HTLV-II) belong to the delta-retrovirus group, while the AIDS viruses human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2) belong to the lentivirus group (for a review see the standard textbook “Retroviruses” of C
In one embodiment, the proviral DNA which may be inserted into the genome of a host cell is the DNA of a retrovirus selected from the group consisting of Mouse mammary tumour 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).
In another embodiment, the retrovirus is a lentivirus selected from the group consisting of Human immunodeficiency virus Type 1 (HIV-1), 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 stated above, HIV, in particular HIV-1 is preferred.
In a more preferred embodiment, the asymmetric target sequence identified in step (a) of the method of the present invention is localised in both the 5′-LTR and the 3′-LTR of a HIV provirus. Preferably, said asymmetric target sequence localised in both the 5′-LTR and the 3′-LTR of a HIV provirus has the sequence set forth as SEQ ID NO: 1 or SEQ ID NO:2.
In a preferred embodiment, the method of molecular directed evolution applied in the method of the present invention is the method of substrate-linked protein evolution (SLiPE; B
By carrying out the method of the invention as described herein, the inventors generated several nucleic acids encoding a tailored recombinase, and the tailored recombinases themselves. The invention thus provides a tailored recombinase comprising a sequence according to any of SEQ ID NO: 40-64 or consisting thereof, or nucleic acids encoding it.
It was surprisingly found that these tailored recombinases preferably comprise certain consensus sequences as shown in
The invention thus provides to a tailored recombinase capable of recombining asymmetric target sequences within the LTR of proviral DNA of a retrovirus strain which may be inserted into the genome of a host cell (i.e., a functional tailored recombinase), which preferably comprises SEQ ID NO: 37. Preferably, said tailored recombinase is capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell. Such a tailored recombinase is e.g. obtainable according to the method of the invention. Preferably, said tailored recombinase comprises SEQ ID NO: 37 and comprises at least one, preferably, 2, 3, 4, 5, 6, or 7 of the following defined amino acids exchanges as compared to the Cre sequence (SEQ ID NO: 36): P12S, P15L, M44V, K86N, G93A, A175S, P307A. These exchanges render the enzyme particularly suited for recombination at a target sequence of SEQ ID NO: 1 or a target sequence having a 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).
In one embodiment, the sequence of the tailored recombinase of the invention, in particular, the tailored recombinase comprising SEQ ID NO: 37 is not disclosed in WO 2008/083931. Preferably, the sequence does not comprise SEQ ID NO: 65 of the present application (which is identical to SEQ ID NO: 3 of WO 2008/083931), or positions 11-351 of SEQ ID NO: 65, and the sequence of the nucleic acid encoding a tailored racombinase of the invention does not encode a protein comprising positions 11-351 of SEQ ID NO: 65. The sequence of the tailored recombinase of the invention comprising SEQ ID NO: 37 also varies from the naturally occurring recombinases such as Cre, Dre, Fre or Zre, which is evident from the feature that it is capable of recombining asymmetric target sequences within the LTR of proviral DNA of a retrovirus strain which may be inserted into the genome of a host cell, and, preferably, that it is capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell.
If the tailored recombinase capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains inserted into the genome of a host cell is to recombine the target sequence of SEQ ID NO: 1, it preferably comprises SEQ ID NO: 38 or SEQ ID NO: 39.
Functional tailored recombinases capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell, which may e.g., be obtained by the method of the invention, may vary from said sequences, but the sequences provide valuable guidance to the skilled person to produce a tailored recombinase capable of recombining asymmetric target sides, such as SEQ ID NO: 1, even without carrying out the method of the invention. The invention also provides a tailored recombinase comprising sequences having at least 97% sequence identity to SEQ ID NO: 38, and/or having at least 98% sequence identity to SEQ ID NO: 38 and/or having at least 99% sequence identity to SEQ ID NO: 38. Preferably, a tailored recombinase has at least 97% sequence identity to SEQ ID NO: 39, and/or at least 98% sequence identity to SEQ ID NO: 39 and/or at least 99% sequence identity to SEQ ID NO: 39. Having a certain percentage of sequence identity to a sequence of SEQ ID NO: 37, 38 or 39 in the context of the present invention means sequence identity to the positions defined in the respective sequence, e.g., with 97% sequence identity, 3% of the defined amino acid positions of said sequences may vary. Preferably, amino acid exchanges with regard to the reference sequences are conservative substitutions, which are well known to the skilled person (eg. Creighton (1984), Proteins. W. H. Freeman and Company (Ed.)). For example, conservative substitutions substitute one amino acid from the group of negatively charged amino acids by another.
The tailored recombinases capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell may also comprise a combination of 2, 3, 4 or more sequences selected from the group consisting of SEQ ID NO: 40-64, e.g., a C-terminal part of any of these sequences, e.g., SEQ ID NO: 40, and the N-terminal part of any other of these sequences, e.g., SEQ ID NO: 64. The C-terminal part may have a length of 1-342 amino acids. In a combination of two sequences, the N-terminal part may have a length of 1-342 amino acids. The combination in any case comprises a TRE consensus motiv, e.g., SEQ ID NO: 37, or preferably, SEQ ID NO: 38 or SEQ ID NO: 39.
The invention further provides composition comprising two or more tailored recombinases comprising different sequences according to SEQ ID NO: 37, preferably, SEQ ID NO: 38 or SEQ ID NO: 39, or nucleic acids encoding them. In one embodiment, the composition comprises two or more, three or more, four or more, five or more, ten or more, 20 or more or 25 recombinases comprising sequences according to any of SEQ ID NO: 40-64 or nucleic acids encoding them. Such compositions may be pharmaceutical compositions as described below.
The invention also provides a nucleic acid encoding a tailored recombinase capable of recombining asymmetric target sequences within the LTR of proviral DNA of a plurality of retrovirus strains which may be inserted into the genome of a host cell, the tailored recombinase comprising an amino acid sequence as defined above. In preferred embodiments, said an amino acid sequence has at least 97% sequence identity to a sequence according to SEQ ID NO: 38, at least 98% sequence identity to a sequence according to SEQ ID NO: 38 or at least 99% sequence identity to SEQ ID N: 38, or comprises SEQ ID NO: 38. The invention also provides a nucleic acid encoding a tailored recombinase comprising an amino acid sequence having at least 97% sequence identity to a sequence according to SEQ ID NO: 39, at least 98% sequence identity to a sequence according to SEQ ID NO: 39 or at least 99% sequence identity to SEQ ID N: 38, or an amino acid sequence comprising SEQ ID NO: 39. The invention also provides a nucleic acid encoding a tailored recombinase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 40-64 or a combination of said sequences.
In the context of the invention, a nucleic acid or a protein comprising a sequence may also consist of this sequence.
It may also comprise further sequences, e.g., a signal sequence providing for expression/localisation in a specific cellular compartment such as a nuclear localization signal (e.g., the signal in positions 2-9 of SEQ ID NO: 65). If a protein is to be used in a pharmaceutical composition, it is especially preferred to express it as a fusion potein with a protein transduction domain such as the tat protein transduction domain, which allows for protein transduction of target cells. Preferably, a tailored recombinase of the invention which is 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 invention may encode such a fusion protein. For example, the following protein transduction domains may be used in a fusion protein with a tailored recombinase of the invention, which preferably further includes a nuclear localization signal:
-
- Basic domain of HIV-1 Tat transactivator (Fawell S, Seery J, Daikh Y, Moore C, Chen L L, Pepinsky B, Barsoum J., Tat-mediated delivery of heterologous proteins into cells., Proc Natl Acad Sci USA. 1994 Jan. 18; 91(2):664-8.)
- Homeodomain of Drosohila antennapedia (Antp) (Derossi D, Joliot A H, 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.)
- 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.)
- Cell permeable translocation motif (TLM) of PreS2 surface antigen of 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.).
In case the protein is to be purified, a tag facilitating purification of a protein such as a His tag, may also be added.
The codon usage of the nucleic acid of the invention encoding a Tre recombinase as defined above can be chosen by the skilled person. For example, a codon usage suitable for expression in a human cell may be chosen, in particular, if expression in a human cell is intended, e.g., for therapeutical purposes. Codon usage may also be based on codon usage of e.g., Cre recombinase.
The tailored recombinase or nucleic acid encoding said tailored recombinase may be obtained by the method of the invention as described herein, or it may be obtainable by this method. It may also be obtained based on the sequences disclosed herein, optionally, by combining and/or further varying these sequences, optionally testing for activity in recombination of asymmetric target sites, such as SEQ ID NO: 1 or SEQ ID NO: 2.
The invention further provides for a composition, e.g., a library, comprising two or more of the nucleic acids encoding a tailored recombinase as defined above, e.g., encoding two or more tailored recombinases comprising different sequences according to SEQ ID NO: 37, preferably, different sequences according to SEQ ID NO: 38 or SEQ ID NO: 39. In one embodiment, the composition comprises nucleic acids encoding tailored recombinases 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 sequences according to any of SEQ ID NO: 40-64 or combinations of these sequences. Such compositions, in particular compositions wherein the nucleic acid is an expression vector, may be particularly suitable as pharmaceutical compositions as described below.
In the method of the present invention, the nucleic acid encoding the at least one tailored recombinase that is active on the asymmetric target sequence within the LTR of the retroviral DNA is cloned into an expression vector. Expression vectors are genetic constructs for expressing the proteins encoded by the nucleic acids within the vector. Such expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the tailored recombinase of the present invention.
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 utilise promoters, polyadenylation signals, and enhancers.
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. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the tailored recombinase. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
The expression vector used in the present invention may be a retroviral vector, a lentiviral vector, a spumavirus vector or an adenoviral vector. However, in a preferred embodiment the expression vector is a lentiviral vector selected from the group consisting of HIV-1-, SIV-, FIV- or EIAV-derived lentiviral vectors. Lentiviral vectors are for example described by S
In preferred embodiments of the present invention the expression vector comprises a cellular, bacterial, a viral or a hybrid promoter.
In general, for the purpose of the present invention, the promoter may be a constitutive or an inducible promoter. Further, the promoters may be either a naturally occurring promoter, such as a bacterial, cellular or a viral promoter, or a hybrid promoter. Hybrid promoters, which combine elements of more than one promoter, are known in the art, and are useful in the present invention. Further, the promoter used in the present invention may also be a derivative of a naturally occurring promoter. A “derivative” of a naturally occurring promoter as used herein may be a combination of cis-active elements obtained from promoters or sequences of different origin or, alternatively, may be obtained by deletion or mutation of cis-active elements within a specific naturally occurring promoter (E
In an embodiment of the present invention, the constitutive promoter or derivative thereof is selected or derived from the group consisting of promoters of cytomegalovirus, Rous sarcoma virus, murine leukemia virus-related retroviruses, phosphoglycerokinase gene, murine spleen focus-forming virus or human elongation factor 1 alpha.
In a further embodiment of the present invention, the inducible promoter or derivative thereof is selected or derived from the group consisting of the LTR or derivatives thereof derived from lentiviruses, spumaviruses and deltaretroviruses.
In this context, the term “LTR” refers to both the 5′ and the 3′ long terminal repeats of provirus having promoter function (for a review see the standard textbook “Retroviruses” (C
Preferably, the inducible promoter or derivative thereof is selected or derived from the LTR or derivatives thereof derived from HIV-1, HIV-2, MVV, EIAV, CAEV, SIV, FIV, BIV, HTLV-I and HTLV-II.
The present invention further provides a method for preparing a tailored recombinase, wherein said 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 said tailored recombinase from the nucleic acid encoding the recombinase inserted into the expression vector obtained in the aforementioned method for preparing an expression vector encoding a tailored recombinase in a suitable host cell.
Preferably, the recombinases finally obtained are tested in mammalian cells to ensure that they function in a mammalian cell environment. Further, to obtain good expression in mammalian cells the recombinases may be optimised for expression in these cells (e.g. codon usage optimisation using methods well known in the art. See for example S
Expression of the nucleic acid encoding the tailored recombinase cloned into an expression vector, e.g., according to step (l) of the method for preparing an expression vector encoding a tailored recombinase, can be carried out using for example bacterial, insect or mammalian expression systems. However, other expression systems known in the art may also be employed. Methods of introducing exogenous nucleic acid 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(s) in liposomes, and direct microinjection of the DNA into nuclei.
Fusion proteins are prepared by methods well known in the art. For example, the expression vector the nucleic acid encoding the tailored recombinase is cloned into already comprises a nucleic 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 fusion protein.
The host cells used for expressing the tailored recombinase from the expression vector are preferably host cells including prokaryotic cells, such as for example bacterial cells or yeast cells, or eukaryotic cells, such as for example insect cells or mammalian cells. The host cell may be a hematopoietic cell, e.g., an adult hematopoietic stem cell or a T-cell, e.g., a CD4+ cell. The cell may be derived from a subject infected with the retrovirus, and the cell may be administered back to the subject after transformation, and, optionally, cultivation and/or propagation.
The present invention further provides a method for preparing a transformed adult stem cell, wherein said 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 en expression vector encoding a tailored recombinase in vitro into a suitable adult stem cell.
In a further aspect, the present invention is directed to the nucleic acid as obtainable from the aforementioned method of the present invention. Nucleic acids encoding a tailored recombinase defined by a sequence are also provided herein.
A “nucleic acid” as used herein is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.
In its broadest aspect, the nucleic acid obtained by the method of the present invention or defined herein is a nucleic acid encoding a tailored recombinase, wherein the tailored recombinase recombines asymmetric target sequences within the DNA of a provirus from a plurality of virus strains which may be inserted into the genome of a host cell, leading to excision of the provirus from the genome of the host cell, wherein the asymmetric target sites are different from the target sites of the wild-type recombinase.
In a further aspect, the present invention is also directed to the expression vector as obtainable from the aforementioned method of the present invention, and to an expression vector comprising the nucleic acid encoding a tailored recombinase as defined herein.
The term “protein” as used herein includes proteins, polypeptides, and peptides. As will be appreciated by those in the art, the nucleic acid sequences of the invention can be used to generate protein sequences. A further aspect of the invention is the tailored recombinase protein as obtainable, e.g., from the aforementioned method of the present invention, which recombinase may optionally be a fusion protein comprising a functional recombinase. In one embodiment, the tailored recombinase protein may 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 method of the present invention, wherein the tailored recombinase is linked to a second polypeptide.
In one embodiment, the tailored recombinase protein is prepared as a fusion polypeptide to increase expression. In a further embodiment, the tailored recombinase protein is made as a fusion polypeptide to allow introduction of the polypeptide into living cells. Typically, purified proteins cannot enter into cells, because they are not able to pass the cell membrane due to their size. However, fusion of specific peptides sequences to proteins can result in the uptake of these fusion proteins into cells. In the cell the protein can then perform its function. Site specific recombinases, including Cre recombinase, have been successfully delivered into cells with this approach (P
A further aspect of the present invention is directed to the adult stem cell as obtainable from the aforementioned method for preparing a transformed adult stem cell of the present invention. The stem cells are preferably infected or transfected with the expression vector according to the invention. 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 (HSC) are bone marrow-derived CD34+ cells, which can e.g. be purified from G-CSF-mobilised peripheral blood of donors (e.g., HIV-infected patients) by routine leukapheresis (S
In the state of the art, the term “stem cells” designates cells which (a) have the capability of self-renewal and (b) the capability to form at least one and often a number of specialised cell types due to their asymmetrical division capability (D
In a further embodiment, the expression vector of the present invention is used for transforming T-cells, e.g., CD4+ primary cells (blood cells) of retrovirus (e.g., HIV)-infected patients.
In a further step of the method of the present invention, the expression vector comprising the nucleic acid sequence encoding a tailored recombinase of the invention, the recombinase protein, the fusion protein or the adult stem cell obtained by the methods of the present invention are formulated as a pharmaceutical composition for use in prevention and/or treatment of a retrovirus infection and/or for the reduction of the viral load in a subject infected by a retrovirus, e.g., HIV, in particular, HIV-1.
A further object of the invention is the pharmaceutical composition obtained by the aforementioned method. The pharmaceutical composition is preferably present in the form of a solution suitable for intravenous application (infusion).
The pharmaceutical preparation may further comprise one or more pharmaceutically acceptable carrier(s), excipient(s) and/or adjuvant(s). Carriers, excipients and adjuvants suitable for use in a pharmaceutical composition are known in the art.
The pharmaceutical composition of the present invention preferably reduces the virus load in a subject infected by a retrovirus below 5.000 genome equivalents/ml plasma, preferably below 500 genome equivalents/ml plasma and more preferably below 50 genome equivalents/ml plasma when administered to the subject. Thus, the pharmaceutical composition of the present invention comprising an expression vector encoding a tailored recombinase (or the tailored recombinase as a protein or fusion polypeptide or a stem cell comprising the expression vector) is capable of reducing the virus load in a subject infected with a retrovirus by eradicating the genetic reservoir of retroviruses within hosts cells, thereby preventing further life cycles of the virus.
The term “virus load” as used herein refers, e.g., to the HIV RNA equivalents (i.e. genomes) that are associated with 1 ml of the patient's plasma (D
- 1) HIV RNA reverse transcription-polymerase chain reaction (RT-PCR): Amplicor™ HIV-1 Monitor Test; Roche Diagnostics
- 2) Branched chain DNA (bDNA): Versant™ HIV RNA Assay; Bayer Diagnostics; and
- 3) Nucleic acid sequence-based amplification (NASBA): NucliSens™ Assay; bioMerieux.
In a preferred embodiment, the pharmaceutical composition of the present invention is capable of reducing the virus load in a subject infected by a retrovirus below 5.000 genome equivalents/ml plasma, preferably below 500 genome equivalents/ml plasma and more preferably below 50 genome equivalents/ml plasma. Patient with a virus load of below 5000 genome equivalents/ml plasma are considered to be relatively well adjusted to the medicinal treatment. However, the goal in current AIDS therapy is a reduction of the viral load below the detection limit of the virus load assays, which is currently below about 50 genome equivalents/ml plasma.
The pharmaceutical composition preferably reduces the viral load of retroviruses selected from the group consisting of the Mouse mammary tumour 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). In yet a further preferred embodiment retrovirus to be treated with the pharmaceutical of the present invention is a lentivirus. Said lentivirus is preferably selected from the group consisting of Human immunodeficiency virus Type 1 (HIV-1), Human immunodeficiency virus Type 2 (HIV-2), Simian immunodeficiency virus (SIV), Feline immunodeficiency virus (Fly), Bovine immunodeficiency virus (BIV), Maedi-visna virus (MVV), Equine infectious anemia virus (EIAV) and Caprine arthritis encephalitis virus (CAEV). Most preferably, the retrovirus is HIV, in particular HIV-1. However, it is obvious to the person skilled in the art that the present invention is also applicable to retroviral infections by other retroviruses than those mentioned above.
The subject infected by a retrovirus, to whom the pharmaceutical composition is to be 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 being.
In general, an effective amount of the expression vector, the tailored recombinase or the transformed cell of the invention is to be administered to the subject. Administration may be, e.g., intravenous or intramuscular administration.
In one embodiment, the pharmaceutical composition is formulated for concomitant administration with other active agents of the highly active antiretroviral therapy (HAART). The highly active antiretroviral therapy HAART is a combination therapy targeting the viral reverse transcriptase, protease and fusion (G
In another embodiment, the pharmaceutical composition is formulated for administration concomitant or subsequent to global immune activation therapy or specific activation of provirus gene expression. The premise of immune activation therapy is based on the hypothesis that deliberate activation of latently HIV-infected cells may accelerate eradication of persistent viral reservoirs. Eradication would occur via immune clearance by programmed death of those cells actively expressing HIV-1 (pro-apoptotic) products (K
In view of the fact that immune activation conducted to deliberately activate HAART-resistant latent reservoirs did unfortunately fail to permanently eliminate HIV-1 and viral rebound (for reviews see K
However, global immune activation therapy or specific activation of provirus gene expression or similar therapy strategies greatly benefits from the concurrent removal of proviral DNA, thereby reducing in the patient the pool of infected cells.
The present invention also provides a method of treatment and/or prevention of a retrovirus infection, in particular, a HIV infection, in a subject. In one embodiment, the sequence of the retrovirus infecting the subject is analyzed in a sample obtained from the subject, and at least one expression vector encoding a tailored recombinase, at least one tailored recombinase or at least one cell transformed with said expression vector, e.g., one adult stem cell, is to be administered to the subject, if the proviral DNA from the subject comprises the asymmetric target sequence identified in step (a) on which the recombinase has been selected. The sample obtained from the subject may be a blood sample, e.g., comprising infected CD4+ cells.
(B) Successful recombinases were furthermore enriched by performing several rounds of substrate-linked protein evolution on the subsites of the 3rd subset, followed by several rounds of substrate-linked evolution cycles on some intermediate substrates (C) and finally on the 1st subsites (D) in order to get recombinases specific for the asymmetric target site SEQ ID NO:1. In (E) the recombination activity after the 8th evolution cycle with relatively high amounts of transcription activator L-arabinose (L-ara; 200 μg/ml) is assayed, in (F) recombination activity was measured after the 16th evolution cycle with lower transcriptional induction at only 50 μg/ml L-ara. Recombined product is marked with one triangle, non-recombined with two triangles. (G) and (H) show the respective recombination activity after the indicated evolution cycles, the indicated L-ara concentrations and on the indicated subsites. After the final evolution cycle (cycle 43), the recombinase population with the highest activity was isolated and enriched, resulting in the active library. Single clones of recombinases were selected and were subjected to further analyses.
The asymmetric target sequences were identified by a method divided in 3 steps: generation of a position weight matrix based on known recognition sites of a recombinase, provision of genomic sequences that are to be searched for recognition sites, and a binary search for potential target sites in the provided sequences and scoring of the resulting hits based on the initial position weight matrix, wherein the nucleotides are transformed into binary space.
SEQ ID NO:1 was found to be present in 348 of 379 HIV-1 strains of subtype B. Furthermore, SEQ ID NO:1 was found to be present in 32 of 40 HIV-1 strains of subtype A. It is located in the R region of the LTR.
SEQ ID NO:2 was found to be present in 288 of 379 HIV-1 strains of subtype B. SEQ ID NO:2 was not found to be present in any of the searched 40 HIV-1 strains of subtype A. It is located in the U3 region of the LTR.
Example 2 Generation of a Tailored Recombinase Recognising and Recombining an Asymmetric Target Sequence within the LTR of HIV-1To start the evolution process, HIV-1 LTR sequences were selected which are highly conserved among HIV-1 strains. SEQ ID NO:1 and SEQ ID NO:2 were found to comply with these criteria and to represent asymmetric target sequences for which a tailored recombinase can be selected. In the following, the generation of a tailored recombinase recognizing SEQ ID NO:1 is described in detail.
A simplified outline of the evolution strategy and the progress made to date is outlined in
The start library of recombinases was generated by pooling and family shuffling Cre and several known Cre-like recombinases, namely a library of Cre mutants (Cre/Fre) (Buchholz & Stewart, Nature 2001), a previously generated Tre library (Sarkar et al., Science 2007), Dre (Sauer et al., Nucl Acids Res 2004), Zre (isolated from Salmonella enterica, Genbank accession number NZ_ABEW01000015), and Shew (Shewanella sp. strain ANA-3, Genbank accession number CP000470) (
A first subset of two target nucleic acids comprising target sequences was generated, wherein the first target sequence is designated subsite 1 and comprises SEQ ID NO:8, and wherein the second target sequence is designated subsite 2 and comprises SEQ ID NO:12.
A second subset of target nucleic acids comprising modified target sequences was then generated on the basis of subsites 1 and 2. As shown in
The enriched subsite libraries are combined and shuffled to obtain the libraries for continuing the evolution cycling process on the 2nd subset of target sequences, namely subsite 1 and subsite 2. The libraries of successful recombinases are then enriched as described above before combining and shuffling them for the final stage of evolution cycling that is the evolution on the target site to be specifically recognized by the Tre recombinase. The nucleic acid encoding the recombinase may be obtained from the library; and cloned into an expression vector.
Materials and methods are used as described in WO 2008/083931 and BUCHHOLZ & STEWART, 2001, if not specified otherwise.
The obtained TRE recombinases adapted to recognize and recombine the asymmetric target sequences SEQ ID NO:1 and SEQ ID NO:2 are sequenced and their ability to recombine proviral DNA of different HIV-1 target strains confirmed.
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Claims
1.-17. (canceled)
18. A method for preparing an expression vector encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences of SEQ ID NO:1 within the long terminal repeat (LTR) of proviral DNA of a plurality of HIV-1 strains, comprising the steps of
- (a) identifying SEQ ID NO:1 sequences with a homology of at least 30% to the left half-site sequence and the right half-site sequence of at least one known recombinase target site in the sequence of the LTR of proviral DNA of a plurality of retrovirus strains, wherein the homologous sequences are separated by a spacer of 5-12 nucleotides, and represent an asymmetric target sequence within the LTR of proviral DNA, and wherein the asymmetric target sequence is found in a plurality of retrovirus strains;
- (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 said 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”;
- (c) determining the nucleotides within the sequences of step (b) deviating from the corresponding homologous left half-site and right half-site sequences of the at least one known homologous target site of step (a);
- (d) generating a first subset of two target nucleic acids comprising target sequences, wherein the first target sequence is designated subsite 1 and comprises, adjacent to each other and in 5′ to 3′ order, half-site sequence 1 of step (b), the spacer sequence of the asymmetric target sequence and an inverted repeat of half-site sequence 1, and wherein the second target sequence is designated subsite 2 and comprises, adjacent to each other and in 5′ to 3′ order, an inverted repeat of half-site sequence 2, the spacer sequence of the asymmetric target sequence and half-site sequence 2 of step (b);
- (e) generating a second subset of target nucleic acids comprising modified target sequences on the basis of the target sequences in the first subset of step (d), wherein, in sequences based on subsite 1, in the left half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the right half-site of said modified target sequence is formed by an inverted repeat of said modified left half-site sequence, which is separated from said modified left half-site sequence by the spacer sequence of the asymmetric target sequence, and wherein, in sequences based on subsite 2, in the right half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the left half-site of said modified target sequence is formed by an inverted repeat of said modified right half-site sequence, which is separated from said modified right half-site sequence by the spacer sequence of the asymmetric target sequence, such that in all modified half-site sequences originating from one target sequence of the first subset of step (d) taken together, all deviating nucleotides can be found, whereas none of said modified half-site sequences alone comprises all deviating nucleotides,
- (f) separately applying molecular directed evolution on at least one recombinase recognising a known homologous target site according to step (a) using each nucleic acid of the second subset obtained in step (e) as a substrate;
- (g) shuffling the recombinase libraries evolved in step (f), wherein all recombinase libraries evolved on sequences based on subsite 1 are combined and shuffled, and wherein all recombinase libraries evolved on sequences based on subsite 2 are combined and shuffled;
- (h) applying molecular directed evolution on the shuffled libraries obtained in step (g) using each nucleic acid of the subset according to step (d) as a substrate;
- (i) shuffling the recombinase libraries evolved in step (h);
- (j) applying molecular directed evolution on the shuffled library obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as a substrate, until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of the proviral DNA of step (a);
- (k) isolating the nucleic acid encoding the least one recombinase obtained in step (j) from the library; and
- (l) optionally, cloning the nucleic acid obtained in step (k) into a suitable expression vector.
19. The method according to claim 18, wherein the at least one known recombinase whose target sequence is used in step (a) and upon which molecular directed evolution is applied in step (f) is part of a recombinase library.
20. The method according to claim 18, wherein the molecular directed evolution employed is substrate-linked protein evolution.
21. The method according to claim 18, wherein the expression vector in step (l) is selected from the group consisting of retroviral vectors, lentiviral vectors, spumavirus vectors and adenoviral vectors.
22. The method according to claim 18, further comprising the step of preparing the expression vector as a pharmaceutical composition.
23. A method for preparing a tailored recombinase, comprising steps (a) to (l) according to claim 18 and the further step of expressing the tailored recombinase from the nucleic acid encoding the recombinase inserted into the expression vector in a suitable host cell, wherein the recombinase is optionally expressed as a fusion polypeptide comprising the amino acid sequence of the tailored recombinase.
24. The method according to claim 23, further comprising the step of preparing the tailored recombinase as a pharmaceutical composition.
25. A method for preparing a transformed cell, comprising steps (a) to (l) according to claim 18 and the further step of introducing the expression vector in vitro into a cell.
26. The method according to claim 25, wherein the cell is an adult stem cell.
27. The method according to claim 25, further comprising the step of preparing the transformed cell as a pharmaceutical composition.
28. A nucleic acid encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences of SEQ ID NO:1 within the long terminal repeat (LTR) of proviral DNA of a plurality of HIV-1 strains, wherein the amino acid sequence of the tailored recombinase has at least 99% sequence identity to a sequence according to SEQ ID NO: 38, and wherein said tailored recombinase comprises defined amino acid exchanges P12S, P15L, M44V, K86N, G93A, A175S, ad P307A as compared to the Cre sequence of SEQ ID NO:36.
29. A nucleic acid according to claim 28, wherein the amino acid sequence of the tailored recombinase comprises SEQ ID NO: 38.
30. The nucleic acid according to claim 28, wherein the amino acid sequence of the tailored recombinase is selected from the group consisting of SEQ ID NOS: 40-64.
31. The nucleic acid according to claim 28, wherein the amino acid sequence of the tailored recombinase is selected from a combination of sequences from the group consisting of SEQ ID NO: 40-64.
32. The nucleic acid according to claim 28, wherein amino acid exchanges at undefined positions with regard to the sequence of Cre recombinase specified in SEQ ID NO:36 are conservative substitutions.
33. The nucleic acid according to claim 28, wherein the nucleic acid is an expression vector.
34. The nucleic acid according to claim 28, wherein the amino acid sequence of the tailored recombinase comprises the sequence according to SEQ ID NO: 39.
35. A pharmaceutical composition comprising a nucleic acid of claim 28, a tailored recombinase encoded by a nucleic acid of claim 28, and/or a transformed cell comprising a nucleic acid of claim 28.
36. A tailored recombinase encoded by a nucleic acid of claim 28, which is optionally expressed as a fusion protein.
37. A transformed cell comprising a nucleic acid of claim 28.
38. The transformed cell according to claim 37, wherein the cell is a stem cell from the hematopoietic lineage.
39. A nucleic acid of claim 28 as obtainable from a method that comprises:
- (a) identifying sequences with a homology of at least 30% to the left half-site sequence and the right half-site sequence of at least one known recombinase target site in the sequence of the LTR of proviral DNA of a plurality of HIV strains, wherein the homologous sequences are separated by a spacer of 5-12 nucleotides, and represent an asymmetric target sequence within the LTR of proviral DNA, and wherein the asymmetric target sequence is SEQ ID NO:1 and is found in a plurality of HIV strains;
- (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 said known target site (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 (half-site sequence 2);
- (c) determining the nucleotides within the sequences of step (b) deviating from the corresponding homologous left half-site and right half-site sequences of the at least one known homologous target site of step (a);
- (d) generating a first subset of two target nucleic acids comprising target sequences, wherein the first target sequence is designated subsite 1 and comprises, adjacent to each other and in 5′ to 3′ order, half-site sequence 1 of step (b), the spacer sequence of the asymmetric target sequence and an inverted repeat of half-site sequence 1, and wherein the second target sequence is designated subsite 2 and comprises, adjacent to each other and in 5′ to 3′ order, an inverted repeat of half-site sequence 2, the spacer sequence of the asymmetric target sequence and half-site sequence 2 of step (b);
- (e) generating a second subset of target nucleic acids comprising modified target sequences on the basis of the target sequences in the first subset of step (d), wherein, in sequences based on subsite 1, in the left half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the right half-site of said modified target sequence is formed by an inverted repeat of said modified left half-site sequence, which is separated from said modified left half-site sequence by the spacer sequence of the asymmetric target sequence, and wherein, in sequences based on subsite 2, in the right half-site sequence, a portion of the nucleotides deviating from the corresponding homologous half-site sequence of the at least one known target-site of step (a) is replaced by the native nucleotides found in said known target-site, until said half-site sequence contains one, two or three nucleotides deviating from said known target site, wherein the left half-site of said modified target sequence is formed by an inverted repeat of said modified right half-site sequence, which is separated from said modified right half-site sequence by the spacer sequence of the asymmetric target sequence, such that in all modified half-site sequences originating from one target sequence of the first subset of step (d) taken together, all deviating nucleotides can be found, whereas none of said modified half-site sequences alone comprises all deviating nucleotides,
- (f) separately applying molecular directed evolution on at least one recombinase recognising a known homologous target site according to step (a) using each nucleic acid of the second subset obtained in step (e) as a substrate to form recombinase libraries;
- (g) shuffling the recombinase libraries evolved in step (f), wherein all recombinase libraries evolved on sequences based on subsite 1 are combined and shuffled, and wherein all recombinase libraries evolved on sequences based on subsite 2 are combined and shuffled;
- (h) applying molecular directed evolution on the shuffled libraries obtained in step (g) using each nucleic acid of the subset according to step (d) as a substrate to form recombinase libraries;
- (i) shuffling the recombinase libraries evolved in step (h);
- (j) applying molecular directed evolution on the shuffled libraries obtained in step (g) using a nucleic acid comprising the asymmetric target sequence of step (a) as a substrate, until at least one recombinase is obtained that is active on the asymmetric target sequence within the LTR of the proviral DNA of step (a); and
- (k) isolating the nucleic acid encoding the least one recombinase obtained in step (j) from the library.
40. A tailored recombinase encoded by a nucleic acid of claim 39, which is optionally expressed as a fusion protein.
41. A transformed cell comprising a nucleic acid of claim 39.
42. The transformed cell according to claim 41, wherein the cell is a stem cell from the hematopoietic lineage.
43. A pharmaceutical composition comprising a nucleic acid of claim 39, a tailored recombinase encoded by a nucleic acid of claim 39, and/or a transformed cell comprising a nucleic acid of claim 39.
44. A method for treating or preventing retrovirus infection in a subject, comprising administering to a subject a pharmaceutical composition according to claim 35, wherein proviral DNA found in a sample obtained from the subject comprises the asymmetric target sequence on which the recombinase has been selected.
45. A method for treating or preventing retrovirus infection in a subject, comprising administering to a subject a pharmaceutical composition according to claim 43, wherein proviral DNA found in a sample obtained from the subject comprises the asymmetric target sequence identified in step (a) on which the recombinase has been selected.
46. A nucleic acid encoding a tailored recombinase, which tailored recombinase is capable of recombining asymmetric target sequences of SEQ ID NO:1 within the long terminal repeat (LTR) of proviral DNA of a plurality of HIV-1 strains, wherein the amino acid sequence of the tailored recombinase has at least 97% sequence identity to a sequence according to SEQ ID NO: 39, and wherein said tailored recombinase comprises the defined amino acid exchanges P12S, P15L, M44V, K86N, G93A, A175S, ad P307A as compared to the Cre sequence of SEQ ID NO:36.
Type: Application
Filed: Dec 4, 2014
Publication Date: Apr 16, 2015
Inventors: Joachim Hauber (Hamburg), Jan Chemnitz (Oederquart), Frank Buchholz (Dresden), Janet Chusainow (Dresden)
Application Number: 14/560,795
International Classification: C12N 9/12 (20060101); C12N 15/10 (20060101);
