Patent ID: 8871516
Filing Date: 2014-10-28
Classification: A61P,C12N

Abstract:
1. A method for preparing an expression vector encoding a single tailored recombinase, which tailored recombinase recombines asymmetric target sequences within the LTR of proviral DNA inserted into the genome of a host cell comprising the steps of (a) determining the sequence of the LTR of the proviral DNA, identifying therein sequences with homology of at least 30% to left half-site and right half-site sequences of known target sites of recombinases, wherein the homologous sequences of the LTR of the proviral DNA are separated by a spacer of 5-12 nucleotides, and wherein the homologous sequences of the LTR of the proviral DNA with highest homology to a known target site comprise the asymmetric target sequences; (b) preparing a first and second synthetic sequence, wherein the first synthetic sequence comprises the sequence of the asymmetric target sequences of step (a) homologous to the left half-site of said known target site (half-site sequence 1), and wherein the second synthetic sequence comprises the sequence of the asymmetric target sequences of step (a) homologous to the right half-site (half-site sequence 2); (c) determining the nucleotides within the synthetic sequences of step (b) deviating from the homologous left half-site and right half-site sequences of the known target site of the recombinase of step (a); (d) generating a first subset of two target sequences on the basis of the synthetic sequences of step (b), wherein the first target sequence in the first subset comprises an inverted repeat consisting of half-site sequence 1 and half-site sequence 1′ separated by the spacer sequence, and wherein the second target sequence in the first subset comprises an inverted repeat consisting of half-site sequence 2′ and half-site sequence 2 separated by the spacer sequence, wherein half-site sequences 1′ and 2′ are inverted repeats of the respective half-site sequences 1and 2of step (b); (e) generating a second subset of target sequences on the basis of the target sequences in the first subset of step (d), wherein each of the half-site sequences together with the respective spacer sequence of the target sequences in the first subset of step (d) is used to generate an independent target sequence of the second subset by forming an inverted repeat such that the spacer sequence separates both sequences forming the inverted repeat, wherein the sequences of both half-site sequences originating from one of the target sequences in the first subset of step (d) are altered during their synthesis and prior to using same for generating the inverted repeat yielding the complete target sequence such that (1) in the left half-site sequence, a portion of the nucleotides deviating from the homologous half-site sequence of the known target-site of step (a) is replaced by the native nucleotides found in the known target- site, and in the fight half-site sequence, the rest of the nucleotides deviating from the homologous left half-site is replaced by the native nucleotides found in the known target-site, and (2) in both 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 half half-site sequences alone comprises all deviating nucleotides; (f) generating further subsets of target sequences starting from the target sequences in the second subset obtained in step (e) by repeating the process of step (e) each time generating a new subset of target sequences, until the half-site sequences forming the inverted repeats within each generated target sequence contain one, two or three nucleotides deviating from the homologous half-site sequence of the known target site; (g) applying molecular directed evolution on the recombinase recognizing the known homologous target site chosen in step (a) using the target sequences of the final subset obtained in step (f) containing one, two or three nucleotides deviating from the homologous half-site sequence of said known homologous target site as a substrate; (h) shuffling the recombinase libraries evolved in step (g); (i) applying molecular directed evolution on the shuffled library obtained in step (h) using the target sequences of the next higher subset according to step (f); (j) repeating steps (h) and (i) until one recombinase is achieved by molecular directed evolution 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 one recombinase obtained in step (j) from the library; and (l) cloning the nucleic acid obtained in step (k) into an expression vector, thereby preparing an expression vector encoding a single recombinase that is active on the asymmetric target sequence, wherein the single recombinase that is active on the asymmetric target sequence has only a residual activity or no activity on