Mutant HSV, materials and methods for generation of mutant HSV
The present invention includes a method of generating a mutant Herpes Simplex Virus (HSV). In one embodiment, the generated HSV genome includes nucleic acid encoding a nucleic acid sequence of interest. In one step, a nucleic acid vector is provided which includes a nucleic acid encoding first and second site specific recombination sequences and a nucleic acid encoding a nucleic acid sequence of interest between said site specific recombination sequences an HSV is provided, the genome of which comprises third and fourth site specific recombination sequences In another step, the nucleic acid vector and HSV are contacted together with one or more recombinase enzymes capable of catalyzing site specific recombination between the site specific recombination sequences of said nucleic acid vector and said HSV. Another step includes identifying HSV containing the nucleic acid sequence of interest. In some embodiments, the methods are conducted in a cell-free system.
1. A method of generating mutant Herpes Simplex Viruses (HSV) comprising a nucleic acid sequence of interest (NOI), the method comprising the steps of:
(i) providing a nucleic acid vector, wherein said vector comprises nucleic acid encoding the site specific att recombination sequences attL1 and attL2 and a NOI, wherein said NOI is between said site specific recombination sequences;
(ii) providing a recombinant HSV, the genome of said recombinant HSV comprising the site specific att recombination sequences attR1 and attR2;
(iii) contacting said nucleic acid vector of (i) with said recombinant HSV of (ii) together with one or more recombinase enzymes capable of catalyzing site specific recombination between the att site specific recombination sequences of said nucleic acid vector and said recombinant HSV, wherein attL1 and attR1 sites and attL2 and attR2 sites, respectively, are recombined, thereby generating mutant HSV;
(iv) performing at least one round of plaque purification of the mutant HSV from step (iii) and identifying mutant HSV which comprise the NOI, wherein the recombination reaction of step (iii) results in at least 75% of the mutant HSV containing the NOI after one round of plaque purification, and wherein steps (i)-(iii) are conducted in a cell-free system.
2. The method of claim 1 , wherein the recombinant HSV of (ii) is generated by a method comprising the steps of:
a) providing a nucleic acid vector comprising nucleic acid encoding a nucleotide sequence having 70-100% sequence identity or complementarity with a selected region of an HSV genome;
b) inserting said attR1 and attR2 att recombination sequences in the nucleotide sequence of a) to generate a modified nucleic acid vector;
c) contacting said modified nucleic acid vector from b) with the HSV genome of step a) under conditions in which homologous recombination between said nucleic acid vector and said HSV genome may occur;
d) identifying recombinant HSV comprising said attR1 and attR2 att recombination sequences in the recombinant HSV genome.
3. The method of claim 1 , wherein the nucleic acid vector of (i) and/or the recombinant HSV of (ii) further comprise a nucleotide sequence coding for a detectable marker, wherein said detectable marker is located between the site specific att recombination sequences of (i) and/or (ii).
4. The method of claim 1 , wherein the recombinant HSV of (ii) is not part of an artificial chromosome.
5. The method of claim 2 , wherein the nucleic acid vector of (b) comprises a nucleotide sequence coding for a detectable marker, which is located between the site specific att recombination sequences.