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US 2004/0067486 A1Published application
Hepatitis c virus replicons and replicon enhanced cells
AssigneeIstituto di Ricerche di Biologia Molecolare P. Angeletti S.p.A.
InventorsGiacomo Paonessa, Giovanni Migliaccio, Raffaele De Francesco
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Abstract
The present invention features nucleic acid containing one or more adaptive mutations, and HCV replicon enhanced cells. Adaptive mutations are mutations that enhance HCV replicon activity. HCV replicon enhanced cells are cells having an increased ability to maintain an HCV replicon.
Claims39
A nucleic acid molecule comprising a region selected from the group consisting of:
- a) an altered HCV NS3 encoding region coding for one or more NS3 mutations, wherein at least one of said NS3 mutations, identified by reference to the amino acid sequence numbering of SEQ. ID. NO. 1, is selected from the group consisting of:
- amino acid 1095 being Ala,
- amino acid 1202 being Gly, and
- amino acid 1347 being Thr;
- b) an altered HCV NS5A encoding region coding for one or more NS5A mutations, wherein at least one of said NS5A mutations, identified by reference to the amino acid sequence numbering of SEQ. ID. NO. 1, is selected from the group consisting of:
- amino acid 2041 being Thr,
- a Lys insertion between residue 2039 and 2040.
- amino acid 2173 being Phe,
- amino acid 2197 being Phe,
- amino acid 2198 being Ser,
- amino acid 2199 being Thr, and
- amino acid 2204 being Arg;
- and
- c) an altered encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) region containing one or more EMCV IRES mutations, wherein at least one of said EMCV IRES mutations, identified by reference to the nucleotide number of SEQ. ID. NO. 3, is an insertion at nucleotide 1736 of adenine.
11 more elements
A nucleic acid molecule comprising a region selected from the group consisting of:
- a) an altered HCV NS3 encoding region containing one or more NS3 mutations, wherein at least one of said NS3 mutations, identified by reference to the nucleotide numbering of SEQ. ID. NO. 2, is selected from the group consisting of:
- nucleotide 3625 being cytosine,
- nucleotide 3946 being guanine,
- nucleotide 4380 being adenine,
- b) an altered HCV NS5A encoding region containing one or more NS5A mutations, wherein at least one of said NS5A mutations, identified by reference to the nucleotide numbering of SEQ. ID. NO. 2, is selected from the group consisting of:
- an insertion of 3 adenine residues between nucleotide 6458 and 6459,
- nucleotide 6463 being cytosine,
- nucleotide 6859 being thymine or uracil,
- nucleotide 6931 being thymine or uracil,
- nucleotide 6934 being cytosine,
- nucleotide 6936 being adenine, and
- nucleotide 6953 being adenine or guanine;
- and
- c) an altered encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) region containing one or more EMCV IRES mutations, wherein at least one of said EMCV IRES mutations, identified by reference to the nucleotide number of SEQ. ID. NO. 3, is an insertion at nucleotide 1736 of adenine.
11 more elements
A nucleic acid molecule comprising the nucleic acid base sequence of bases 1-7989 of SEQ. ID. NO. 3, or the RNA version thereof, consisting of one or more different modifications selected from the group consisting of:
- a) nucleotides 5335-5337 modified to code for arginine;
- b) nucleotides 5242-5244 modified to code for phenylalanine;
- c) nucleotides 5314-5316 modified to code for phenylalanine;
- d) nucleotides 5317-5319 modified to code for serine;
- e) nucleotides coding for lysine inserted after nucleotide 4843;
- f) nucleotides 2329-2331 modified to code for glycine, nucleotides 2764-2766 modified to code for threonine, nucleotides 5242-5244 modified to code for phenylalanine, and an extra adenosine inserted after nucleotide 1736;
- g) nucleotides 4846-4848 modified to code for threonine, and nucleotides 5242-5244 modified to modified to code for phenylalanine;
- h) nucleotides 4846-4848 modified to code for threonine, and nucleotides 5314-5316 modified to code for phenylalanine;
- i) nucleotides 4846-4848 modified to code for threonine, and nucleotides 5317-5319 modified to code for serine;
- j) nucleotides 2329-2331 modified to code for glycine, and nucleotides coding for lysine inserted after nucleotides 4843;
- k) nucleotides 5314-5316 modified to code for phenylalanine and nucleotides 5320-5322 modified to code for threonine;
- l) nucleotides 4846-4848 modified to code for threonine, nucleotides 5314-5316 modified to code for phenylalanine, and nucleotides 5320-5322 modified to code for threonine;
- m) nucleotides 4846-4848 modified to code for threonine, nucleotides 5314-5316 modified to code for phenylalanine, and an extra adenosine inserted after nucleotide 1736;
- and
- n) nucleotides 5314-5316 modified to code for phenylalanine, nucleotides 5320-5322 modified to code for threonine, and an extra adenosine inserted after nucleotide 1736;
- and
- o) nucleotides 5320-5322 modified to code for threonine.
14 more elements
A method of making an HCV replicon enhanced cell comprising the steps of:
- a) introducing and maintaining a HCV replicon in a cell;
- and
- b) curing said cell of said HCV replicon to produce said replicon enhanced cell.
A method of making an HCV replicon enhanced cell containing a functional HCV replicon comprising the steps of:
- a) introducing and maintaining a first HCV replicon in a cell;
- b) curing said cell of said first replicon to produce a cured cell;
- and
- c) introducing and maintaining a second HCV replicon into said cured cell, wherein said second HCV replicon may be the same or different than said first HCV replicon.
1 more element
Dependencies are inferred from the claim text.