Novel nucleic acid molecule

Abstract

The present invention is directed generally to an isolated nucleic acid molecule encompassing a neocentromere or a functional derivative thereof or a latent, synthetic or hybrid form thereof and its use inter alia in developing a range of eukaryotic artificial chromosomes including mammalian (e.g. human) and non-mammalian artificial chromosomes. Such artificial chromosomes are useful in a range of genetic therapies.

1 . An isolated nucleic acid molecule comprising a sequence of nucleotides derived from a eukaryotic chromosome and encompassing a neocentromere or a functional derivative synthetic or hybrid form thereof which nucleic acid molecule or its derivatives, synthetic forms or hybrid forms when introduced into a compatible cell is capable of replicating, acting as an extra-chromosomal element and segregating with cell division.  
     
     
         2 . An isolated nucleic acid molecule according to  claim 1  wherein the eukaryotic chromosome is a mammalian chromosome.  
     
     
         3 . An isolated nucleic acid molecule according to claim) wherein the chromosome is a human chromosome.  
     
     
         4 . An isolated nucleic acid molecule according to  claim 2  wherein the nucleic acid molecule is capable of associating with centromeric binding proteins (CENP)-A and -C or antibodies thereto.  
     
     
         5 . An isolated nucleic acid molecule according to  claim 4  wherein the chromosome is human chromosome 10 or a modified form of human chromosome 10 or its non-human mammalian or non-mammalian equivalent.  
     
     
         6 . An isolated nucleic acid molecule according to  claim 5  wherein the nucleotide sequence corresponds to a region mapping between q24 and q26 on chromosome 10.  
     
     
         7 . An isolated nucleic acid molecule according to  claim 5  wherein a modified from of human chromosome 10 is a mardel (10) chromosome.  
     
     
         8 . An isolated nucleic acid molecule according to  claim 6  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 40%-similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 3 under low stringency conditions at 42° C.  
     
     
         9 . An isolated nucleic acid molecule according to  claim 7  comprising a nucleotide sequence substantially as set forth in SEQ D NO: 4 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 4 under low stringency conditions at 42° C.  
     
     
         10 . An isolated nucleic acid molecule according to  claim 7  comprising a nucleotide sequence substantially as set forth in one or more of SEQ ID NOs: 5-29 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to one or more of SEQ ID NOs: 5-29 under low stringency conditions at 42° C.  
     
     
         11 . An isolated nucleic acid molecule according to  claim 1  wherein the length of the nucleic acid molecule is from about 50 bp to about 1500 kbp.  
     
     
         12 . An isolated nucleic acid molecule according to  claim 11  wherein the length of the nucleic acid molecule is from about 1 kbp to about 1000 kbp.  
     
     
         13 . An isolated nucleic acid molecule according to  claim 12  wherein the length of the nucleic acid molecule is from about 10 kbp to about 500 kbp.  
     
     
         14 . An isolated nucleic acid molecule according to  claim 13  wherein the length of the nucleic acid molecule is from about 10 kbp to about 100 kbp.  
     
     
         15 . An isolated nucleic acid molecule comprising a nucleotide sequence encompassing a neocentromere or a functional derivative, synthetic or hybrid form thereof which when said nucleic acid molecule is in linear form and co-introduced into a cell together with a telomeric sequence, is capable of replicating, remaining as an extra-chromosomal element and segregates with cell division.  
     
     
         16 . An isolated nucleic acid molecule according to  claim 15  wherein the nucleotide sequence is derived from a mammalian chromosome.  
     
     
         17 . An isolated nucleic acid molecule according to  claim 16  wherein said nucleic acid molecule is capable of associating with CENP-A and CENP-C antibodies.  
     
     
         18 . An isolated nucleic acid molecule according to  claim 16  or  17  wherein the mammalian chromosome is human chromosome 10 or a modified form of chromosome 10 or its non-human mammalian or non-mammalian equivalent.  
     
     
         19 . An isolated nucleic acid molecule according to  claim 18  wherein the nucleotide sequence corresponds to a region mapping between q24 and q26 on chromosome 10.  
     
     
         20 . An isolated nucleic acid molecule according to  claim 18  wherein the modified form of human chromosome 10 is mardel (10) chromosome.  
     
     
         21 . An isolated nucleic acid molecule according to  claim 18  comprising a nucleotide sequence substantially as set forth in SEQ M NO: 3 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 3 under low stringency conditions at 42° C.  
     
     
         22 . An isolated nucleic acid molecule according to  claim 19  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 4 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 4 under low stringency conditions at 42° C.  
     
     
         23 . An isolated nucleic acid molecule according to  claim 19  comprising a nucleotide sequence substantially as set forth in one or more of SEQ ID Nos: 5-29 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to one or more of SEQ ID Nos: 5-29 under low stringency conditions at 42° C.  
     
     
         24 . An isolated nucleic acid molecule according to  claim 15  wherein the length of the nucleic acid molecule is from about 50 bp to about 1500 kbp.  
     
     
         25 . An isolated nucleic acid molecule according to  claim 24  wherein the length of the nucleic acid molecule is from about 1 kbp to about 1000 kbp.  
     
     
         26 . An isolated nucleic acid molecule according to  claim 25  wherein the length of the nucleic acid molecule is from about 10 kbp to about 500 kbp.  
     
     
         27 . An isolated nucleic acid molecule according to  claim 26  wherein the length of the nucleic acid molecule is from about 10 kbp to about 100 kbp.  
     
     
         28 . An Isolated nucleic acid molecule or its chemical equivalent encompassing a human neocentromere or a functional derivative thereof or a latent, synthetic, hybrid or its mammalian or non-mammalian homologue.  
     
     
         29 . An isolated nucleic acid molecule according to  claim 28  wherein said nucleic acid molecule when introduced into a compatible cell is a replicating, extra-chromosomal element which segregates with cell division.  
     
     
         30 . An isolated nucleic acid molecule according to  claim 29  wherein the nucleic acid molecule is capable of associating with centromeric binding proteins (CENP)-A and —C or antibodies thereto.  
     
     
         31 . An isolated nucleic acid molecule according to  claim 29  or  30  wherein the chromosome is human chromosome 10 or a modified form of human chromosome 10 or its non-human mammalian or non-mammalian equivalent.  
     
     
         32 . An isolated nucleic acid molecule according to  claim 31  wherein the nucleotide sequence corresponds to a region mapping between q24 and q26 on chromosome 10.  
     
     
         33 . An isolated nucleic acid molecule according to  claim 31  wherein a modified form of human chromosome 10 is a mardel (10) chromosome.  
     
     
         34 . An isolated nucleic acid molecule according to  claim 31  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 3 under low stringency conditions at 42° C.  
     
     
         35 . An isolated nucleic acid molecule according to  claim 32  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 4 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 4 under low stringency conditions at 42° C.  
     
     
         36 . An isolated nucleic acid molecule according to  claim 32  comprising a nucleotide sequence substantially as set forth in one or more of SEQ ID Nos: 5-29 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to one or more of SEQ ID Nos: 5-29 under low stringency conditions at 42° C.  
     
     
         37 . An isolated nucleic acid molecule according to  claim 28  wherein the length of the nucleic acid molecule is from about 50 bp to about 1500 kbp.  
     
     
         38 . An isolated nucleic acid molecule according to  claim 37  wherein the length of the nucleic acid molecule is from about 1 kbp to about 1000 kbp.  
     
     
         39 . An isolated nucleic acid molecule according to  claim 38  wherein the length of the nucleic acid molecule is from about 10 kbp to about 500 kbp.  
     
     
         40 . A genetic construct comprising an origin of replication for a eukaryotic cell and a nucleic acid molecule encompassing a eukaryotic neocentromere or a functional derivative thereof or a latent, synthetic, hybrid form thereof or its mammalian or non-mammalian homologue flanked by telomeric nucleotide sequences functional in the cell in which the genetic construct Is to replicate and wherein said genetic construct when introduced into a cell is a replicating, extra-chromosomal element which segregates with cell division.  
     
     
         41 . A genetic construct according to  claim 40  wherein the eukaryotic neocentromere is a mammalian centromere.  
     
     
         42 . An isolated nucleic acid molecule according to  claim 41  wherein the neocentromere is a human neocentromere.  
     
     
         43 . An isolated nucleic acid molecule according to  claim 42  wherein the nucleic acid molecule is capable of associating with CENP-A and —C or antibodies thereto.  
     
     
         44 . An isolated nucleic acid molecule according to  claim 43  wherein the neocentromere is from human chromosome 10 or a modified form of human chromosome 10 or its non-human mammalian or non-mammalian equivalent.  
     
     
         45 . An isolated nucleic acid molecule according to  claim 44  wherein the human neocentromere maps to a region between q24 and q26 on chromosome 10.  
     
     
         46 . An isolated nucleic acid molecule according to  claim 44  wherein a modified form of human chromosome 10 is a mardel (10) chromosome.  
     
     
         47 . An isolated nucleic acid molecule according to  claim 45  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 3 under low stringency conditions at 42° C.  
     
     
         48 . An isolated nucleic acid molecule according to  claim 46  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 4 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 4 under low stringency conditions at 42° C.  
     
     
         49 . An isolated nucleic acid molecule according to  claim 46  comprising a nucleotide sequence substantially as set forth in one or more of SEQ ID Nos: 5-29 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to one or more of SEQ ID Nos: 5-29 under low stringency conditions at 42° C.  
     
     
         50 . An artificial chromosome for use in gene therapy said artificial chromosome comprising a nucleic acid molecule capable of conferring a phenotypic property on a cell carrying said artificial chromosome wherein said artificial chromosome is a replicating element which segregates with cell division.  
     
     
         51 . An artificial chromosome according to  claim 50  wherein said artificial chromosome is capable of functioning in a mammalian cell.  
     
     
         52 . An artificial chromosome according to  claim 51  wherein said artificial chromosome is capable of functioning in a human cell.  
     
     
         53 . An artificial chromosome according to  claim 52  wherein the chromosome is a human chromosome.  
     
     
         54 . An artificial chromosome according to  claim 53  wherein the chromosome is capable of associating with CENP-A and —C or antibodies thereto.  
     
     
         55 . An artificial chromosome according to  claim 53  or  54  wherein the chromosome is human chromosome 10 or a modified form of human chromosome 10 or its non-human mammalian or non-mammalian equivalent.  
     
     
         56 . An artificial chromosome according to  claim 55  comprising a region mapping between q24 and q26 on chromosome 10.  
     
     
         57 . An artificial chromosome according to  claim 5  wherein a modified form of human chromosome 10 is a mardel (10) chromosome.  
     
     
         58 . An artificial chromosome according to  claim 56  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 3 under low stringency conditions at 42° C.  
     
     
         59 . An artificial chromosome according to  claim 57  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 4 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 4 under low stringency conditions at 42° C.  
     
     
         60 . An artificial chromosome according to  claim 57  comprising a nucleotide sequence substantially as set forth in one or more of SEQ ID Nos: 5-29 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to one or more of SEQ ID Nos: 5-29 under low stringency conditions at 42° C.  
     
     
         61 . An isolated nucleic acid molecule comprising a sequence of nucleotides which defines a eukaryotic neocentromere.  
     
     
         62 . An isolated nucleic acid molecule according to  claim 61  wherein the neocentromere is derived from a mammalian chromosome.  
     
     
         63 . An isolated nucleic acid molecule according to  claim 61  wherein the neocentromere is derived from a human chromosome.  
     
     
         64 . An isolated nucleic acid molecule according to  claim 63  wherein the nucleic acid molecule is capable of associating with centromeric binding proteins (CENP)-A and -C or antibodies thereto.  
     
     
         65 . An isolated acid molecule according to  claim 63  or  64  wherein the chromosome is human chromosome 10 or a modified form of human chromosome 10 or its non-human mammalian or non-mammalian equivalent.  
     
     
         66 . An isolated nucleic acid molecule according to  claim 65  wherein the nucleotide sequence corresponds to a region mapping between q24 and q26 on chromosome 10.  
     
     
         67 . An isolated nucleic acid molecule according to  claim 65  wherein a modified form of human chromosome 10 is a mardel (10) chromosome.  
     
     
         68 . An isolated nucleic acid molecule according to  claim 66  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 3 under low stringency conditions at 42° C.  
     
     
         69 . An isolated nucleic acid molecule according to  claim 67  comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 4 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to SEQ ID NO: 4 under low stringency conditions at 42° C.  
     
     
         70 . An isolated nucleic acid molecule according to  claim 67  comprising a nucleotide sequence substantially as set forth in one or more of SEQ ID NOs: 5-29 or a nucleotide sequence having at least 40% similarity thereto or a nucleotide sequence capable of hybridising to one or more of SEQ ID NOs: 5-29 under low stringency conditions at 42° C.  
     
     
         71 . An isolated nucleic acid molecule according to  claim 61  wherein the length of the nucleic acid molecule is from about 50 bp to about 1500 kbp.  
     
     
         72 . An isolated nucleic acid molecule according to  claim 71  wherein the length of the nucleic acid molecule is from about 1 kbp to about 1000 kbp.  
     
     
         73 . An isolated nucleic acid molecule according to  claim 72  wherein the length of the nucleic acid molecule is from about 10 kbp to about 500 kbp.  
     
     
         74 . An isolated nucleic acid molecule according to  claim 73  wherein the length of the nucleic acid molecule is from about 10 kbp to about 100 kbp.