Synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the invention provides novel synthetic genes and genetic constructs which are capable of repressing, delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto.

Graham, Rice, Waterhouse

A method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention utilises recombinant DNA technology to post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, organ or whole organism, thereby producing novel phenotypes. Novel synthetic genes and genetic constructs which are capable or repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.

Waterhouse, Graham, Wang, Rice

A method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the invention utilises recombinant DNA technology post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, organ or whole organism, thereby producing novel phenotypes. Novel synthetic genes and genetic constructs which are capable or repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.

Graham, Rice, Waterhouse, Wang

A genetic construct which is capable of delaying, repressing or otherwise reducing the expression of a target gene in a vertebrate animal cell, tissue or organ which is transfected with said genetic construct, wherein said genetic construct comprises one or more dispersed or foreign nucleic acid molecules which include multiple copies of a nucleotide sequence which is substantially identical to the target gene, or a region of said target gene, and wherein at least one of said copies is in the sense orientation and wherein at least one other of said copies is in the antisense orientation.
20.  A genetic construct which is capable of delaying, repressing or otherwise reducing the expression of a target gene in a vertebrate animal cell, tissue or organ which is transfected with said genetic construct, wherein said genetic construct comprises multiple copies of a nucleotide sequence which is substantially identical to said target gene, or a region of said target gene, and wherein said multiple copies are placed operably under the control of a single promoter sequence which is operable in said cell, tissue or organ, wherein at least one of said copies is placed operably in the sense orientation under the control of said promoter sequence, wherein at least one other of said copies is placed operably in the antisense orientation under the control of said promoter sequence, and wherein the at least one copy that is placed in the sense orientation relative to said promoter and the at least one other copy that is placed in the antisense orientation

1. A double-stranded DNA construct comprising two copies of a structural gene region whose nucleotide sequence is identical to the nucleotide sequence of a region of a target gene in an animal cell,
wherein one of the two copies is in the sense orientation and the other of the two copies is in the antisense orientation operably under the control of a single promoter sequence which is operable in the cell, and wherein
the copy of the structural gene region in the sense orientation and the copy of the structural gene region in the antisense orientation are arranged as an interrupted palindrome sequence such that the copy of the structural gene region in the sense orientation and the copy of the structural gene region in the antisense orientation are separated and linked by nucleotides other than the nucleotides of the copy of the structural gene region in the sense orientation and the copy of the structural gene region in the antisense orientation.
26. An animal cell having a DNA comprising …
51. A method of modulating the expression of a target gene in an animal cell…

Methods for reducing the phenotypic expression of a nucleic acid of interest in eukaryote cells by providing aberrant RNA molecules, preferably unpolyadenylated RNA molecules comprising at least one target specific nucleotide sequence homologous to the nucleic acid of interest, preferably a sense strand, into the nucleus of plant cells.

Waterhouse, Wang, Graham, (Smith)

15.  A eukaryotic cell, comprising a nucleic acid of interest, which is normally capable of being phenotypically expressed, further comprising a chimeric DNA molecule comprising the following operably linked parts:
a)  a promoter, operative in said eukaryotic cell;
b)  a DNA region, which when transcribed, yields an RNA molecule with at least one RNA region with a nucleotide sequence comprising
i.  a sense nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence of the nucleic acid of interest; and
ii.  an antisense nucleotide sequence including at least 20 consecutive nucleotides, having 100% sequence identity with the complement of said at least 20 consecutive nucleotides of said sense nucleotide sequence;
  wherein the RNA is capable of forming an artificial hairpin RNA structure with a double stranded RNA stem by base-pairing between the regions with sense and antisense nucleotide sequence; and
c)  a DNA region involved in transcription termination and polyadenylation
wherein the phenotypic expression of said nucleic acid of interest is reduced.
16.  A eukaryotic cell, comprising a nucleic acid of interest, which is normally capable of being phenotypically expressed, further comprising a chimeric RNA molecule comprising at least one RNA region with a nucleotide sequence comprising
i.  a sense nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence of the nucleic acid of interest; and
ii.  an antisense nucleotide sequence including at least 20 consecutive nucleotides, having 100% sequence identity with the complement of said at least 20 consecutive nucleotides of said sense nucleotide sequence;
wherein said RNA is capable of forming an artificial hairpin RNA with a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence such that at least said 20 consecutive nucleotides of the sense sequence basepair with said 20 consecutive nucleotides of the antisense sequence.

EP1068311
Accepted
27 April 2011

Currently under opposition at the EPO

A chimeric RNA molecule comprising at least one RNA region with a nucleotide sequence comprising
i.  a sense nucleotide sequence of at least 10 consecutive nucleotides having between 75 and 100% sequence identity with at least part of a coding region of a nucleic acid of interest; said nucleic acid of interest being a gene incorporated in the genome of a eukaryotic cell and
ii.  an antisense nucleotide sequence including at least 10 consecutive nucleotides, having between 75% to 100% sequence identity with the complement of said at least 10 consecutive nucleotides of said sense nucleotide sequence;

(105)

(Licensed to Tacere Therapeutics for HCV)
A genetic construct comprising a multi-promoter expression cassette comprising at

A genetic construct comprising a multi-promoter expression cassette comprising at least three promoter/RNAi/terminator components wherein each promoter/RNAi/terminator component comprises a promoter element, a terminator element and a sequence encoding an RNAi species operably linked to the promoter element and the terminator element, and wherein at least one of the RNAi species is encoded by SEQ ID NO: 22.

1725660
Granted
11 July 2011

A genetic construct comprising a multi-promoter expression cassette comprising at least three promoter/RNAi/terminator components wherein each promoter/RNAi/terminator component comprises a promoter element, a terminator element and a sequence encoding an RNAi species operably linked to the promoter element and the terminator element, wherein the RNAi species target a transcribed nucleic acid sequence and at least one of the RNAi species is encoded by SEQ ID NO: 22.

7727970
Granted
June 1, 2010

1.  A method of inhibiting the level of one or more Hepatitis C virus nucleic acid targets that are expressed in a cell comprising contacting the cell with a genetic construct comprising a multi-promoter expression cassette, the multi-promoter expression construct comprising at least three promoter/RNAi/terminator components wherein each promoter/RNAi/terminator component comprises a promoter element, a terminator element and an RNAi sequence, the RNAi sequence operably linked to the promoter element and terminator element, and wherein the RNAi sequences are SEQ ID NO: 6, 19 and 22.
2.  A method of treating an animal cell, tissue or organ, to inhibit the level of one or more Hepatitis C virus nucleic acid targets expressed in a cell, said method comprising introducing to said animal cell, tissue or organ a genetic construct comprising a multi-promoter expression cassette, the multi-promoter expression construct comprising at least three promoter/RNAi/terminator components wherein each promoter/RNAi/terminator component comprises a promoter element, a terminator element and an RNAi sequence operably linked to the promoter element and the terminator element, wherein the RNAi species are SEQ ID NO: 6, 19 and 22.
(NB: Product claims being pursued in 12/723466)

A method of inducing, promoting or otherwise facilitating a change in the phenotype of an animal cell or group of animal cells including an animal. The modulation of phenotypic expression is accomplished via genotypic manipulation by inducing, promoting or otherwise facilitating the silencing of expressible genetic sequences thus reducing translation of transcript to protein. Expressible genetic sequences contemplated by the invention include not only genes normally resident in a particular cell (i.e. indigenous genes) but also genes introduced through recombinant means or through infection by pathogenic agents such as viruses.

Graham, Rice, Murphy, Reed

1.  A genetic construct comprising a sequence of nucleotides substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal cell and a nucleotide sequence complementary to said target endogenous nucleotide sequence wherein the nucleotide sequences identical and complementary to said target endogenous nucleotide sequence are separated by a spacer sequence wherein upon introduction of said genetic construct to said animal cell, an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a proteinaceous product.
3.  A genetic construct comprising:
(i)  a nucleotide sequence substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal cell;
(ii)  a single nucleotide sequence substantially complementary to said target endogenous nucleotide sequence defined in (i);
(iii)  an intron nucleotide sequence separating said nucleotide sequence of (i) and (ii);
wherein upon introduction of said construct to said animal cell, an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation.
4.  A genetic construct comprising:
(i)  a nucleotide sequence substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal cell;
(ii)  a nucleotide sequence substantially complementary to said target endogenous nucleotide sequence defined in (i);
(iii)  an intron nucleotide sequence separating said nucleotide sequence of (i) and (ii);

1.  A genetic construct comprising a sequence of nucleotides substantially identical to a target endogenous sequence of nucleotides in the genome or a vertebrate animal cell and a nucleotide sequence complementary to said target endogenous nucleotide sequence wherein the nucleotide sequences identical and complementary to said target endogenous nucleotide sequences are separated by a spacer sequence wherein upon introduction of said genetic construct to said animal cell, an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a proteinaceous product.
12.  A genetic construct comprising:-
(i)  a nucleotide sequence substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal cell;
(ii)  a single nucleotide sequence substantially complementary to said target endogenous nucleotide sequence defined in (i);
(iii)  an intron nucleotide sequence separating said nucleotide sequence of (i) and (ii);

1.  A genetic construct comprising a sequence of nucleotides substantially identical to a target endogenous sequence of nucleotides in the genome or a vertebrate animal cell and a nucleotide sequence complementary to said target endogenous nucleotide sequence wherein the nucleotide sequences identical and complementary to said target endogenous nucleotide sequences are separated by a spacer sequence wherein upon introduction of said genetic construct to said animal cell, an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for translation into a proteinaceous product.
12.  A genetic construct comprising:-
(i)  a nucleotide sequence substantially identical to a target endogenous sequence of nucleotides in the genome of a vertebrate animal cell;
(ii)  a single nucleotide sequence substantially complementary to said target endogenous nucleotide sequence defined in (i);
(iii)  an intron nucleotide sequence separating said nucleotide sequence of (i) and (ii);
wherein upon introduction of said construct to said animal cell, an RNA transcript resulting from transcription of a gene comprising said endogenous target sequence of nucleotides exhibits an altered capacity for transcription.

(LONG HAIR PIN)
A ribonucleic acid (RNA) for use as interfering RNA in gene silencing techniques to silence a target gene comprising in a 5’ to 3’ direction at least four sequences being a first and second effector sequence 17 to 21 nucleotides in length; a sequence substantially complementary to the second effector sequence; and a sequence substantially complementary to the first effector sequence; wherein the complementary sequences are capable of forming double stranded regions with their respective effector sequences and wherein at least one of the four sequences is substantially identical to the predicted transcript of a region of the target gene; and the RNA further comprising a spacing sequence of one or more nucleotides,  the spacing sequence being located between and spacing the first effector sequence and the second effector sequence, or between the sequence substantially complementary to the second effector sequence and the sequence substantially complementary to the first effector sequence.

1.  A ribonucleic acid (RNA) for use as interfering RNA in gene silencing techniques to silence one or more target genes comprising in a 5' to 3' direction at least four sequences being:
a first effector sequence;
a second effector sequence;
a sequence substantially complementary to the second effector sequence; and
a sequence substantially complementary to the first effector sequence
wherein the first and second effector sequences are each 17 to 30 nucleotides in length, and
wherein the complementary sequences are capable of forming double stranded regions with their respective effector sequences, and wherein at least one of the four sequences is substantially identical to the predicted transcript of a region of the target gene,
the RNA further comprising a spacing sequence of 1 to 20 nucleotides, the spacing sequence being located between and spacing the first effector sequence and the second effector sequence, or between the sequence substantially complementary to the second effector sequence and the sequence substantially complementary to the first effector sequence.
12.  A double stranded nucleic acid construct including a sequence encoding a ribonucleic acid (RNA) suitable for use as interfering RNA in gene silencing techniques to silence one or more target genes, the construct comprising in a 5' to 3' direction at least four sequences being:
a first effector-encoding sequence;
a second effector-encoding sequence;
a sequence encoding RNA that is capable of forming double stranded regions with RNA encoded by the second effector-encoding sequence; and
a sequence encoding RNA that is capable of forming double stranded regions with RNA encoded by the first effector- encoding sequence,
wherein the first and second effector-encoding sequences are each 17 to 30 nucleotides in length, and wherein at least one of these sequences is substantially identical to a region of the target gene,

1.  A ribonucleic acid (RNA) for use as interfering RNA in gene silencing techniques to silence a target gene comprising in a 5’ to 3’ direction at least a first effector sequence, a second effector sequence, a sequence substantially complementary to the second effector sequence and a sequence substantially complementary to the first effector sequence, wherein the complementary sequences are capable of forming double stranded regions with their respective effector sequences and wherein at least one of these sequences is substantially identical to the predicted transcript of a region of the target gene, the RNA further comprising a spacing sequence of one or more nucleotides wherein any two of the sequences are spaced by the spacing sequence.
18.  A nucleic acid construct including a sequence encoding a ribonucleic acid (RNA) suitable for use as interfering RNA in gene silencing techniques to silence a target gene, the construct comprising in a 5¢ to 3¢ direction at least a first effector-encoding sequence, a second effector-encoding sequence, a sequence substantially complementary to the second effector-encoding sequence and a sequence substantially complementary to the first effector-encoding sequence, wherein the transcripts of the complementary sequences are capable of forming double stranded regions with the transcripts of their respective effector-encoding sequences and wherein at least one of these sequences is substantially identical to a region of the target gene.

.  A ribonucleic acid (RNA) for use as interfering RNA in gene silencing techniques to silence a target gene comprising in a 5’ to 3’ direction at least four sequences being:
a first effector sequence;
a second effector sequence;
a sequence substantially complementary to the second effector sequence; and
a sequence substantially complementary to the first effector sequence;
wherein the first and second effector sequences are each 10 to 200 nucleotides in length; and
wherein the complementary sequence are capable of forming double stranded regions with their respective effector sequences and wherein at least one of the four sequences is substantially identical to the predicted transcript of a region of the target gene
the RNA further comprising a spacing sequence of one or more nucleotides wherein any two of the effector sequences and the sequences substantially complementary to the effector sequences are spaced by the spacing sequence.
17.  A double stranded nucleic acid construct including a sequence encoding a ribonucleic acid (RNA) suitable for use as interfering RNA in gene silencing techniques to silence a target gene, the construct comprising in a 5’ to 3’ direction at least four sequences being:
a first effector sequence;
a second effector sequence;
a sequence encoding RNA that is capable of forming double stranded regions with RNA encoded by the second effector-encoding sequence; and
a sequence encoding RNA that is capable of forming double stranded regions with RNA encoded by the first effector-encoding sequence;
wherein the first and second effector-encoding sequences are each 10 to 200 nucleotides in length, and
wherein at least one of the four sequences is substantially identical to a region of the target gene,

1.  A ribonucleic acid (RNA) for use as interfering RNA in gene silencing techniques to silence a target gene comprising in a 5’ to 3’ direction at least four sequences being:
a first effector sequence;
a second effector sequence;
a sequence substantially complementary to the second effector sequence; and
a sequence substantially complementary to the first effector sequence;
wherein the first and second effector sequences are each 10 to 200 nucleotides in length; and
wherein the complementary sequence are capable of forming double stranded regions with their respective effector sequences and wherein at least one of the four sequences is substantially identical to the predicted transcript of a region of the target gene
the RNA further comprising a spacing sequence of one or more nucleotides wherein any two of the effector sequences and the sequences substantially complementary to the effector sequences are spaced by the spacing sequence.
17.  A double stranded nucleic acid construct including a sequence encoding a ribonucleic acid (RNA) suitable for use as interfering RNA in gene silencing techniques to silence a target gene, the construct comprising in a 5’ to 3’ direction at least four sequences being:
a first effector sequence;
a second effector sequence;
a sequence encoding RNA that is capable of forming double stranded regions with RNA encoded by the second effector-encoding sequence; and
a sequence encoding RNA that is capable of forming double stranded regions with RNA encoded by the first effector-encoding sequence;
wherein the first and second effector-encoding sequences are each 10 to 200 nucleotides in length, and
wherein at least one of the four sequences is substantially identical to a region of the target gene,

Compositions and methods suitable for expressing 1-x RNAi agents against a gene or genes in cells, tissues or organs of

21.  A genetic construct capable of modifying expression of one or more genes, the genetic construct comprising a 1-x RNAi expression cassette encoding x stem-loop structures, wherein x is two or more, and the stem-loop structures are separated from one another by one or more spacer regions, wherein the stem-loop structures are RNAi agents and at least one stem-loop structure is encoded by SEQ ID NO: 39.
30.  A 1-x RNAi expression cassette encoding x stem-loop structures, wherein x is two or more, and the stem-loop structures are separated from one another by one or more spacer regions, wherein the stem-loop structures are RNAi agents and at least one stem-loop structure is encoded by SEQ ID NO: 39.

1.  A 1-x RNAi expression cassette for targeting multiple regions of a HCV genome comprising:
a promoter;
x RNAi agent coding sequences, wherein x is at least two; and
sequences coding for spacer regions;
wherein transcription of the RNAi agent coding sequences produces RNAi agents which form stem-loop structures, the stem loop structures being spaced  from one another by the spacer regions.
8.  A genetic construct comprising a 1-x RNAi expression cassette for targeting multiple regions of a HCV genome comprising:
a promoter;
RNAi agent coding sequences wherein x is two or more; and
sequences coding for spacer regions;
wherein transcription of the RNAi agent coding sequences produces RNAi agents which form stem-loop structures, the stem loop structures being spaced from one another by the spacer regions.

1.  A 1-x RNAi expression cassette for targeting multiple regions of a HCV genome comprising:
a promoter;
x RNAi agent coding sequences, wherein x is at least two; and
sequences coding for spacer regions;
wherein transcription of the RNAi agent coding sequences produces RNAi agents which form stem-loop structures, the stem loop structures being spaced  from one another by the spacer regions.
8.  A genetic construct comprising a 1-x RNAi expression cassette for targeting multiple regions of a HCV genome comprising:
a promoter;
RNAi agent coding sequences wherein x is two or more; and
sequences coding for spacer regions;
wherein transcription of the RNAi agent coding sequences produces RNAi agents which form stem-loop structures the stem loop structures being spaced from one another by the spacer regions.

200680010811.3
April 2012:
Ready for allowance.

An expression construct comprising: one or more enhancer elements selected from the group consisting of ApoE enhancer elements and SynEnh enhancer elements; one or more liver-specific promoters; and one or more RNAi constructs that provide one or more RNAi agents.

8008468
Granted
30 August 2011

 1.  A DNA directed RNA interference (ddRNAi) expression construct comprising:
one or more enhancer elements selected from the group consisting of ApoE enhancer elements and SynEnh enhancer elements;
one or more liver-specific promoters; and
one or more RNAi constructs that provide three or more RNAi agents;

(STANFORD)

Methods and compositions for expressing a gene or nucleotide sequence of interest. The compositions include an expression cassette that includes a synthetic enhancer, a transthyretin promoter, and a nucleotide sequence operably under the control of the synthetic enhancer and the transthyretin promoter. The expression cassette may be used in an AAV vector, such as a self-complementary AAV vector.

8129510
Granted
6th March 2012

An expression cassette, comprising:
a synthetic enhancer;
a transthyretin promoter; and
a nucleotide sequence operably under the control of the synthetic enhancer and the transthyretin promoter,
wherein the synthetic enhancer and the transthyretin promoter comprise a sequence having at least 95% sequence identity over the entire length of SEQ ID NO:3.

A method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention utilises recombinant DNA technology to post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, organ or whole organism, thereby producing novel phenotypes. Novel synthetic genes and genetic constructs which are capable or repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.

Waterhouse, Graham, Wang, Rice

90/007,247

Earliest Date Priority
19 Jun 1998

A method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the invention utilises recombinant DNA technology post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, organ or whole organism, thereby producing novel phenotypes. Novel synthetic genes and genetic constructs which are capable or repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto are also provided.

Graham, Wang, Waterhouse, Rice

Methods for reducing the phenotypic expression of a nucleic acid of interest in eukaryote cells by providing aberrant RNA molecules, preferably unpolyadenylated RNA molecules comprising at least one target specific nucleotide sequence homologous to the nucleic acid of interest, preferably a sense strand, into the nucleus of plant cells.

Waterhouse, Wang, Graham, (Smith)

(105)

(Not available for HCV)

 genetic construct comprising a multi-promoter expression cassette comprising at least three promoter/RNAi/terminator components wherein each promoter/RNAi/terminator component comprises a promoter element, a terminator element and an RNAi species operably linked to the promoter element and the terminator element, and wherein each of the RNAi species is different from one another.

Roelvink, Suhy, Kolykhalov,

A method of inducing, promoting or otherwise facilitating a change in the phenotype of an animal cell or group of animal cells including an animal. The modulation of phenotypic expression is accomplished via genotypic manipulation by inducing, promoting or otherwise facilitating the silencing of expressible genetic sequences thus reducing translation of transcript to protein. Expressible genetic sequences contemplated by the invention include not only genes normally resident in a particular cell (i.e. indigenous genes) but also genes introduced through recombinant means or through infection by pathogenic agents such as viruses.

Graham, Rice, Murphy, Reed

A ribonucleic acid (RNA) for use as interfering RNA in gene silencing techniques to silence a target gene comprising in a 5’ to 3’ direction at least four sequences being a first and second effector sequence 17 to 21 nucleotides in length; a sequence substantially complementary to the second effector sequence; and a sequence substantially complementary to the first effector sequence; wherein the complementary sequences are capable of forming double stranded regions with their respective effector sequences and wherein at least one of the four sequences is substantially identical to the predicted transcript of a region of the target gene; and the RNA further comprising a spacing sequence of one or more nucleotides,  the spacing sequence being located between and spacing the first effector sequence and the second effector sequence, or between the sequence substantially complementary to the second effector sequence and the sequence substantially complementary to the first effector sequence.

Graham, Rice, Roelvink, Suhy,, Kolkykhalov, Harrison, Reed.

Compositions and methods suitable for expressing 1-x RNAi agents against a gene or genes in cells, tissues or organs of interest in vitro and in vivo so as to treat diseases or disorders.

Roelvink, Suhy, Kolykhalov, Couto