U.S. patent application number 12/602005 was filed with the patent office on 2011-10-06 for rna interference target for treating aids.
This patent application is currently assigned to XIAMEN UNIVERSITY. Invention is credited to Tong Cheng, Ji Miao, Ningshao Xia, Jun Zhang, Tao Zhang, Yali Zhang.
Application Number | 20110243904 12/602005 |
Document ID | / |
Family ID | 40093162 |
Filed Date | 2011-10-06 |
United States Patent
Application |
20110243904 |
Kind Code |
A1 |
Cheng; Tong ; et
al. |
October 6, 2011 |
RNA INTERFERENCE TARGET FOR TREATING AIDS
Abstract
The present invention relates to RNA interference target
sequence targeting HIV for the treatment of AIDS. Based on the
target sequence, recombinant expression vectors, packaging vectors
and cells were constructed, which express a siRNA and/or a miRNA
and/or a ribozyme and/or an antisense oligonucleotide targeting
HIV. Also provided is the use of the recombinant expression
vectors, packaging vectors and recombinant cells in the manufacture
of a medicament for the treatment of AIDS.
Inventors: |
Cheng; Tong; (Fujian
Province, CN) ; Zhang; Tao; (Fujian Province, CN)
; Zhang; Yali; (Fujian Province, CN) ; Miao;
Ji; (Fujian Province, CN) ; Zhang; Jun;
(Fujian Province, CN) ; Xia; Ningshao; (Fujian
Province, CN) |
Assignee: |
XIAMEN UNIVERSITY
Fujian Province
CN
BEIJING WANTAI BIOLOGICAL PHARMACY ENTERPRISE CO., LTD.
Changping District, Beijing
CN
|
Family ID: |
40093162 |
Appl. No.: |
12/602005 |
Filed: |
June 2, 2008 |
PCT Filed: |
June 2, 2008 |
PCT NO: |
PCT/CN08/01074 |
371 Date: |
February 24, 2010 |
Current U.S.
Class: |
424/93.21 ;
435/243; 435/320.1; 435/325; 435/366; 435/419; 435/455; 514/44A;
536/23.1; 536/24.5 |
Current CPC
Class: |
A61P 31/18 20180101;
C12N 15/1132 20130101; C12N 2310/12 20130101; C12N 2310/11
20130101; C12N 2310/14 20130101 |
Class at
Publication: |
424/93.21 ;
536/24.5; 435/320.1; 435/325; 435/419; 435/243; 435/366; 435/455;
536/23.1; 514/44.A |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C07H 21/02 20060101 C07H021/02; C12N 15/85 20060101
C12N015/85; C12N 5/10 20060101 C12N005/10; C12N 1/00 20060101
C12N001/00; A61K 31/713 20060101 A61K031/713; A61K 31/7088 20060101
A61K031/7088; A61K 35/12 20060101 A61K035/12; A61P 31/18 20060101
A61P031/18 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2007 |
CN |
200710105818.X |
Claims
1. A RNA interference target sequence targeting HIV, which is
selected from: (1) a sequence set forth in any one of SEQ ID
NOs:1-32, or (2) a sequence that has at least 70%, preferably at
least 80%, 85%, 90%, 95%, 98% or higher identity to the sequence
defined in (1), or (3) a nucleotide sequence that can hybridize
with the sequence defined in (1) under stringent conditions or
highly stringent conditions, or (4) a nucleotide sequence that is
different from the sequence defined in (1) by only 1-3, preferably
1-2, more preferably 1 nucleotide(s), or (5) a fragment or a
complementary sequence of any of the sequences referred to
above.
2. A nucleic acid construct or a vector, such as an expression
vector, comprising the sequence according to claim 1.
3. A siRNA or a miRNA or a ribozyme or an antisense oligonucleotide
obtained based on the RNA interference target sequence according to
claim 1, which can inhibit the expression of the corresponding gene
of HIV and/or the replication of HIV and/or the infection of
HIV.
4. A recombinant expression vector which can express the siRNA or
the miRNA or the ribozyme or the antisense oligonucleotide
according to claim 3.
5. The recombinant expression vector according to claim 4,
comprising a nucleic acid sequence encoding the siRNA or the miRNA
or the ribozyme or the antisense oligonucleotide targeting HIV,
wherein the encoding nucleic acid sequence is operably linked to an
expression controlling sequence so that the siRNA or the miRNA or
the ribozyme or the antisense oligonucleotide can be expressed in
an animal cell, especially a mammalian cell, such as a human cell,
preferably a HIV receptor cell and a stem cell.
6. The recombinant expression vector according to claim 4, which is
a plasmid vector or a virus vector, such as a retrovirus vector,
including a lentivirus vector.
7. (canceled)
8. An isolated cell transformed or transfected or transduced with
the recombinant expression vector according to claim 4.
9. (canceled)
10. A modified cell, including an animal cell, such as a mammalian
cell, preferably a human cell, preferably a HIV receptor cell and a
stem cell, such as a CD4+cell and a CD34+cell, which can express or
comprises the siRNA or the miRNA or the ribozyme or the antisense
oligonucleotide according to claim 3.
11. The modified cell according to claim 10, which carries in its
genome or outside of its genome an encoding nucleic acid sequence
comprising the RNA interference target sequence according to claim
1, wherein the encoding nucleic acid sequence is operably linked to
an expression-controlling sequence so that the siRNA or the miRNA
or the ribozyme or the antisense oligonucleotide can be expressed
in the cell, including an animal cell, such as a mammalian cell,
preferably a human cell, preferably a HIV receptor cell and a stem
cell, such as a CD4+cell and a CD34+cell.
12. A method for producing the cell according to any one of claims
8 and 10, comprising transforming or transfecting or transducing a
cell, including an animal cell, such as a mammalian cell,
preferably a human cell, preferably a HIV receptor cell and a stem
cell, such as a CD4+cell and a CD34+cell, with the recombinant
expression vector according to claim 4.
13. A combination of DNA sequences comprising or consisting of a
first DNA sequence encoding a sense RNA segment and a second DNA
sequence encoding an antisense RNA segment, wherein the sense RNA
segment comprises a RNA sequence encoded by the target sequence
according to claim 1, and the antisense RNA segment can form a
double-stranded RNA with the sense RNA segment, and wherein the
double-stranded RNA can suppress the expression of HIV gene and/or
the replication of HIV and/or infection of HIV.
14. A small interference RNA (siRNA) comprising a sense RNA segment
and an antisense RNA segment, wherein the sense RNA segment
comprises a RNA sequence encoded by the target sequence according
to claim 1, and the antisense RNA segment can form a
double-stranded RNA with the sense RNA segment, and wherein the
double-stranded RNA can suppress the expression of the
corresponding gene of HIV and/or the replication of HIV and/or the
infection of HIV.
15. (canceled)
16. A method for treating HIV infection or a HIV patient or
inhibiting the replication or gene expression of HIV, comprising
administering to a patient a therapeutically effective amount of:
the RNA interference target sequence according to claim 1, or the
nucleic acid construct or the vector according to claim 2, or the
siRNA or the miRNA or the ribozyme or the antisense oligonucleotide
according to claim 3, or the recombinant expression vector
according to claim 4, or the cell according to any one of claims 8
and 10, or the siRNA according to claim 14.
17. (canceled)
18. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to molecular biology, cell
biology and gene therapy. More specifically, it relates to 32 RNA
interference (RNAi) targets which can be used for AIDS treatment,
recombinant expression vectors using the targets, and drugs and
methods for treating AIDS obtained in a variety of ways using these
targets.
BACKGROUND OF THE INVENTION
[0002] Acquired Immune Deficiency Syndrome (AIDS) caused by HIV
infection is one of most significant health threat faced by the
world. Currently, AIDS has almost spread to countries around the
world, and resulted in more than 40 million patients suffering from
it, and nearly 30 million people were killed by it (WHO, Report on
the Global AIDS Epidemic, 2004). In recent years, the spread of
AIDS in China is growing rapidly, and the infected people have
already amounted to 0.84 million. At present, the treatment of HIV
infection is primarily through high-intensity anti-retrovirus
therapy, such as through a combined use of inhibitors against viral
reverse transcriptase and protease. However, due to the high
mutation rate of HIV and its complex pathogenesis, this type of
approach could not completely eradicate the virus in vivo.
Therefore, there is an urgent need to develop a new method of
treatment for dealing with the threat of AIDS.
[0003] RNA interference (RNAi) is a mechanism for inhibiting gene
expression intracellularly mediated by a double-stranded RNA
(dsRNA), and was first proposed in a research on the inhibition of
gene expression in nematode in 1998 (Fire A et al., Nature, 1998,
391: 806-811). Further research discovered that the RNAi exists
widely in higher mammals and almost all eukaryotic organisms, such
as fungi, arabidopsis, hydra, planarian, trypanosoma, zebrafish
etc., and is a widely existing and conservative mechanism for
inhibiting gene expression, which may play a role in the regulation
of gene expression, protection against virus infection and
suppression of the activities of transposon and so on (Dykxhoorn D
M et al., nature molecular biology review, 2003, 4: 457-467).
Working mechanism of RNAi has now almost been elucidated: the
endogenous or exogenous dsRNA molecules are cleaved into small
interfering RNA (siRNA) in the cytoplasm by Dicer belonging to
RNase III. Typical siRNA structural characteristics are: a dsRNA
that is 19-23 nt in length with its 5' end phosphorylated and its
3' end symmetrically overhanged by 2-3 nt and with hydroxyl. siRNA
molecules bind to the protein complex of RNA-inducing silencing
complex (RISC), and the RISC has the helicase and endonuclease
activities. The siRNA molecule is unwound in the complex, and the
antisense strand can match target mRNA according to the principle
of base pairing, and guide the RISC binding to it to enzymatically
digest the target mRNA at a position 10 nt from the 5' end in the
middle of the antisense strand binding region, thereby inhibiting
the expression of the target gene. Currently, main methods for
obtaining siRNA include: plasmid and recombinant virus vector that
can express small hairpin RNA (shRNA), chemical synthesis, in vitro
transcription and so on.
[0004] At present, RNAi has shown a good application prospect in
the research of prevention and treatment of diseases such as viral
disease, including AIDS, and tumor. Studies have shown that siRNA
targeting the mRNA of HIV-1 can inhibit the replication of HIV-1
and viral gene expression in HIV-1 susceptible cell cultured in
vitro (Martinez M A et al., Immunology Trends, 2002, 23: 559-561).
Due to the complicated pathogenic mechanism of HIV, its effective
treatment requires highly efficient inhibition of viral replication
and gene expression. However, due to the different inhibition
efficiency of different targets, not all RNA interference targets
met the requirement of conventional design are able to inhibit the
expression of target genes effectively. Therefore, suitable RNA
interference targets having high inhibition efficiency becomes an
important factor in successful HIV treatment using RNAi technology.
Selection of an appropriate RNA interference target needs
comprehensive considerations in structural features, inhibition
efficiency, non-human gene homology and so on. Assistant methods
available include the following methods that have been put forward
presently: siRNA aided designing software, analysis of the
molecular structure of RNA, nucleic acid sequence analysis and
alignment and experimental experience etc., these also can be
verified through particular inhibition experiment.
[0005] Development of novel and more effective AIDS treatment
methods are expected based on RNA interference technology, and such
kind of treatment method requires a RNA interference target that
can effectively suppress HIV replication and expression to be
provided. The present invention meets this need and provides a RNA
interference target, a recombinant expression vector and so on for
such purpose.
BRIEF DESCRIPTION OF THE INVENTION
[0006] The present invention provides at highly efficient RNA
interference target targeting HIV. The RNA interference target can
be used to construct an expression plasmid, a recombinant viral
vector and a cell which comprises or into which is introduced a
nucleic acid sequence encoding the RNA interference target
according to the present invention, and the RNA interference target
also can be used to obtain a drug for AIDS treatment comprising the
RNA interference target according to the present invention. In at
particular aspect of the present invention, it relates to a RNA
interference target sequence targeting the HIV GAG, POL, VIF, or
VPU gene.
[0007] The RNA interference target provided by the present
invention can target HIV efficiently, inhibit HIV replication and
viral gene expression efficiently. The RNA interference target
provided by the present invention is obtained through a method
comprising: selecting and designing a RNA interference target
sequence that can target HIV, constructing shRNA through designing
appropriate primers, and cloning the shRNA into a pSUPER vector to
obtain a corresponding shRNA expression plasmid, co-transfecting
with the plasmid and a HIV infectious cloning plasmid, and
screening for a highly effective RNA interference target by
analysis such as detection of the expression level of HIV p24
protein and identification of inhibition specificity.
[0008] The present invention provides RNA interference target
sequence specifically targeting HIV, said sequence being selected
from:
(1) a sequence shown in any one of SEQ ID NO: 1-32, or (2) a
sequence that has at least 70%, preferably at least 80%, 85%, 90%,
95%, 98% or higher identity to the sequences defined in (1), or (3)
a nucleotide sequence able to hybridize with the sequence defined
in (1) under stringent conditions or highly stringent conditions,
or (4) a sequence that has only 1-3, preferably 1-2, more
preferably 1 different nucleotides from the sequence defined in
(1), or (5) a fragment or complementary sequence of the above
sequences.
[0009] In a particular aspect of the present invention, the RNA
interference target sequence may target to the HIV GAG, POL, VIF,
or VPU gene.
[0010] The RNA interference targets provided in the present
invention may be DNA or RNA sequences.
[0011] In a preferred embodiment, the RNA interference targets are
selected from siVIF037 (SEQ ID NO:24), siPOL1102 (SEQ ID NO:8),
siPOL1217 (SEQ ID NO:10), siPOL1327 (SEQ ID NO:29), and siPOL2252
(SEQ ID NO:22).
[0012] The present invention also provided a nucleic acid construct
or vector, such as expression vector, which contains the RNA
interference target sequence.
[0013] The present invention also provided a siRNA or a miRNA, or a
ribozyme, or an antisense oligonucleotide, which is obtained from
the above-mentioned RNA interference target sequence and can
inhibit the expression of the HIV corresponding gene and/or
replication and/or infection of HIV.
[0014] The present invention also provided a modified recombinant
expression vector, which can be used to express the HIV-targeting
siRNA and/or miRNA and/or ribozymes and/or anti-sense
oligonucleotide of the present invention.
[0015] In one embodiment, the recombinant expression vector of the
present invention is characterized in that it comprises a nucleic
acid sequence encoding the siRNA and/or the miRNA and/or the
ribozymes and/or the anti-sense oligonucleotide of the RNA
interference target sequence provided by the present invention,
wherein the nucleic acid sequence is operably linked with the
expression controlling sequence, making it possible to express the
siRNA and/or the miRNA and/or the ribozymes and/or the anti-sense
oligonucleotide targeting HIV in animal cells (especially mammalian
cells, such as human cells, HIV receptor cells, preferably
CD4+cells, such as mammalian stem cells, preferably hematopoietic
stem cells).
[0016] The recombinant expression vector according to the present
invention may be a plasmid vector or a viral vector, for example, a
retrovirus vector, including a lentivirus vector. Preferably, the
recombinant expression vector is a retrovirus vector, more
preferably a lentivirus vector.
[0017] The present invention provided a modified packaging vector
(such as a packaging plasmid) used for the production of a
retrovirus vector (for example, a lentivirus vector), which
contains a mutated HIV-derived gene sequence(s) used for expressing
a packaging protein(s). Specifically, the modified gene sequence(s)
can be characterized, independently from one another, in that, the
packaging vectors are mutated as follows:
TABLE-US-00001 a packaging --------GTAGACAGGATGAGGATTA--------
vector: is mutated --------GTAGACAGGACGAAGATTA--------, to: a
packaging --------GGATTTACCACACCAGACA-------- vector: is mutated
--------GGATTTACCACCCCCGACA--------, to: a packaging
--------GCTGGACTGTCAATGACAT-------- vector: is mutated
--------GCTGGACTGTGAACGACAT--------, to: a packaging
--------GCACTAACAGAAGTAGTAC-------- vector: is mutated
--------GCACTAACAGAAGTGGTGC--------, to: and a Packaging
--------TAGTAGCCAGCTGTGATAA-------- vector: is mutated
--------TAGTAGCCAGCTGCGACAA--------. to:
[0018] The present invention also involves isolated cells,
comprising:
(1) the RNA interference target sequence according to the present
invention, or (2) a nucleic acid construct or vector, such as
expression vector, which contains the RNA interference target
sequence according to the present invention.
[0019] The present invention also involves an isolated cell
transformed or transfected or transduced with a recombinant
expression vector which can express the siRNA and/or the miRNA
and/or the ribozyme, and/or the anti-sense oligonucleotide of the
present invention targeting HIV.
[0020] The present invention also involves an isolated cell
transformed or transfected or transduced with or comprises the
packaging vector of the present invention (such as a packaging
plasmid).
[0021] The present invention also involves a tissue and an
organism, such as an animal, that contains the above-mentioned
cell.
[0022] The present invention also involves a modified cell
(including an animal cell, such as a mammalian cell, preferably a
human cell, preferably a HIV receptor cell and a stem cell, such as
a CD4+cell and a CD34+cell), which can express or contain the siRNA
or the miRNA or the ribozyme or the antisense oligonucleotide
according to the present invention.
[0023] The present invention also involves a cell carrying in or
outside of its genome the nucleic acid sequence encoding the RNA
interference target according to the present invention, including a
prokaryotic cell (for example a bacterial cell, such as a E. coli
cell) and a eukaryotic cell (such as a fungal cell, an insect cell,
a plant cell, an animal cell, preferably a mammalian cell, such as
a human cell, preferably a HIV receptor cell and a stem cell, such
as a CD4+cell and a CD34+cell), which contains the nucleic acid
sequence encoding the RNA interference target according to the
present invention, wherein these nucleic acid sequences can be
operably linked with the expression controlling sequence, making it
possible to express the siRNA and/or the miRNA and/or the ribozyme,
and/or the antisense oligonucleotide in the cell.
[0024] The present invention further relates to a cell into which
is introduced the siRNA and/or the miRNA and/or the ribozyme,
and/or the antisense oligonucleotide obtained from the RNA
interference target provided by the present invention, including a
prokaryotic cell (e.g. a bacterial cell, such as an E. coli cell)
and a eukaryotic cell (e.g. a fungal cell, an insect cell, a plant
cell, an animal cell, preferably a mammalian cell, such as a human
cell, preferably a HIV receptor cell and a stem cell, such as a
CD4+cell and a CD34+cell) into which is introduced the siRNA and/or
the miRNA and/or the ribozyme and/or the antisense oligonucleotide
obtained from the RNA interference target of the present
invention.
[0025] In a preferred embodiment, HIV receptor cells introduced
with a shRNA expression element containing a nucleic acid sequence
encoding the RNA interference target obtained from the present
invention can thus acquire an ability to inhibit HIV replication
and viral gene expression.
[0026] The present invention also relates to a tissue and an
organism, such as an animal, comprising the cell mentioned above.
The present invention also relates to a pharmaceutical composition
comprising the cell according to the present invention.
[0027] In another aspect, the present invention also relates to a
method for the preparation of the modified cell according to the
present invention, comprising transforming or transfecting or
transducing the cell (including an animal cell such as a mammalian
cell, preferably a human cell, preferably a HIV receptor cell and a
stem cell, such as a CD4+cell and a CD34+cell) with the recombinant
expression vectors according to the present invention.
[0028] In an embodiment, the method comprises transducing a
mammalian cell, preferably a human HIV receptor cell and a stein
cell, such as a CD4+cell and a CD34+cell with the recombinant
retrovirus vector according to the present invention (for example,
a lentivirus vector, such as the lentivirus Lenti-VIF037,
etc.).
[0029] In the method mentioned above, the cell can be in an
isolated (or ex vivo) form, such as a cell isolated from a
HIV-infected patient or a normal individual, or in vivo, or a cell
strain cultured in vitro.
[0030] The present invention also relates to a combination of DNA
sequences, which comprises or consists of a first DNA sequence
encoding a sense RNA segment and a second DNA sequence encoding an
antisense RNA segment, wherein the sense RNA segment contains a RNA
sequence encoded by the target sequence according to the present
invention, and the antisense RNA segment can form a double-stranded
RNA with the sense RNA segment, said double-stranded RNA can
suppress the expression of HBV gene and/or the replication and/or
infection of HBV.
[0031] The present invention also relates to a small interfering
RNA (siRNA), comprising a sense RNA segment and an antisense RNA
segment, wherein the sense RNA segment contains a RNA sequence
encoded by the target sequence according to the present invention,
and the antisense RNA segment can form a double-stranded RNA with
the sense RNA segment, and said double-stranded RNA can suppress
the expression of HIV corresponding gene and/or replication and/or
infection of HIV.
[0032] The present invention also relates to a use of the siRNA
and/or the miRNA and/or the ribozyme and/or the antisense
oligonucleotide obtained from the RNA interference target provided
by the present invention in the preparation of a drug and/or a
pharmaceutical composition for the treatment of HIV infection or
HIV patients.
[0033] The present invention also relates to a use of the siRNA
and/or the miRNA and/or the ribozyme and/or the antisense
oligonucleotide obtained from the RNA interference target provided
by the present invention in the preparation of a drug and/or a
pharmaceutical composition for the suppression of HIV replication
or HIV gene expression.
[0034] The present invention also relates to a use of the RNA
interference target sequence, or the nucleic acid construct or the
vector, or the recombinant expression vector according to the
present invention in the preparation of a drug for the treatment of
HIV infection or HIV patients.
[0035] The present invention also relates to a use of the modified
cell according to the present invention (including an animal cell,
such as a mammalian cell, preferably a human cell, preferably a HIV
receptor cell and a hematopoietic stem cell, such as a CD4+cell and
a CD34+cell) in the preparation of a drug and/or a pharmaceutical
composition for the treatment of HIV infection or HIV patients.
[0036] The present invention also relates to a use of the siRNA
target sequence according to the present invention in the screening
of anti-HIV drugs.
[0037] The present invention also relates to a method for the
treatment of HIV infection or HIV patients, comprising
administering to an individual in need the RNA interference target
sequence, the nucleic acid construct or the vector, the recombinant
expression vector, the siRNA or the miRNA or the ribozyme or the
antisense oligonucleotide or the cell according to the present
invention.
[0038] The present invention also relates to a use of the vector
and the cell according to the present invention for the treatment
of HIV infection or HIV patients.
[0039] The present invention also relates to a method for the
treatment of HIV infection or HIV patients, comprising
administering to a patient a therapeutically effective amount of
the RNA interference target sequence, the nucleic acid construct or
the vector, the siRNA or the miRNA, or the ribozyme, or the
antisense oligonucleotide, the expression vector, the cell, or the
siRNA according to the present invention.
[0040] The present invention also relates to a method for the
suppression of HIV replication or HIV gene expression, comprising
administering to an individual in need a therapeutically effective
amount of the RNA interference target sequence, the nucleic acid
construct or the vector, the siRNA or the miRNA, or the ribozyme,
or the antisense oligonucleotide, the expression vector, the cell,
or the siRNA according to the present invention.
[0041] The present invention also relates to the RNA interference
target sequence, the nucleic acid construct or the vector, the
siRNA or the miRNA, of the ribozyme, or the antisense
oligonucleotide, the expression vector, the cell, or the siRNA
according to the present invention used for the treatment of HIV
infection or HIV patients, or for the suppression of HIV
replication or HIV gene expression.
[0042] The present invention will be described more specifically
with reference to the following figures. From the detailed
description below, the above-mentioned aspects and other aspects of
the present invention will be obvious.
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 depicts the flow diagram of the construction of
pSUPER-siRNA expression plasmid series.
[0044] FIG. 2 depicts the suppression effect on HIV gene expression
of the siRNA expression plasmids respectively targeting the
obtained 32 RNA interference targets in the co-transfection
experiment with HIV infectious clone plasmid. The results show that
these RNA interference targets can suppress HIV.
[0045] FIG. 3 depicts that the constructed expression vectors
pDEST-VIF037, pDEST-POL1102, pDEST-POL1217, pDEST-POL1327,
pDEST-POL2252 can effectively express the siRNA sequence encoded,
and has the gene targeting specificity. When pGL3-VIF and the
expression vector pDEST-VIF037 were co-transfected, the luciferase
gene expression was effectively inhibited, while there was no
inhibition of luciferase gene expression when pGL3-control and the
expression vector pDEST-VIF037 were co-transfected. When pGL3-POL
was co-transfected respectively with the expression vectors
pDEST-POL1102, pDEST-POL1217, pDEST-POL1327, pDEST-POL2252, the
luciferase gene expression was inhibited, while when the
pGL3-control was co-transfected respectively with the expression
vectors pDEST-POL1102, pDEST-POL1217, pDEST-POL1327, pDEST-POL2252,
the luciferase gene expression was not inhibited.
[0046] FIG. 4 depicts that the expression of mutated lentivirus
packaging vector was not interfered by the lentivirus expression
vector expressing the HIV-targeting siRNA.
[0047] FIG. 5 depicts the inhibitory effect of the HIV receptor
cell MT-4 transduced with a recombinant lentivirus carrying
HIV-targeting siRNA expression sequences on the replication of
HIV-1.sub.NL4-3.
[0048] FIG. 6 depicts the inhibitory effect of the mutated HIV
receptor cell on the replication of HIV-1.sub.NL4-3.
[0049] FIG. 7 depicts the inhibitory effect on HIV of the
synthesized siRNA of RNA interference target targeting HIV. After
transfection, siR-VIF037 can inhibit the replication and expression
of HIV in cells.
[0050] FIG. 8 depicts the inhibitory effect on HIV of the siRNA of
RNA interference target targeting HIV. Said siRNA is synthesized
and modified by 2'-Ome (2'-methoxy) modification and/or
phosphorylation and/or steroid modification. After transfection,
siRpo-VIF037, siRpo-POL1217; siRpoC-VIF037, siRpoC-POL1217 can
inhibit the replication and expression of HIV in cells.
[0051] FIG. 9 depicts that the HIV-targeting siRNA has the ability
to inhibit the replication of HIV in H.sup.2K-PBL-SCID mouse model.
VIF037 chimeric mice can show an ability of anti-HIV infection, and
can significantly reduce the levels of HIV proteins in the serum
comparing to the control group.
DETAILED DESCRIPTION OF THE INVENTION
[0052] Unless otherwise stated, the terms of the present invention
have the conventional meanings in the art.
[0053] The present invention provided a RNA interference target
that can target HIV with high efficiency, comprising any one or
more sequences shown in SEQ ID NO:1-32 or any one or more sequences
that have at least 70% (preferably at least 80%, 85%, 90%, 95%, 98%
or higher) identity to the sequences shown in SEQ ID NO:1-32.
[0054] The identity can be calculated according to the well-known
methods in this art. Preferred examples of algorithms suitable for
determining the percentage of sequence identity and sequence
similarity are BLAST and BLAST 2.0, which were respectively
described in Altschul et al. Nucl. Acid. Res. 1977, Volume 25: page
3389-3402 and Altschul et al. J. Mol. Biol. 1990, Volume 215: page
403-410. Using parameters such as those described herein, BLAST and
BLAST 2.0 can be used to determine the percentage of sequence
identity of the polynucleotides and polypeptides of the present
invention. The software for conducting BLAST analysis can be
obtained by the public from the National Center for Biotechnology
Information.
[0055] In other embodiments, the sequences of the RNA interference
target comprise a polynucleotide sequence that can hybridize with
the polynucleotide of the present invention or the fragment or
complementary sequence thereof under stringent conditions or highly
stringent conditions. Hybridization technology is well-known in the
field of molecular biology. For the purpose of illustration, the
hybridization condition is a stringent condition, for example, a
DNA binding to the filtration membrane is hybridized in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.quadrature., then
is washed one or more times in 0.2.times.SSC/0.1% SDS at about
50-65.quadrature.; or is a highly stringent condition, for example,
a nucleic acid binding to the filtration membrane is hybridized in
6.times.SSC at about 45.quadrature., then is washed one or more
times in 0.1.times.SSC/0.2% SDS at about 68.quadrature.; or is
other stringent hybridization conditions known in the art (See for
example, Ausubel, E. M. et al., 1989, Current Protocols in
Molecular Biology, Volume 1, Green Publishing Associates, Inc. and
John Wiley & Sons, Inc., New York, page 6.3.1-6.3.6 and
2.10.3).
[0056] The present invention also relates to a nucleotide sequence
which can hybridize with any sequence of SEQ ID NO: 1-32 or the
fragment, complementary sequence thereof under stringent conditions
or a highly stringent conditions.
[0057] According to the present invention, the siRNA and/or the
miRNA and/or the ribozyme, and/or the anti-sense oligonucleotide
can be designed to target a gene of interest or a regulatory
sequence, such as a gene, whose expression is to be inhibited, or a
regulatory sequence thereof, in order to inhibit or reduce its
expression. The directed gene or the regulatory sequences thereof
may be any gene whose expression is to be inhibited or reduced or
the regulatory sequence thereof, such as those from the pathogen,
or those participating in the formation and development of cancer,
and especially those targeting HIV. The siRNA, miRNA, ribozyme and
antisense oligonucleotide of the present invention can be designed
in accordance with conventional methods.
[0058] "The siRNA, miRNA, ribozyme and antisense oligonucleotide
obtained from the RNA interference target sequence according to the
present invention" refers to the siRNA, miRNA, ribozyme and
antisense oligonucleotide obtained by ways of designing expression
or designing synthesis, and the target sequence (either DNA or RNA
sequence) with which they interact is or contains the RNA
interference target sequences according to the present
invention.
[0059] For conventional methods of siRNA design, one can refer to
references (such as, Reynolds A et al., Nature Biotechnology, 2004,
volume 22: page 326-330) or publicly available information on the
web sites of Amhion or Qiagen Inc. etc., or the description in
example 1. For conventional methods of miRNA design, one can refer
to the paper of Lo H L et al, Gene Therapy, 2007, Volume 14: page
1503-1512. The methods for selecting target sequences are similar
to the methods for designing siRNA. For example, the designed sense
strand comprising the target sequence and the corresponding
antisense strand can replace other sequences of the pri-microRNA,
and this enables the constructed miRNA to prevent the expression of
the mRNA comprising the target sequence. For conventional methods
of ribozyme design, one can refer to the paper of Haseloff J et al,
Nature, 1988, Volume 334: page 585-591. For example, nucleotide
sequences complementary to sequences upstream and downstream of the
target sequence can be placed upstream and downstream of the
conservative core sequence of the ribozyme (such as the hammerhead
structure) respectively, so that the constructed ribozyme can
cleave the nucleic acids containing the target sequence at the
target sequence. For conventional methods of antisense
oligonucleotides design, one can refer to the paper of Matveeva O V
et al, Nucleic Acid Research, 2003, Volume 31: page 4989-4994.
[0060] Promoters used in the present invention can be any promoter
suitable for expressing desired genes in cells of interest, and can
be constitutive or inducible promoters, and also can be complex
promoters, such as dual promoters.
[0061] "Operably linked" means that the way of linking the linked
molecules allows to perform the desired functions. For example, the
operable linking of an expression-controlling sequence and a
gene-coding sequence allows the expression-controlling sequence to
control the expression of the gene-coding sequence.
[0062] "Expression controlling sequences" is known to the art as
controlling sequences required for gene expression, and it
generally must comprise a promoter, and often also comprises a
transcription termination sequence, and can also comprise other
sequences, such as an enhancer sequence. For the siRNA, miRNA,
ribozyme and antisense oligonucleotide etc., gene expression refers
to transcription, and can also include post-transcriptional
processing; and for protein-coding sequences, it usually refers to
transcription and translation, resulting in mature proteins.
[0063] The present invention provided a RNA interference target
that can highly effectively target HIV and the siRNA, miRNA,
ribozyme and antisense oligonucleotide designed according to the
target. The siRNA, miRNA, ribozyme and antisense oligonucleotide
according to the present invention comprise modified products
produced by chemically modifying the constitution moieties, such as
phosphate backbone and/or ribose and/or base etc., of the siRNA,
miRNA, ribozyme and antisense oligonucleotide. The modification
methods are known in the art, which can be thio-modification and/or
sterol modification and/or PEG-modification and/or
glyco-modification and/or LNA-modification etc. One can refer to,
such as Dykxhoorn D M et al, Annual Review of Biomedical
Engineering, 2006, Volume 8: page 377-402 and Behlke M A et al,
Molecular Therapy, 2006, Volume 13: page 644-670.
[0064] In a particular embodiment, the present invention relates to
a small interfere RNA (siRNA) comprising a sense RNA segment and an
antisense RNA segment, the sense RNA segment contains a RNA
sequence encoded by the RNA interfere target according to the
present invention, the antisense RNA segment can form a
double-stranded RNA with the sense RNA segment and the
double-stranded RNA can suppress the expression of HIV
corresponding gene and/or the replication and/or infection of
HIV.
[0065] According to the present invention, the terms "small
molecule RNA", "small interfering RNA" or "siRNA" can be used
interchangeably, which all refer to RNAs that can suppress the
expression of the target HIV gene and contain sense RNA segment
region and antisense RNA segment region.
[0066] Related, the present invention also provided a combination
of DNA sequences which comprises or consists of a first DNA
sequence encoding the sense RNA segment and a second DNA sequence
encoding the antisense RNA segment, wherein the sense RNA segment
comprises a RNA sequence encoded by the target sequence of the
present invention, and the antisense RNA segment can form a
double-stranded RNA with the sense RNA segment, and the
double-stranded RNA can suppress (by RNA interference) the
expression of HIV gene and/or the replication and/or infection of
HIV.
[0067] In such aspect of the present invention, the sense RNA
segment and antisense RNA segment could reside in two different RNA
strands or a single RNA strand, for example, in one single strand
comprising the sense RNA segment and the antisense RNA segment.
[0068] For example, the siRNA according to the present invention
can be a hairpin single-stranded RNA molecule, wherein a
double-stranded RNA region is formed between the complementary
regions of the sense RNA segment and the antisense RNA segment.
[0069] The length of the sense RNA segment and the antisense RNA
segment is preferably 8-50 nt, preferably 10-30 nt (more preferably
15-27 nt, 19-23 nt, such as 19 nt, 20 nt or 21 nt). But it could
also be longer or shorter.
[0070] The complementary region in the double-stranded RNA formed
between the sense RNA segment and the antisense RNA segment has at
least 10 bp (preferably 15 bp, more preferably 18 bp, e.g. 19 bp,
20 bp or 21 bp). Preferably, the complementary region between the
sense RNA segment and the antisense RNA segment comprises 19, 20 or
21 complementary base pairs.
[0071] In an embodiment, few bases of mismatch, such as 1-5, e.g. 1
or 2 or 3 or 4 mismatches are allowed to exist between the sense
RNA segment and the antisense RNA segment. In a preferred
embodiment, the sense RNA segment and the antisense RNA segment
match perfectly.
[0072] In an embodiment, the siRNA according to the present
invention is a double-stranded RNA molecule with 10-30 bp,
preferably 15-27 bp, more preferably 19-23 bp. There are at least
10, preferably 15, more preferably 18 pairs of complementary bases
in the double-stranded RNA.
[0073] In a preferred embodiment, the GC content of the sense RNA
segment and the antisense RNA segment is 35-75%, for example
40-60%, 45-55%, 48-52%, e.g. about 50%.
[0074] In a preferred embodiment, there is no significant identity
between the sense and antisense RNA segment and a known human gene
and the expression fragment of the gene. The significant identity
means that there is at least 60%, such as 70%, 80%, 90%
identity.
[0075] Preferably, the ratio between the sum of the number of bases
guanine (G) and cytosine (C) in the 19 nucleotide sequence from the
5' end and the number of the 19 nucleotides except for TT in the 3'
end (G/C ratio) is 35% to 75% in the sense RNA segment. There is no
significant identity between the antisense RNA segment and its
mutant having one-nucleotide-mutation and known human genes and
gene expression fragments.
[0076] In an embodiment of the recombinant expression vector
according to the present invention, the recombinant expression
vector according to the present invention comprises a nucleic acid
sequence encoding the RNA interference target according to the
present invention, these nucleic acid sequences are operably linked
with expression controlling sequence, thereby making it possible to
express the HIV-targeting siRNA and/or miRNA and/or ribozyme and/or
antisense oligonucleotide in an animal cell, especially in a
mammalian cell, such as a human cell, e.g. a HIV receptor cell and
a stem cell.
[0077] Similarly, in the method for preparing the modified cell of
the present invention, the modified cell of the invention can be
obtained by transforming or transfecting or transducing the cell
(including an animal cell, such as a mammalian cell, preferably a
human cell, preferably a HIV receptor cell and a stem cell, such as
a CD4+cell and a CD34+cell) with an expression vector comprising a
nucleic acid sequence encoding the RNA interference target
according to the present invention, as long as the cells finally
obtained comprise the nucleic acid sequence encoding the RNA
interference target according to the present invention.
[0078] The modified cell according to the present invention can
also be obtained by introducing into the cell the siRNA and/or the
miRNA and/or the ribozyme and/or the antisense oligonucleotide
obtained from the RNA interference target provided by the present
invention, as long as the cells obtained comprise the siRNA and/or
the miRNA and/or the ribozyme and/or the antisense oligonucleotide
obtained from the RNA interference target according to the present
invention.
[0079] The recombinant expression vector according to the present
invention may either be a plasmid vector or a virus vector, such as
a retrovirus vector, including a lentivirus vector. Preferably, the
recombinant expression vector is a retrovirus vector, more
preferably a lentivirus vector.
[0080] The modified cell according to the present invention is
preferably a mammalian cell, preferably a human cell, preferably a
HIV receptor cell, such as a CD4+cell, preferably a stem cell,
especially a hematopoietic stem cell, such as a CD34+cell. The cell
carries in or outside of its genome a nucleic acid sequence
encoding the RNA interference target according to the present
invention, which is operably linked with expression controlling
sequence, and thereby can express the siRNA and/or the miRNA and/or
the ribozyme and/or the antisense oligonucleotide in the cells.
[0081] The recombinant vector and the modified cell according to
the present invention can be used to treat HIV infection.
[0082] In a particular embodiment, the present invention relates
to:
1. RNA interference target sequences targeting HIV:
TABLE-US-00002 siGAG0942: AAATTGGATGACAGAAACC, (SEQ ID NO. 1)
siGAG1091: CTGAAGCAATGAGCCAAGT, (SEQ ID NO. 2) siGAG1273:
GATTGTACTGAGAGACAGGCT, (SEQ ID NO. 3) siPOL0922:
TGGAAAGGATCACCAGCAA, (SEQ ID NO. 4) siPOL0927: AGGATCACCAGCAATATTC,
(SEQ ID NO. 5) siPOL0937: GCAATATTCCAGTGTAGCA, (SEQ ID NO. 6)
siPOL1026: GTATGTAGGATCTGACTTA, (SEQ ID NO. 7) siPOL1102:
GGATTTACCACACCAGACA, (SEQ ID N0. 8) siPOL1131: GAAAGAACCTCCATTCCTT,
(SEQ ID NO. 9) siPOL1217: GCTGGACTGTCAATGACAT, (SEQ ID NO. 10)
siPOL1223: CTGTCAATGACATACAGAA, (SEQ ID NO. 11) siPOL1402:
CCGGTACATGGAGTGTATT, (SEQ ID NO. 12) siPOL1411:
GGAGTGTATTATGACCCAT, (SEQ ID NO. 13) siPOL1468:
GGCCAATGGACATATCAAA, (SEQ ID NO. 14) siPOL1470:
CCAATGGACATATCAAATT, (SEQ ID NO. 15) siPOL1544:
CCCACACTAATGATGTGAA, (SEQ ID NO. 16) siPOL1548:
CACTAATGATGTGAAACAA, (SEQ ID NO. 17) siPOL1550:
ACACTAATGATGTGAAACAATT, (SEQ ID NO. 18) siPOL1734:
GAAGTTATGGTACCAGTTA, (SEQ ID NO. 19) siPOL1762:
CCCATAATAGGAGCAGAAA, (SEQ ID NO. 20) siPOL2008:
TCAGAGTTAGTCAGTCAAA, (SEQ ID NO. 21) siPOL2252:
TAGTAGCCAGCTGTGATAA, (SEQ ID NO. 22) siVIF009: CAGATGGCAGGTGATGATT,
(SEQ ID NO. 23) siVIF037: GTAGACAGGATGAGGATTA, (SEQ ID NO. 24)
siVIF038: TAGACAGGATGAGGATTAA, (SEQ ID NO. 25) siGAG0432:
TCAGGCCATATCACCTAGA, (SEQ ID NO. 26) siGAG0738:
AATAGGATGGATGACACAT, (SEQ ID NO. 27) siGAG1438:
GGAGCCGATAGACAAGGAA, (SEQ ID NO. 28) siPOL1327:
GCACTAACAGAAGTAGTAC, (SEQ ID NO. 29) siVIF090: TATTTCAAGGAAAGCTAAG,
(SEQ ID NO. 30) siVIF344: TTTCAGAATCTGCTATAAG, (SEQ ID NO. 31)
siVPU164: GAGTGAAGGAGAAGTATCA; (SEQ ID NO. 32)
2. an expression vector, preferably a lentivirus vector, can be
used to modify HIV receptor cell or hematopoietic stem cell: A. a
recombinant lentivirus vector that can express the MGMT (P140K)
gene and one or more siRNAs and/or miRNAs, and/or HIV-targeting
ribozymes, B. a modified packaging vector for producing lentivirus
vector, such as a packaging plasmid, comprising a mutated
HIV-derived gene sequence for expressing a packaging protein,
examples of modified sequences are:
TABLE-US-00003 a packaging --------GTAGACAGGATGAGGATTA--------
vector: is mutated --------GTAGACAGGACGAAGATTA--------, to: a
packaging --------GGATTTACCACACCAGACA-------- vector: is mutated
--------GGATTTACCACCCCCGACA--------, to: a packaging
--------GCTGGACTGTCAATGACAT-------- vector: is mutated
--------GCTGGACTGTGAACGACAT--------, to: a packaging
--------GCACTAACAGAAGTAGTAC-------- vector: is mutated
--------GCACTAACAGAAGTGGTGC--------, to: a packaging
--------TAGTAGCCAGCTGTGATAA-------- vector: is mutated
--------TAGTAGCCAGCTGCGACAA--------, to:
The use of the lentivirus in a HIV receptor cell and/or a
hematopoietic stem cell, A. to stably express anti-HIV molecules,
for example, a siRNA specifically blocking the replication of HIV
in the HIV receptor cell and/or the hematopoietic stem cell, or B.
to prevent the hematopoietic stem cell from being killed by
BG/BCNU; 3. a modified cell, such as a HIV receptor cell and a
hematopoietic stem cell, which comprises a nucleic acid sequence
encoding the RNA interference target according to the present
invention and can express the siRNA and/or the miRNA and/or the
ribozyme and/or the antisense oligonucleotide; or is introduced
with the siRNA and/or the miRNA and/or the ribozyme and/or the
antisense oligonucleotide obtained from the RNA interference target
according to the present invention.
[0083] The sequence of the RNA interference target (SEQ ID
NO:1-32).
EXAMPLES
Example 1
Design and Construction of the siRNA Expression Plasmid
[0084] Design of the RNA interference target sequence targeting HIV
was carried out by: [0085] selecting a highly conservative region
for the design of the siRNA sequence by "DNA walking" with HIV
reference sequence as a target sequence; [0086] conducting BLAST
search in the GenBank with the preliminarily selected siRNA
sequence; and [0087] selecting sequences that have three or more
different bases from non-targeting sequences as candidate
sequences.
[0088] Construction of the siRNA expression plasmid: the expression
vector of the siRNA is pSUPER vector (oligoengine company Cat. No
VEC-PBS-0001/0002). For more information about the construction
procedure, please refer to Experimental Protocol for the pSUPER
vector from the company (www.oligoengine.com). A brief construction
procedure is shown in the FIG. 1. Primers carrying the RNA
interference sequence were synthesized, complementary primers were
annealed and then ligated into the pSUPER vector digested with
BgIII and HindIII, and the correct siRNA expression plasmid was
confirmed by restriction enzyme digestion and sequencing.
[0089] Construction of the control siRNA expression plasmid:
siRNA-luc (5'-GTGCGCTGCTGGTGCCAAC-3') which is a siRNA sequence
specifically targeting luciferase and siRNA-Nk
(5'-TGCATCGGAAAATAGATGT-3') which is an unrelated siRNA sequence
not matching with HIV and human gene were taken as controls. The
synthesis of the primers, construction into the pSUPER vector,
obtaining of corresponding siRNA expression plasmid after
restriction enzyme digestion and sequencing were carried out using
the method described above.
Example 2
Screening by Co-Transfection Assay for a RNA Interference Target
which Can Effectively Inhibiting HIV
[0090] pNL4-3 plasmid (from Pasteur Institute; may also use other
HIV-1 infectious cloning plasmid), a HIV-1 infectious cloning
plasmid, has an ability to express HIV viral proteins and viral
particles after being transfected into suitable Mammalian cells
(for example, 293FT cells). P24 is a capsid protein of the HIV
virus, which can indicate the expression level of the virus protein
and virus particle by detecting the content of the p24 protein in
the supernatant of the cell culture, and is positively correlated
with the virus titer. Therefore, the efficiency of different siRNAs
in inhibiting the replication of HIV-1 can be determined by
co-transfecting the siRNA expression plasmids with HIV infectious
cloning plasmid (pNL4-3 plasmid) in the 293FT cells, and detecting
the expression level of p24 protein in the cells after
co-transfection.
[0091] 293FT cells (Invitrogen, Catalog #R700-07) were cultured in
24-well cell culture plates to about 70% confluence. After 12 h,
cells were transfected with 0.1 .mu.g/well pNL4-3 plasmid and 1
.mu.g/well siRNA expression plasmid, with Lipofectamine 2000
(Invitrogen Cat. No 11668-027) as the transfection reagent. For the
method of transfection, see the instructions of the reagent. Cell
culture supernatant was collected respectively 48 h after
co-transfection, and after gradient dilution, the activity of the
p24 protein in the supernatant of the cell culture was examined
using Murex HIV Antigen Mab (Cat. No. 8E77-02). The efficiency of
each of the siRNAs in inhibiting HIV was calculated using as
control the contents of p24 protein in the supernatant of the cell
culture of the 293FT cells co-transfected with control siRNA
expression plasmid and HIV infectious cloning plasmid. Through
comparison, 32 RNA interference targets with the ability of highly
effective inhibition were obtained. FIG. 2 shows the inhibitory
efficiency of the siRNA expression plasmids constructed
respectively with the 32 RNA interference target sequences on HIV
viral gene expression after being transfected into the cell.
[0092] The RNA interference target sequences that can be used to
effectively suppress HIV are listed below:
TABLE-US-00004 siGAG0942: AAATTGGATGACAGAAACC (SEQ ID NO. 1)
siGAG1091: CTGAAGCAATGAGCCAAGT (SEQ ID NO. 2) siGAG1273:
GATTGTACTGAGAGACAGGCT (SEQ ID NO. 3) siPOL0922: TGGAAAGGATCACCAGCAA
(SEQ ID NO. 4) siPOL0927: AGGATCACCAGCAATATTC (SEQ ID NO. 5)
siPOL0937: GCAATATTCCAGTGTAGCA (SEQ ID NO. 6) siPOL1026:
GTATGTAGGATCTGACTTA (SEQ ID NO. 7) siPOL1102: GGATTTACCACACCAGACA
(SEQ ID NO. 8) siPOL1131: GAAAGAACCTCCATTCCTT (SEQ ID NO. 9)
siPOL1217: GCTGGACTGTCAATGACAT (SEQ ID NO. 10) siPOL1223:
CTGTCAATGACATACAGAA (SEQ ID NO. 11) siPOL1402: CCGGTACATOGAGTGTATT
(SEQ ID NO. 12) siPOL1411: GGAGTGTATTATGACCCAT (SEQ ID NO. 13)
siPOL1468: GGCCAATGGACATATCAAA (SEQ ID NO. 14) siPOL1470:
CCAATGGACATATCAAATT (SEQ ID NO. 15) siPOL1544: CCCACACTAATGATGTGAA
(SEQ ID NO. 16) siPOL1548: CACTAATGATGTGAAACAA (SEQ ID NO. 17)
siPOL1550: ACACTAATGATGTGAAACAATT (SEQ ID NO. 18) siPOL1734:
GAAGTTATGGTACCAGTTA (SEQ ID NO. 19) siPOL1762: CCCATAATAGGAGCAGAAA
(SEQ ID NO. 20) siPOL2008: TCAGAGTTAGTCAGTCAAA (SEQ ID NO. 21)
siPOL2252: TAGTAGCCAGCTGTGATAA (SEQ ID NO. 22) siVIF009:
CAGATGGCAGGTGATGATT (SEQ ID NO. 23) siVIF037: GTAGACAGGATGAGGATTA
(SEQ ID NO. 24) siVIF038: TAGACAGGATGAGGATTAA (SEQ ID NO. 25)
siGAG0432: TCAGGCCATATCACCTAGA (SEQ ID NO. 26) siGAG0738:
AATAGGATGGATGACACAT (SEQ ID NO. 27) siGAG1438: GGAGCCGATAGACAAGGAA
(SEQ ID NO. 28) siPOL1327: GCACTAACAGAAGTAGTAC (SEQ ID NO. 29)
siVIF090: TATTTCAAGGAAAGCTAAG (SEQ ID NO. 30) siVIF344:
TTTCAGAATCTGCTATAAG (SEQ ID NO. 31) siVPU164: GAGTGAAGGAGAAGTATCA
(SEQ ID NO. 32)
[0093] Through the above experiment, the 32 RNA interference
targets according to the present invention were confirmed to be
useful in efficiently inhibiting HIV expression.
[0094] In the following experiments, siVIF037, siPOL1102,
siPOL1217, siPOL1327, siPOL2252 were selected as examples from the
RNA interference targets listed above for the further construction
of the recombinant lentivirus that can express siRNAs targeting
siVIF037, siPOL1102, siPOL1217, siPOL1327, siPOL2252 respectively.
For the construction method, see example 3 and example 4.
Example 3
Construction of the Expression Vector and the Lentivirus Packaging
Vector Expressing siRNA
Expression Vector:
[0095] The expression vector of the lentivirus system pDEST-MR
(patent application number: 200510112917.1; Publication Number:
CN1948475) used in this example comprises the MGMT (P140K) gene
controlled by the mPGK promoter and an expression cassette for
expressing siRNA controlled by the H1 promoter.
Method for Constructing the Expression Vectors pDEST-VIF037,
pDEST-POL1102, pDEST-POL1217, pDEST-POL1327, pDEST-POL2252:
[0096] Gene fragments VIF037, POL1102, POL1217, POL1327, POL2252
(including as examples the RNA interference target sequences
siVIF037 (SEQ ID NO: 24), siPOL1102 (SEQ ID NO: 8), siPOL1217 (SEQ
ID NO: 10), siPOL1327 (SEQ ID NO: 29), siPOL2252 (SEQ ID NO: 22)
shown in example 2, but may also include other RNA interference
target sequences provided by the present invention) were
synthesized respectively, and Age I site was added at the 5' end of
the fragments, Sma I site was added at the 3' end of the fragments;
gene fragments digested with Age I and Sma I were ligated with the
plasmid pDEST-MR digested with the same enzymes, thereby
constructing the expression vectors pDEST-VIF037, pDEST-POL1102,
pDEST-POL1217, pDEST-POL1327, pDEST-POL2252.
[0097] The expression effectivity of the constructed recombinant
lentivirus expression vectors pDEST-VIF037, pDEST-POL1102,
pDEST-POL1217, pDEST-POL1327, pDEST-POL2252 was examined. Reporter
plasmids pGL3-VIF and pGL3-POL were constructed by inserting the
VIF and POL gene sequence respectively between the stop codon and
the PolyA of the luciferase gene in the pGL3-control plasmid
(purchased from Promega Corporation). Co-transfection inhibition
assay was conducted with the expression plasmid siRNA-luc targeting
luciferase and siRNA-Nk targeting unrelated sequences as controls.
The results were shown in FIG. 3, when the pGL3-VIF was
co-transfected with the expression vector pDEST-VIF037, the
expression of the luciferase gene was effectively inhibited,
however, when the pGL3-control was co-transfected with the
expression vector pDEST-VIF037, the expression of the luciferase
gene was not inhibited; and when pGL3-POL was co-transfected with
the expression vectors pDEST-POL1102, pDEST-POL1217, pDEST-POL1327,
pDEST-POL2252 respectively, the expression of the luciferase gene
was effectively inhibited, but when the pGL3-control was
co-transfected with the expression vectors pDEST-POL1102,
pDEST-POL1217, pDEST-POL1327, pDEST-POL2252 respectively, the
expression of the luciferase gene was not inhibited. The results
show that the constructed expression vectors pDEST-VIF037,
pDEST-POL1102, pDEST-POL1217, pDEST-POL1327, pDEST-POL2252 can
express the encoded siRNA sequence, and have gene targeting
specificity.
Modification of the Lentivirus Packaging Vector:
[0098] Because lentivirus vector is mainly from HIV-1, the in vitro
packaging of the lentivirus vector needs several HIV-1 proteins,
such as the products of HIV POL and GAG gene. Because it was needed
to use lentivirus as an expression vector of the siRNA targeting
HIV-1, in order to prevent the siRNA expressed in the expression
vector from inhibiting the expression of lentivirus packaging
vectors, that is, in order to obtain recombinant lentivirus
normally, in this example, corresponding mutations were introduced
into the HIV-1-derived gene sequence in the packaging vector. Thus,
the mRNA transcribed from the packaging vector in the packaging
cells will not be degraded by the siRNA needed to be expressed (for
example, the siRNAs selected as examples targeting siVIF037,
siPOL1102, siPOL1217, siPOL1327, siPOL2252 respectively in this
example).
[0099] The mutations of the sequences of the packaging vectors are
as follows:
TABLE-US-00005 a packaging --------GTAGACAGGATGAGGATTA--------
vector: is mutated --------GTAGACAGGACGAAGATTA--------, to: a
Packaging --------GGATTTACCACACCAGACA-------- vector: is mutated
--------GGATTTACCACCCCCGACA--------, to: a Packaging
--------GCTGGACTGTCAATGACAT-------- vector: is mutated
--------GCTGGACTGTGAACGACAT--------, to: a Packaging
--------GCACTAACAGAAGTAGTAC-------- vector: is mutated
--------GCACTAACAGAAGTGGTGC--------, to: and a Packaging
--------TAGTAGCCAGCTGTGATAA-------- vector: is mutated
--------TAGTAGCCAGCTGCGACAA--------. to:
[0100] In order to verify whether the expression of the mutated
packaging vector will be affected by the siRNA expressed by the
expression vector, co-transfection verification assay was
conducted, and the results were shown in FIG. 4. The expression of
the mutated packaging vector will not be affected by the expression
vectors pDEST-VIF037, pDEST-POL1102, pDEST-POL1217, pDEST-POL1327,
pDEST-POL2252.
Example 4
Construction of Recombinant Lentivirus Expressing siRNA and its
Efficiency of Gene Transfer for HIV Receptor Cells
[0101] Apart from the expression vector plasmid expressing the
HIV-targeting siRNA and the mutated packaging vector plasmid
pLP1-M1, other plasmids needed for the construction of the
recombinant lentivirus were pLP2 and VSVG purchased from
Invitrogen, with a product name of pLenti4/V5-DEST Gateway Vector
Kit, and a Product Number of No. V469-10.
Method for Preparing the Recombinant Lentivirus:
[0102] (1) A large amount of the four plasmids pVSVG, pLP1-M1,
pLP2, and expression vector plasmid (such as the exemplary
pDEST-VIF037 plasmid, pDEST-POL1102 plasmid, pDEST-POL1217 plasmid,
pDEST-POL1327 plasmid, pDEST-POL2252 plasmid in this example) were
extracted by cesium chloride-ethidium bromide density gradient
centrifugation (for extraction methods, please see "Molecular
cloning", J. Sambrook, D W Russell, the Science Press, 2002); (2)
293FT cells were cultured in DMEM medium (in which 10% FBS, 2 mM
L-glutamine, 0.1 mM MEM Non-Essential Amino Acids and 1%
penicillin-streptomycin were added); (3) The 293FT cells were
cultured in a cell culture plate with a diameter of 10 cm, to about
70% confluence. After 12 h, co-transfection of the 4 plasmids 10
.mu.g pLP1, 10 .mu.g pLP2, 10 .mu.g pVSVG, and 20 .mu.g expression
vector plasmid were mediated by the calcium phosphate transfection
method (for the detail of the method, please see "Molecular
cloning", J. Sambrook, D W Russell, Science Press, 2002); (4) The
supernatant of the cell culture was collected 48 hours after
transfection, and filtrated with a 0.45 .mu.m filtration membrane,
and was centrifuged with a SW28 rotor (BECKMAN company) at 25,000
rpm for 90 min at 4.quadrature.; (5) The supernatant was discarded
and the precipitate was dissolved with 500 .mu.L PBS; (6) The virus
collecting fluid was aliquoted and stored at -80.quadrature. for
later use.
[0103] MT-4 cells are human-derived CD4+ T lymphocytes, which can
support the replication of HIV-1. The MT-4 cells were transduced
with the recombinant lentiviruses Lenti-VIF037, Lenti-POL1102,
Lenti-POL1217, Lenti-POL1327, Lenti-POL2252 respectively with
moi=40, and the expression efficiency of the MGMT (P140K) gene in
the target cells was detected with immunofluorescence staining and
flow cytometry after culturing for 1 week (Table 1). The results
showed that recombinant lentivirus Lenti-VIF037, Lenti-POL 1102,
Lenti-POL1217, Lenti-POL1327, Lenti-POL2252 could effectively
transduce MT-4 cells.
TABLE-US-00006 TABLE 1 Recombinant lentivirus Transduction
efficiency for MT-4 cells Lenti-VIF037 65.02% Lenti-POL1102 71.21%
Lenti-POL1217 70.63% Lenti-POL1327 62.25% Lenti-POL2252 75.18%
[0104] Table 1 shows the transduction efficiency of recombinant
lentiviruses for the CD4+ HIV receptor cell MT-4.
Example 5
HIV Inhibition Effect of the HIV-Targeting siRNA Introduced into
the HIV Receptor Cells by Recombinant Lentivirus
[0105] The inhibitory effect of siRNA on HIV was confirmed using a
HIV in vitro cell infection model.
[0106] MT-4 cell, which is a human-derived CD4+ T lymphocyte
strain, can support the infection and replication of HIV, and can
also be used for the in vitro culture of HIV.
[0107] HIV-1.sub.NL4-5 is a B subtype, T cell-philic HIV-1 virus,
which can effectively infect MT-4 cells and replicate therein.
Challenge Test:
[0108] MT-4 cells were transduced with lentiviruses Lenti-VIF037,
Lenti-POL1102, Lenti-POL1217, Lenti-POL1327, Lenti-POL2252
respectively with moi=40, and the medium was changed after
centrifuging at 600 g for 60 min; the MT-4 cells were transduced
with control recombinant lentivirus Lenti-luc carrying siRNA
expression element targeting luciferase gene (the sequence of the
siRNA expression element targeting luciferase gene was the same as
in example 1; the control virus was prepared according to the
method in example 3 and example 4) with moi=40, and the medium was
changed after centrifuging at 600 g for 60 min;
The transduced MT-4 cells were cultured at 37.quadrature. for 48 h,
and were challenged with different doses of HIV-1.sub.NL4-3 (100 pg
and 500 pg) respectively, the medium was changed 12 h after
infection; The supernatant of the cell culture was collected at
different time points after infection, and the content of p24
protein in the supernatant of the cell culture was detected using
Murex HIV Antigen Mab (Cat. No. 8E77-02) detection kit. The
controls in the challenge test were untransduced MT-4 cells and
MT-4 cells transduced with the control recombinant lentivirus
Lenti-luc.
[0109] The results were shown in FIG. 5, the MT-4 cells transduced
with recombinant lentiviruses Lenti-VIF037, Lenti-POL1102,
Lenti-POL1217, Lenti-POL1327, Lenti-POL2252 all exhibited the
ability to inhibit the replication of HIV-1. This showed that the
anti-HIV siRNAs were expressed in the HIV receptor cells transduced
with the recombinant lentivirus vector carrying the siRNA
expression sequence targeting HIV, which resulted in a resistance
to HIV infection.
Example 6
Obtaining of the HIV Receptor Cells which can Stably Express the
siRNA Targeting HIV by Modification
[0110] Clonal selection of the MT-4 cells transduced with the
recombinant lentiviruses Lenti-VIF037, Lenti-POL1102,
Lenti-POL1217, Lenti-POL1327, Lenti-POL2252 was respectively
conducted using limiting dilution. Due to the stable integration
ability of the lentivirus vector, the HIV-targeting siRNA
expression element sequence and the drug screening gene MGMT
(P140K) expression element sequence carried by the lentivirus
vector may be integrated into the genome of the target cells.
Through screening, the expression efficiency of the MGMT (P140K)
gene in the selected cells was detected by immunofluorescense
staining and flow cytometry. The results were shown in Table 2,
MGMT (P140K) gene expression could be detected in more than 99% of
the cells after clonal selection. The modified cells were named
MT-4-VIF037 cells, MT-4-POL1102 cells, MT-4-POL1217 cells,
MT-4-POL1327 cells, MT-4-POL2252 cells respectively.
TABLE-US-00007 TABLE 2 Cells Expression Efficiency of the MGMT
(P140K) gene MT-4-VIF037 99.90% MT-4-POL1102 99.25% MT-4-POL1217
99.92% MT-4-POL1327 99.30% MT-4-POL2252 99.21%
[0111] Table 2 shows the expression efficiency of the drug
screening gene MGMT (P140K) in the modified HIV receptor cells.
Example 7
HIV Inhibition Effect of the Modified HIV Receptor Cells in High
Dosage HIV Challenge Test
[0112] The modified HIV receptor cells that can express
HIV-targeting siRNA were subjected to high dosage HIV challenge
test. In the test, challenging dosage of HIV-1.sub.NL4-3 was
increased to 2500 pg and 12500 pg respectively. The supernatant of
the cell culture was collected respectively at different time
points after challenging, and the content of p24 protein in the
supernatant of the cell culture was detected with Murex HIV Antigen
Mab (Cat. No. 8E77-02) detection kit. The results showed (FIG. 6)
that, a significant HIV inhibition effect of the modified HIV
receptor cells able to stably express the HIV-targeting siRNA
(MT-4-POL1102 cells, MT-4-POL1217 cells, MT-4-POL1327 cells,
MT-4-POL2252 cells, MT-4-VIF037 cells in the present experiment)
could be obtained comparing to the control cells. The results
demonstrated that the modified HIV receptor cells carrying the
siRNA expression element targeting HIV had the ability of
inhibiting the replication and expression of the HIV virus, and
therefore could generate resistance to HIV infection.
Example 8
Inhibitory Effect of the Chemically Synthesized siRNA on HIV
[0113] From the RNA interference targets described above, siVIF037
was selected as an example (the RNA interference target sequence
siVIF037 (SEQ ID NO.24) shown in example 2 was included herein as
an example, but other RNA interference target sequences provided by
the present invention can also be included), and a siRNA that can
target siVIF037 was synthesized, wherein the sense RNA segment of
the siRNA comprises the RNA sequence encoded by the target sequence
siVIF037 (SEQ ID NO.24) according to the present invention, and the
antisense RNA segment can form a double-stranded RNA with the sense
RNA segment (in this example, the antisense strand was perfectly
complementary to the sense strand, but a few, such as 1 or 2 or 3
or 4 mismatches can be allowed between the antisense and sense
strand), dTdT was added to the 3' ends of the sense RNA segment and
the antisense RNA segment respectively. The synthesized siRNA was
named as siR-VIF037. The siRNA targeting luciferase gene (siR-luc)
(the target sequence of the siR-luc was the same as in example 1)
was synthesized as a control with the same method.
[0114] The method for synthesizing the siRNA is briefly described
below:
the sense RNA segment and antisense RNA segment of the siRNA were
synthesized respectively by .beta.-acetonitrile-phosphoramidite
chemosynthesis with automatic DNA synthesizer, the synthesized
sense RNA segment and antisense RNA segment were mixed with equal
molar ratio, and the desired siRNA was obtained through
denaturation and annealing processes in a PCR amplifier.
Experimental Methods:
[0115] 293FT cells were cultured in 24-well cell culture plate to
about 70% confluence. After 12 h, the cells were transfected with
0.1 .mu.g/well pNL4-3 plasmid and 5 ng/well of the chemically
synthesized siRNA, with Lipofectamine 2000 (Invitrogen Cat. No
11668-027) as the transfection reagent, for the transfection
method, see the manual of the transfection reagent. The supernatant
of the cell culture was collected 48 h after co-transfection, and
after gradient dilution, the activity of the p24 protein in the
supernatant of the cell culture was detected with Murex HIV Antigen
Mab (Cat. No. 8E77-02). The inhibitory efficiency of the siR-VIF037
on HIV was calculated with the p24 protein content in the
supernatant of the 293FT cell culture as control, these 293FT cells
had been co-transfected with the siR-luc and HIV infectious cloning
plasmid.
[0116] As shown in FIG. 7, the synthesized siR-VIF037 could inhibit
the replication and expression of HIV in the cells.
Example 9
Inhibitory Effect of the Chemically Synthesized and Modified siRNA
on HIV
[0117] From the RNA interference targets described above, siVIF037
and siPOL1217 were selected as examples (the RNA interference
target sequences siVIF037 (SEQ ID NO.24) and siPOL1217 (SEQ ID
NO.10) shown in example 2 were included herein as examples, but
other RNA interference target sequences provided by the present
invention can also be included), siRNAs targeting siVIF037 and
siPOL1217 were synthesized respectively, wherein the sense RNA
segments of the siRNAs comprise the RNA sequences encoded by the
target sequences siVIF037 (SEQ ID NO.24) and siPOL1217 (SEQ ID
NO.10) of the present invention, and the antisense RNA segments can
form double-stranded RNAs with the sense RNA segments, dTdT was
added respectively at the 3' ends of the sense RNA segments and the
antisense RNA segments. At the same time, different modifications
were used in the synthesis process, wherein siRpo-VIF037 and
siRpo-POL1217 are siRNAs targeting siVIF037 and siPOL1217
respectively and were modified by 2'-OMe modification (2'-methoxy
modification) and phosphorylation (Method of synthesis: the sense
RNA segment and antisense RNA segment of the siRNA were synthesized
respectively by .beta.-acetonitrile-phosphoramidite chemosynthesis
with automatic DNA synthesizer, wherein the three bases at the 5'
end and the three bases before dTdT at the 3' end of the sense RNA
segments and antisense RNA segments were synthesized with 2'-OMe
modified single-nucleotide, and the terminal base of the 5' end of
the antisense RNA segment was subjected to phosphorylation; the
synthesized sense RNA segments and antisense RNA segments were
mixed with equal molar ratio, and the desired siRNA was obtained
through the denaturation and annealing processes in a PCR
amplifier); siRpoC-VIF037 and siRpoC-POL1217 are siRNAs targeting
siVIF037 and siPOL1217 respectively and they were modified by
2'-OMe modification and phosphorylation and sterol modification
(the synthesis method is as above, except that when the sense RNA
segment was synthesized, the three bases before dTdT at the 3' end
were not subjected to 2'-OMe modification, but a Glass supporter
comprising cholesterol-aminocaproic-acid-pyrrolidine linker was
used as a synthesis support and the bases before dTdT at the 3' end
was linked to cholesterol group through phosphorothioate, the
remaining steps were the same as the method described above for
synthesizing siRpo-VIF037 and siRpo-POL1217). At the same time,
siRNAs (siRpo-Nk and siRpoC-Nk) with the same modifications
targeting the unrelated RNA interference target siRNA-Nk (the
sequence is the same as in example 1) not matching with HIV and
human genes were synthesized as control. The 2'-OMe modification
and/or phosphorylation and/or sterol modification made to the
synthesized siRNAs targeting the RNA interference target sequences
siVIF037 (SEQ ID NO.24) and siPOL1217 (SEQ ID NO.10) shown in
example 2 were included herein as examples, but other modifications
of different types made to the synthesized siRNA targeting other
RNA interference target sequences provided by the present invention
can also be included.
Experimental Methods:
[0118] 293FT cells were cultured in 24-well cell culture plate to
about 70% confluence. After 12 h, the cells were transfected with
0.1 .mu.g/well pNL4-3 plasmid and 5 ng/well of the synthesized and
modified siRNA, with Lipofectamine 2000 (Invitrogen Cat. No
11668-027) as the transfection reagent, for the transfection
method, see the manual of the transfection reagent. The supernatant
of the cell culture was collected 48 h after co-transfection, and
after gradient dilution, the activity of the p24 protein in the
supernatant of the cell culture was detected with Murex HIV Antigen
Mab (Cat. No. 8E77-02). The inhibitory efficiency of siRpo-VIF037,
siRpo-POL1217, siRpoC-VIF037, siRpoC-POL1217 on HIV was calculated
with the p24 protein content in the supernatant of the 293FT cell
culture as control, these 293FT cells had been co-transfected with
siRpo-Nk, siRpoC-Nk, and pNL4-3 plasmid.
[0119] The results are shown in FIG. 8, in which the synthesized
siRpo-VIF037, siRpo-POL1217, siRpoC-VIF037, siRpoC-POL1217 all
could inhibit the replication and expression of HIV in the
cells.
Example 10
Examination of the Inhibitory Effect of the siRNA on HIV in the
H.sup.2K-PBL-SCID Mouse Model
[0120] From the RNA interference targets described above, siVIF037
was selected as an example (the RNA interference target sequence
siVIF037 (SEQ ID NO.24) shown in example 2 was included herein as
an example, but other RNA interference target sequences provided by
the present invention can also be included), the plasmid
pDEST-H.sup.2K-VIF037 having the expression cassette of the
H.sup.2K gene and an expression cassette able to express the siRNA
targeting siVIF037 was constructed based on the lentivirus
expression vector plasmid pDEST-MR (see example 3). At the same
time, the expression cassette in the plasmid pDEST-H.sup.2K-VIF037
expressing the siRNA targeting siVIF037 was further substituted
with an expression cassette expressing the siRNA targeting
luciferase (with the same target sequence as in example 1), and the
pDEST-H.sup.2K-luc plasmid was obtained as control. With
pDEST-H.sup.2K-VIF037 and pDEST-H.sup.2K-luc, lentiviruses
Lenti-H.sup.2K-VIF037 and Lenti-H.sup.2K-luc were prepared
respectively (see the method in example 4).
[0121] 40 ml of human whole blood was taken, and PBMC cells were
isolated using Ficoll-paque plus (GE Healthcare cat. NO 17-1440-03)
(for the method, please see the operation manual), and cultured in
the AIM-V medium (GIBCO cat. NO 12055) with the stimulation of PHA
for 48 h. Then, the PHA-stimulated PBMC cells were transduced with
lentiviruses Lenti-H.sup.2K-VIF037 and Lenti-H.sup.2K-luc (the
method for transduction is the same as in example 5). After
transduction, the cells were cultured in AIM-V medium containing
IL-2 for another 72 h.
[0122] The above cells infected by lentiviruses
Lenti-H.sup.2K-VIF037 and Lenti-H.sup.2K-luc and stably expressing
the exogenous marker gene H.sup.2K were isolated respectively with
the MAcSelect Kk transfected cell selection kit (Miltenyi Biotec
cat. NO 130-091-986) (for the method, see the operation manual).
The selected positive cells were cultured in the AIM-V medium
containing IL-2 for another 96 h.
[0123] Severe combined immune deficiency (SCID) mice (sterile
grade, 7 to 8 weeks old, the weight was about 16.about.20 g, three
mice per group) were injected intraperitoneally with 0.5 ml
paraffin oil 1 week before they were used, and then each mouse was
injected intraperitoneally with the PBMC cells (5.times.10.sup.5/g
body weight) obtained from the above steps to obtain
H.sup.2K-PBL-SCID chimeric mice. If the human PBMC cells in the
mice were derived from the cells transduced with
Lenti-H.sup.2K-VIF037, then these mice were named as VIF037
chimeric mice for short; if the human PBMC cells were derived from
cells transduced with Lenti-H.sup.2K-luc, then these mice were
called luc chimeric mice for short.
[0124] HIV-1.sub.NL4-3 was injected intraperitoneally into the
VIF037 chimeric mice and the luc chimeric mice respectively after
12 h. HIV-1.sub.NL4-5 is the B subtype, T cell-philic HIV-1 virus.
7d and 14d after virus infection, 200 .mu.L blood was collected by
removing the eyeball, and after gradient dilution, the activity of
p24 protein in the supernatant of the cell culture was detected
with Murex HIV Antigen Mab (Cat. No, 8E77-02). The activity of the
p24 protein in luc chimeric mice was served as the experimental
control.
[0125] The results were shown in FIG. 9, in which the VIF037
chimeric mice had the ability to inhibit the replication of
HIV.
[0126] Those skilled in the art should know that although specific
embodiments of the invention were described for the purpose of
exemplary illustration, various modifications can be made without
departing from the spirit and scope of the present invention.
Therefore, the scope of the present invention should not be viewed
to be limited by the embodiments and examples of the present
invention. The scope of the present invention is only limited by
the claims attached below. All the documents referred to by the
present application are incorporated herein by reference in their
entirety.
Sequence CWU 1
1
32119DNAArtificial SequenceRNA interference target sequence
targeting HIV 1aaattggatg acagaaacc 19219DNAArtificial SequenceRNA
interference target sequence targeting HIV 2ctgaagcaat gagccaagt
19321DNAArtificial SequenceRNA interference target sequence
targeting HIV 3gattgtactg agagacaggc t 21419DNAArtificial
SequenceRNA interference target sequence targeting HIV 4tggaaaggat
caccagcaa 19519DNAArtificial SequenceRNA interference target
sequence targeting HIV 5aggatcacca gcaatattc 19619DNAArtificial
SequenceRNA interference target sequence targeting HIV 6gcaatattcc
agtgtagca 19719DNAArtificial SequenceRNA interference target
sequence targeting HIV 7gtatgtagga tctgactta 19819DNAArtificial
SequenceRNA interference target sequence targeting HIV 8ggatttacca
caccagaca 19919DNAArtificial SequenceRNA interference target
sequence targeting HIV 9gaaagaacct ccattcctt 191019DNAArtificial
SequenceRNA interference target sequence targeting HIV 10gctggactgt
caatgacat 191119DNAArtificial SequenceRNA interference target
sequence targeting HIV 11ctgtcaatga catacagaa 191219DNAArtificial
SequenceRNA interference target sequence targeting HIV 12ccggtacatg
gagtgtatt 191319DNAArtificial SequenceRNA interference target
sequence targeting HIV 13ggagtgtatt atgacccat 191419DNAArtificial
SequenceRNA interference target sequence targeting HIV 14ggccaatgga
catatcaaa 191519DNAArtificial SequenceRNA interference target
sequence targeting HIV 15cccacactaa tgatgtgaa 191619DNAArtificial
SequenceRNA interference target sequence targeting HIV 16cccacactaa
tgatgtgaa 191719DNAArtificial SequenceRNA interference target
sequence targeting HIV 17cactaatgat gtgaaacaa 191822DNAArtificial
SequenceRNA interference target sequence targeting HIV 18acactaatga
tgtgaaacaa tt 221919DNAArtificial SequenceRNA interference target
sequence targeting HIV 19gaagttatgg taccagtta 192019DNAArtificial
SequenceRNA interference target sequence targeting HIV 20cccataatag
gagcagaaa 192119DNAArtificial SequenceRNA interference target
sequence targeting HIV 21tcagagttag tcagtcaaa 192219DNAArtificial
Sequencetcagagttag tcagtcaaa 22tagtagccag ctgtgataa
192319DNAArtificial SequenceRNA interference target sequence
targeting HIV 23cagatggcag gtgatgatt 192419DNAArtificial
SequenceRNA interference target sequence targeting HIV 24gtagacagga
tgaggatta 192519DNAArtificial SequenceRNA interference target
sequence targeting HIV 25tagacaggat gaggattaa 192619DNAArtificial
SequenceRNA interference target sequence targeting HIV 26tcaggccata
tcacctaga 192719DNAArtificial SequenceRNA interference target
sequence targeting HIV 27aataggatgg atgacacat 192819DNAArtificial
SequenceRNA interference target sequence targeting HIV 28ggagccgata
gacaaggaa 192919DNAArtificial SequenceRNA interference target
sequence targeting HIV 29gcactaacag aagtagtac 193019DNAArtificial
SequenceRNA interference target sequence targeting HIV 30tatttcaagg
aaagctaag 193119DNAArtificial SequenceRNA interference target
sequence targeting HIV 31tttcagaatc tgctataag 193219DNAArtificial
SequenceRNA interference target sequence targeting HIV 32gagtgaagga
gaagtatca 19
* * * * *