WO2023125823A1 - 靶向HIV的siRNA和shRNA及其相应的组合、表达盒、细胞及其应用 - Google Patents

靶向HIV的siRNA和shRNA及其相应的组合、表达盒、细胞及其应用 Download PDF

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WO2023125823A1
WO2023125823A1 PCT/CN2022/143442 CN2022143442W WO2023125823A1 WO 2023125823 A1 WO2023125823 A1 WO 2023125823A1 CN 2022143442 W CN2022143442 W CN 2022143442W WO 2023125823 A1 WO2023125823 A1 WO 2023125823A1
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targeting
sequence
seq
shrna
gene
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French (fr)
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李萃
张悦
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北京三诺佳邑生物技术有限责任公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine, in particular to HIV-targeting RNA or a combination thereof, siRNA or a combination thereof, shRNA or a combination thereof, an shRNA coding sequence or a combination thereof, expression cassettes, vectors, cells, pharmaceutical compositions and applications thereof.
  • HIV Human Immunodeficiency virus
  • HIV belongs to the Retroviridae family and is an RNA retrovirus, an enveloped virus composed of two single-stranded positive-sense RNA molecules (+ssRNA). After the HIV virus invades the cell, the viral RNA is reverse-transcribed into single-stranded cDNA under the action of its own reverse transcriptase, and double-stranded cDNA is synthesized in the host cell, integrated with the chromosome of the host cell, and produced by transcription and synthesis system of the host cell Pathogenic mRNA/gRNA.
  • CD4-positive cells such as CD4 + T cells.
  • CD4 + T cells The main target of HIV is CD4-positive cells, such as CD4 + T cells.
  • anti-AIDS drugs approved for clinical use in the world.
  • None of the existing drugs can completely remove the HIV virus in the human body.
  • the "cocktail therapy" effect of several drug combinations will be better than single drug treatment, but it cannot completely eliminate HIV virus. After drug treatment, HIV can still reactivate and expand in CD4+ cells carrying HIV genome.
  • RNA interference is a mechanism to inhibit gene expression, through the specific RNA degradation process induced by double-stranded short interfering RNA (siRNA), which occurs after gene transcription, that is, the regulation of genes at the mRNA level, which can efficiently Specifically block the expression of the target gene.
  • siRNA small interfering RNA
  • siRNA sometimes called short interfering RNA (short interfering RNA) or silencing RNA (silencing RNA)
  • siRNA short interfering RNA
  • silencing RNA silencing RNA
  • shRNA is the abbreviation of short hairpin RNA, translated as "short hairpin RNA", shRNA includes two short inverted repeat sequences. It is currently known that shRNA is mainly involved in the phenomenon of RNA interference (RNAi), regulating gene expression in a specific manner. After the shRNA enters the cell, it is unwound by the RNA helicase in the host cell into a sense RNA strand and an antisense RNA strand.
  • RNAi RNA interference
  • RISC silencing complex
  • siRNAs nef, tat, gag, vif, env, rev, LTR
  • this method of interference is only effective in the short term.
  • siRNAs Due to the high mutation rate of the HIV viral genome and the high specificity of siRNAs, these siRNAs are easily lost, making it difficult for RNAi to inhibit HIV replication in the long term (Das et al., 2004; Westerhout et al., 2005).
  • the present invention provides an HIV-targeting RNA, siRNA, shRNA or shRNA coding sequence, which can significantly and effectively inhibit the replication of HIV strains and drug-resistant mutant strains.
  • the present invention also provides a combination of RNA, siRNA, shRNA or shRNA coding sequences, which can not only efficiently inhibit the replication of HIV strains or drug-resistant mutant virus strains, but also effectively inhibit the escape of HIV for a long time.
  • the production of HIV mutant strains has a good therapeutic effect on HIV-positive patients.
  • Exemplary RNAs, siRNAs, shRNAs and their coding sequences involved in the present invention are shown in Table 1 and Table 2.
  • the present invention provides an HIV-targeting RNA sequence, which targets the sequence shown in any one of SEQ ID NO: 1-5.
  • the RNA sequence provided by the invention is shown in any one of SEQ ID NO: 6-10 or 20-24.
  • the present invention provides an siRNA targeting HIV, the siRNA includes a sense strand and an antisense strand reversely complementary thereto, consisting of the following sequence:
  • the sense strand sequence is as shown in SEQ ID NO: 9, and the antisense strand sequence is as shown in SEQ ID NO: 23; or
  • the present invention provides shRNA targeting HIV or its coding sequence
  • the sequence of the shRNA includes:
  • sequence of the shRNA is as shown in any one of SEQ ID NO: 25-29.
  • the coding sequence of the shRNA is as shown in any one of SEQ ID NO: 11-15.
  • the present invention also provides combinations of RNAs as described above.
  • the RNA combination comprises:
  • RNA sequence targeting the Gag gene and the RNA sequence targeting the LTR gene, wherein the RNA sequence targeting the Gag gene is shown in SEQ ID NO: 6 or 20, and the RNA sequence targeting the LTR gene is as shown in SEQ ID NO: 9 or 23;
  • RNA sequence targeting the Gag gene and the RNA sequence targeting the LTR gene, wherein the RNA sequence targeting the Gag gene is shown in SEQ ID NO: 7 or 21, and the RNA sequence targeting the LTR gene is as shown in SEQ ID NO: 9 or 23;
  • RNA sequence targeting the Gag gene and the RNA sequence targeting the LTR gene, wherein the RNA sequence targeting the Gag gene is shown in SEQ ID NO: 8 or 22, and the RNA sequence targeting the LTR gene is as shown in SEQ ID NO: 9 or 23; or
  • RNA sequence targeting the Gag gene and the RNA sequence targeting the Nef gene, wherein the RNA sequence targeting the Gag gene is shown in SEQ ID NO: 7 or 21, and the RNA sequence targeting the Nef gene is as shown in SEQ ID NO: 10 or 24.
  • the present invention also provides combinations of siRNAs as described above.
  • the siRNA combination comprises:
  • siRNA targeting the Gag gene and siRNA targeting the LTR gene wherein the sense strand sequence of the siRNA targeting the Gag gene is shown in SEQ ID NO: 6, and the antisense strand sequence is shown in SEQ ID NO: 20, The sense strand sequence of the siRNA targeting the LTR gene is shown in SEQ ID NO: 9, and the antisense strand sequence is shown in SEQ ID NO: 23;
  • siRNA targeting the Gag gene and siRNA targeting the LTR gene wherein the sense strand sequence of the siRNA targeting the Gag gene is shown in SEQ ID NO: 7, and the antisense strand sequence is shown in SEQ ID NO: 21,
  • the sense strand sequence of the siRNA targeting the LTR gene is shown in SEQ ID NO: 9, and the antisense strand sequence is shown in SEQ ID NO: 23;
  • siRNA targeting the Gag gene and siRNA targeting the LTR gene wherein the sense strand sequence of the siRNA targeting the Gag gene is shown in SEQ ID NO: 8, and the antisense strand sequence is shown in SEQ ID NO: 22,
  • the sense strand sequence of the siRNA targeting the LTR gene is shown in SEQ ID NO: 9, and the antisense strand sequence is shown in SEQ ID NO: 23; or
  • siRNA targeting the Gag gene and siRNA targeting the Nef gene wherein the sense strand sequence of the siRNA targeting the Gag gene is shown in SEQ ID NO: 7, and the antisense strand sequence is shown in SEQ ID NO: 21,
  • the sense strand sequence of the siRNA targeting the Nef gene is shown in SEQ ID NO: 10
  • the antisense strand sequence is shown in SEQ ID NO: 24.
  • the present invention also provides combinations of shRNAs targeting HIV.
  • the shRNA combination comprises:
  • the present invention also provides combinations of shRNA coding sequences as described above.
  • the combination of the shRNA coding sequence comprises:
  • the present invention also provides an expression cassette comprising the above coding sequence.
  • the present invention also provides a vector comprising the above coding sequence, or the above expression cassette.
  • the present invention also provides cells comprising the above-mentioned RNA or a combination thereof, siRNA or a combination thereof, shRNA or a combination thereof, an shRNA coding sequence or a combination thereof, an expression cassette or a vector.
  • the present invention also provides a pharmaceutical composition comprising the above-mentioned RNA or a combination thereof, siRNA or a combination thereof, shRNA or a combination thereof, an shRNA coding sequence or a combination thereof, an expression cassette, a vector or a cell.
  • the shRNA coding sequence described in the present invention is a DNA sequence.
  • Synthetic Created in a laboratory by artificial means, for example synthetic nucleic acids can be chemically synthesized in a laboratory.
  • Isolated An "isolated" biological component (e.g., a nucleic acid molecule, protein, virus, or cell) has been removed from other biological components in the cells or tissues of an organism in which the component occurs naturally, or in the organism itself (e.g. other chromosomal and extrachromosomal DNA and RNA, proteins and cells) are substantially isolated or purified.
  • Nucleic acid molecules and proteins that have been "isolated” include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acid molecules and proteins.
  • a recombinant nucleic acid molecule refers to a nucleic acid molecule that has a sequence that does not occur in nature, or that has a sequence prepared by the artificial combination of two sequence fragments that would otherwise be separate. This artificial combination can be achieved by chemical synthesis or by artificial manipulation (eg, by genetic engineering techniques) of isolated nucleic acid molecule fragments.
  • a recombinant virus is a virus that contains non-naturally occurring sequences or sequences prepared by the artificial combination of sequences from at least two different sources.
  • the term “recombinant” also includes nucleic acids, proteins and viruses that are altered only by the addition, substitution or deletion of a portion of a native nucleic acid molecule, protein or virus.
  • recombinant SIV refers to a SIV particle in which a recombinant nucleic acid molecule (eg, shRNA-encoding recombinant nucleic acid molecule) is packaged.
  • Sequence identity The identity or similarity between two or more nucleic acid sequences or between two or more amino acid sequences is expressed in terms of the identity or similarity between their sequences. Sequence identity can be measured in terms of percent identity; the higher the percent, the more identical the sequences. Sequence similarity can be measured in terms of percentage similarity (taking into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity/similarity when aligned using standard methods.
  • orthologous protein or cDNA comes from a more closely related species (for example, human and mouse sequences) than when it comes from a more distantly related species (for example, human and nematode (C. elegans) sequences), this The homology is more pronounced.
  • the length of the sequence identity comparison can be over the full length of the genome, the full length of the coding sequence of a gene, or fragments of at least about 500 to 5000 nucleotides are desired. However, in smaller fragments (e.g., having at least about 9 nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides)
  • the identity of can also be desired.
  • identity refers to "identity", “homology” or “similarity” between two different sequences.
  • An “aligned” sequence or “alignment” refers to a plurality of nucleic acid sequences or protein (amino acid) sequences, which typically contain corrections for missing or additional bases or amino acids compared to a reference sequence.
  • Sequence alignment programs are available for amino acid sequences, such as the "Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME” and “Match-Box” programs. Typically, any of these programs are used with default settings, although those skilled in the art can change these settings as desired. Alternatively, one skilled in the art may employ another algorithm or computer program that provides at least the level of identity or alignment as provided by the reference algorithm or program. See, eg, J.D. Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).
  • Multiple sequence alignment programs can also be used with nucleic acid sequences. Examples of such programs include “Clustal W”, “CAPSequence Assembly”, “BLAST”, “MAP” and “MEME”, which can be accessed through a Web server on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, also use the Vector NTI app. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, Fasta TM (a program in GCG Version 6.1) can be used to compare polynucleotide sequences. Fasta( TM) provides an alignment and percent sequence identity of the region of best overlap between the query and search sequences. For example, Fasta TM can be used to determine percent sequence identity between nucleic acid sequences using its default parameters (word size of 6, and NOPAM factor for scoring matrix) provided in GCG Version 6.1 (herein incorporated by reference) sex.
  • a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, when necessary to join two protein coding regions, in the same reading frame.
  • a vector is a nucleic acid molecule that allows the insertion of foreign nucleic acid without disrupting the vector's ability to replicate and/or integrate in a host cell.
  • a vector may contain nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication.
  • a vector may also contain one or more selectable marker genes and other genetic elements.
  • An expression vector is a vector that contains the necessary regulatory sequences to allow the transcription and translation of an inserted gene. In some embodiments herein, the vector is a SIV vector.
  • Replication deficient virus or "viral vector” means a synthetic or artificial virion in which the expression cassette containing the relevant gene is packaged in a viral capsid or envelope, in which any Viral genome sequences are replication deficient; that is, they are unable to produce progeny virions, but retain the ability to infect target cells.
  • the genome of the viral vector does not include genes encoding the enzymes required for replication (the genome can be engineered to be "no content" - containing only relevant genes flanking the signals required for amplification and packaging of the artificial genome). genetically modified), but these genes can be provided during production. Therefore, it is considered safe for use in gene therapy since replication and infection by progeny virions does not occur except in the presence of viral enzymes required for replication.
  • Such replication defective viruses may be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrating or non-integrating) or another suitable source of virus.
  • “Lentivirus” belongs to the subgenus of retrovirus, represented by human immunodeficiency virus (HIV), which can infect dividing cells and integrate into its genome, and also has the ability to infect neuronal cells, macrophages, liver cells, cardiomyocytes and capacity of stem cells.
  • "Lentiviral vector” is a viral vector derived from primate immunodeficiency virus, which has been widely used in gene therapy research as an effective tool for gene transfer. The lentiviral vector carrying the exogenous gene realizes the expression of the exogenous gene in the cell or living tissue by infecting the cell or living tissue.
  • Exemplary lentiviral vectors include primate recombinant lentiviral vectors, such as human immunodeficiency virus (human immunodeficiency virus, HIV) or simian immunodeficiency virus (simian immunodeficiency virus, SIV), or non-primate recombinant lentiviral vector.
  • "Simian immunodeficiency virus (SIV) vector” refers to a vector in which the sequence necessary for the function of a viral vector is a sequence derived from the SIV gene in the nucleic acid molecule in the virus particle, and the necessary sequence refers to the sequence from the 5' end in sequence. The second is the R region of 5'LTR, U5 region, packaging signal ( ⁇ ), RRE, U3 region and R region sequences outside the promoter region of 3'LTR. .
  • Promoter A region of DNA that directs/causes the transcription of a nucleic acid (eg, gene).
  • a promoter includes the necessary nucleic acid sequences near the start site of transcription. Typically, a promoter is located near the gene it transcribes.
  • a promoter region also optionally includes distal enhancer or repressor elements, which can be located as many as several thousand base pairs from the start site of transcription.
  • Pharmaceutically acceptable carriers that can be used in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.
  • Preventing a disease (eg, HIV infection) means inhibiting the full onset of the disease.
  • Treatment refers to a therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after the onset of the disease.
  • “Ameliorating” means reducing the number or severity of signs or symptoms of disease.
  • Administration/administration Providing or administering an agent, such as a therapeutic agent (eg, recombinant SIV), to a subject by an effective route.
  • a therapeutic agent eg, recombinant SIV
  • routes of administration include, but are not limited to, injection (eg, subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intracatheter, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.
  • Therapeutically effective amount The amount of a particular drug or therapeutic agent (eg, recombinant SIV) sufficient to achieve the desired effect in a subject or cell treated with the agent.
  • An effective amount of an agent depends on a variety of factors including, but not limited to, the subject or cell being treated, and the mode of administration of the therapeutic composition.
  • Subject Living multicellular vertebrate organisms, including classes of humans and non-human mammals.
  • the first aspect of the present invention provides an HIV-targeting RNA sequence, wherein the RNA sequence targets the sequence shown in any one of SEQ ID NO: 1-5.
  • RNA sequence is shown in any one of SEQ ID NO: 6-10 or 20-24.
  • the second aspect of the present invention provides an siRNA targeting HIV, the siRNA includes a sense strand and an antisense strand reversely complementary thereto, wherein the siRNA targets Gag gene, LTR gene and/or or the Nef gene.
  • the siRNA targets the sequence shown in any one of SEQ ID NO: 1-5.
  • the positive-sense strand sequence of the siRNA targeting HIV's Gag gene is selected from a sequence consisting of consecutive 19-23 nucleotides in the sequence shown in any one of SEQ ID NO: 1-3;
  • the positive-sense strand sequence of the siRNA targeting the LTR gene of HIV is selected from the sequence consisting of consecutive 19-26 nucleotides in SEQ ID NO: 4;
  • the positive-sense strand sequence of the siRNA targeting the Nef gene of HIV is selected from the sequence of SEQ ID NO: 5 A sequence consisting of 19-25 nucleotides in a row.
  • the antisense strand of the siRNA is a reverse complementary sequence to the sense strand.
  • the siRNA targeting the Gag gene consists of the following sequence:
  • the sense strand sequence of the siRNA targeting the LTR gene is shown in SEQ ID NO: 9
  • the antisense strand sequence is shown in SEQ ID NO: 23.
  • the sense strand sequence of the siRNA targeting Nef gene is shown in SEQ ID NO: 10
  • the antisense strand sequence is shown in SEQ ID NO: 24.
  • the third aspect of the present invention provides a shRNA and its coding sequence, said shRNA targets Gag gene, LTR gene and/or Nef gene of HIV.
  • the shRNA is an shRNA formed by linking the sense strand sequence of the above-mentioned siRNA and the reverse complementary antisense strand sequence through an intermediate non-complementary linking sequence, wherein the sense strand sequence and the antisense strand sequence Constitute the stem region in the stem-loop structure, and the non-complementary linking sequence in the middle constitutes the loop region in the stem-loop structure.
  • the shRNA is processed in vivo to produce siRNA as described above.
  • the sequence of the shRNA targeting the Gag gene comprises:
  • the sequence of the shRNA targeting the LTR gene includes the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 23.
  • the sequence of the shRNA targeting the Nef gene includes the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 24.
  • the shRNA sequence is shown in any one of SEQ ID NO: 25-29; the coding sequence of the shRNA is shown in any one of SEQ ID NO: 11-15.
  • RNA sequences targeting HIV is also provided.
  • the combination includes at least one of the aforementioned RNA sequences.
  • the combination includes at least two of the RNA sequences targeting the Gag gene, the RNA sequences targeting the LTR gene, or the RNA sequences targeting the Nef gene.
  • the combination of RNA sequences targeting HIV comprises:
  • RNA sequences targeting the Gag gene and RNA sequences targeting the Nef gene (ii) RNA sequences targeting the Gag gene and RNA sequences targeting the Nef gene, or
  • the RNA sequence of the Gag gene targeting HIV is selected from a sequence consisting of consecutive 19-23 nucleotides in the sequence shown in any one of SEQ ID NO: 1-3 or reverse complementary thereto The sequence, more preferably, the sequence shown in any one of SEQ ID NO: 6-8 or 20-22;
  • the RNA sequence targeting the LTR gene of HIV is selected from the continuous 19-26 in SEQ ID NO: 4 A sequence composed of nucleotides or a reverse complementary sequence thereto, more preferably, the sequence shown in SEQ ID NO: 9 or 23;
  • the RNA sequence targeting the Nef gene of HIV is selected from the sequence in SEQ ID NO: 5 A sequence consisting of 19-25 nucleotides or a reverse complementary sequence thereto, more preferably, the sequence shown in SEQ ID NO: 10 or 24.
  • the HIV-targeting RNA sequence includes:
  • RNA sequence targeting the Gag gene and the RNA sequence targeting the LTR gene, wherein the RNA sequence targeting the Gag gene is shown in SEQ ID NO: 6 or 20, and the RNA sequence targeting the LTR gene is as shown in SEQ ID NO: 9 or 23;
  • RNA sequence targeting the Gag gene and the RNA sequence targeting the LTR gene, wherein the RNA sequence targeting the Gag gene is shown in SEQ ID NO: 7 or 21, and the RNA sequence targeting the LTR gene is as shown in SEQ ID NO: 9 or 23;
  • RNA sequence targeting the Gag gene and the RNA sequence targeting the LTR gene, wherein the RNA sequence targeting the Gag gene is shown in SEQ ID NO: 8 or 22, and the RNA sequence targeting the LTR gene is as shown in SEQ ID NO: 9 or 23; or
  • RNA sequence targeting the Gag gene and the RNA sequence targeting the Nef gene, wherein the RNA sequence targeting the Gag gene is shown in SEQ ID NO: 7 or 21, and the RNA sequence targeting the Nef gene is as shown in SEQ ID NO: 10 or 24.
  • the fifth aspect of the present invention also provides an siRNA combination targeting HIV.
  • the combination includes at least one of the aforementioned siRNAs.
  • the siRNA combination includes at least two of the siRNA targeting the Gag gene, the siRNA targeting the LTR gene or the siRNA targeting the Nef gene.
  • the siRNA targeting HIV comprises:
  • siRNA targeting the Gag gene and siRNA targeting the LTR gene (i) siRNA targeting the Gag gene and siRNA targeting the LTR gene
  • siRNA targeting the Gag gene and siRNA targeting the Nef gene (ii) siRNA targeting the Gag gene and siRNA targeting the Nef gene, or
  • siRNA targeting LTR gene and siRNA targeting Nef gene (iii) siRNA targeting LTR gene and siRNA targeting Nef gene.
  • the positive-sense strand sequence of the siRNA targeting the Gag gene is selected from a sequence consisting of consecutive 19-23 nucleotides in the sequence shown in any one of SEQ ID NO: 1-3, preferably as SEQ ID NO :
  • the antisense strand sequence is a sequence reversely complementary to the sense strand
  • the siRNA sense strand sequence targeting the LTR gene is selected from the consecutive 19-26 cores in SEQ ID NO: 4
  • the antisense strand sequence is a sequence reversely complementary to the sense strand
  • the sense strand sequence of the siRNA targeting the Nef gene is selected from the continuous sequences in SEQ ID NO: 5 A sequence consisting of 19-25 nucleotides, preferably the sequence shown in SEQ ID NO: 10, the antisense strand sequence is a reverse complementary sequence to the sense strand.
  • the HIV-targeting siRNA combination includes:
  • siRNA targeting the Gag gene and siRNA targeting the LTR gene wherein the sense strand sequence of the siRNA targeting the Gag gene is shown in SEQ ID NO: 6, and the antisense strand sequence is shown in SEQ ID NO: 20, The sense strand sequence of the siRNA targeting the LTR gene is shown in SEQ ID NO: 9, and the antisense strand sequence is shown in SEQ ID NO: 23;
  • siRNA targeting the Gag gene and siRNA targeting the LTR gene wherein the sense strand sequence of the siRNA targeting the Gag gene is shown in SEQ ID NO: 7, and the antisense strand sequence is shown in SEQ ID NO: 21,
  • the sense strand sequence of the siRNA targeting the LTR gene is shown in SEQ ID NO: 9, and the antisense strand sequence is shown in SEQ ID NO: 23;
  • siRNA targeting the Gag gene and siRNA targeting the LTR gene wherein the sense strand sequence of the siRNA targeting the Gag gene is shown in SEQ ID NO: 8, and the antisense strand sequence is shown in SEQ ID NO: 22,
  • the sense strand sequence of the siRNA targeting the LTR gene is shown in SEQ ID NO: 9, and the antisense strand sequence is shown in SEQ ID NO: 23; or
  • the sixth aspect of the present invention also provides a combination of shRNA targeting HIV or a combination of shRNA coding sequences.
  • the shRNA combination includes at least one of the aforementioned shRNAs.
  • the combination of shRNA coding sequences includes at least one of the aforementioned shRNA coding sequences.
  • the shRNA combination or the combination of shRNA coding sequences includes the shRNA targeting the Gag gene or its coding sequence, the shRNA targeting the LTR gene or its coding sequence, or the targeting Nef gene. shRNA or at least two of its coding sequences.
  • the HIV-targeting shRNA combination or shRNA coding sequence combination includes: (i) shRNA targeting Gag gene or its coding sequence, and shRNA targeting LTR gene or its coding sequence , or (ii) the shRNA targeting the Gag gene or its coding sequence, and the shRNA targeting the Nef gene or its coding sequence, or (iii) the shRNA targeting the LTR gene or its coding sequence, and the shRNA targeting the Nef gene or its coding sequence.
  • the sequence of the shRNA targeting the Gag gene comprises: (i) the sequence shown in SEQ ID NO: 6 and SEQ ID NO: 20; (ii) the sequence shown in SEQ ID NO: 7 and SEQ ID NO: 21 or (iii) the sequence shown in SEQ ID NO: 8 and SEQ ID NO: 22; the sequence of the shRNA targeting the LTR gene includes the sequence shown in SEQ ID NO: 9 and SEQ ID NO: 23; the targeting The sequence of the shRNA of the Nef gene includes the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 24.
  • the sequence of the shRNA targeting the Gag gene is shown in any one of SEQ ID NO: 25-27; the sequence of the shRNA targeting the LTR gene is shown in SEQ ID NO: 28; the sequence of the shRNA targeting the Nef gene The sequence of the shRNA is shown in SEQ ID NO: 29.
  • the coding sequence of the shRNA targeting the Gag gene is shown in any one of SEQ ID NO: 11-13; the coding sequence of the shRNA targeting the LTR gene is shown in SEQ ID NO: 14; the target The coding sequence of the shRNA to the Nef gene is shown in SEQ ID NO: 15.
  • the HIV-targeting shRNA combination or shRNA coding sequence combination includes:
  • the shRNA targeting the Gag gene or its coding sequence and the shRNA targeting the LTR gene or its coding sequence, wherein the shRNA targeting the Gag gene includes sequences shown in SEQ ID NO: 6 and SEQ ID NO: 20, the target The shRNA to the LTR gene includes sequences shown in SEQ ID NO: 9 and SEQ ID NO: 23;
  • the shRNA targeting the Gag gene or its coding sequence includes sequences shown in SEQ ID NO: 7 and SEQ ID NO: 21, the target The shRNA to the LTR gene includes sequences shown in SEQ ID NO: 9 and SEQ ID NO: 23;
  • the shRNA targeting the Gag gene or its coding sequence includes sequences shown in SEQ ID NO: 8 and SEQ ID NO: 22, the target The shRNA to the LTR gene includes sequences shown in SEQ ID NO: 9 and SEQ ID NO: 23; or
  • the HIV-targeting shRNA combination or shRNA coding sequence combination includes:
  • the seventh aspect of the present invention provides an expression cassette, wherein the expression cassette can express the shRNA as described above.
  • the expression cassette comprises the coding sequence or a combination of coding sequences of the aforementioned shRNA.
  • the expression cassette comprises a coding sequence of shRNA or a combination thereof, and the shRNA comprises:
  • the expression cassette comprises a sequence as shown in any one of SEQ ID NO: 11-15 or a combination thereof.
  • the expression cassette further comprises a promoter.
  • the promoter is operably linked to the above coding sequence.
  • the promoter used in the present invention can be any RNA polymerase II promoter or RNA polymerase III promoter; preferably an RNA polymerase III promoter.
  • the RNA polymerase III promoter includes H1 promoter, U6 promoter, 7SK promoter.
  • the RNA polymerase II promoter or RNA polymerase III promoter also includes any synthetic or engineered DNA fragment.
  • the promoter sequence contained in the expression cassette is selected from H1 and/or U6 promoters.
  • the shRNA targeting Gag is expressed by the H1 promoter.
  • the coding sequence of the Gag-targeting shRNA is operably linked to the H1 promoter.
  • the expression of shRNA targeting LTR or Nef is driven by U6 promoter.
  • the coding sequence of the shRNA targeting LTR or Nef is operably linked to the U6 promoter.
  • the eighth aspect of the present invention provides a vector comprising the RNA or its combination according to the present invention, siRNA or its combination, or comprising the shRNA or its combination according to the present invention, or the shRNA coding sequence or its combination; or comprising an expression cassette according to the present invention.
  • the vector contains the combination of RNAs described in the present invention, and the combination of RNAs is contained in the same vector. In another embodiment of the invention, said combination of RNAs are contained in different vectors.
  • the vector contains the combination of siRNAs described in the present invention, and the combination of RNAs is contained in the same vector. In another embodiment of the invention, said combination of RNAs are contained in different vectors.
  • the vector comprises a combination of shRNA coding sequences described in the present invention, and the combination of shRNA coding sequences is contained in the same vector. In another embodiment of the present invention, the combination of shRNA coding sequences is contained in different vectors.
  • the vector comprises one or more expression cassettes described in the present invention.
  • the multiple expression cassettes are included in the same vector.
  • said plurality of expression cassettes are contained in different vectors.
  • the carrier comprises:
  • expression cassette 1 it comprises the coding sequence of the shRNA of H1 promotor and targeting Gag, and described coding sequence is as shown in any one in SEQ ID NO:11-13;
  • expression cassette 2 it comprises U6 and promotes sub and the coding sequence of the shRNA targeting LTR, said coding sequence is shown in SEQ ID NO: 14; or
  • expression box 1 it comprises the coding sequence of the shRNA of H1 promoter and targeting Gag, and described coding sequence is shown in SEQ ID NO:12; And expression box 2, it comprises U6 promoter and targeting Nef The coding sequence of shRNA, described coding sequence is shown in SEQ ID NO:15.
  • the vector can be a DNA vector, a viral vector, a liposome, a nanoparticle or an exosome.
  • the DNA vector is selected from the group consisting of DNA plasmid vectors, molecular conjugates bound thereto, and polymers bound thereto.
  • the viral vector is selected from the group consisting of adeno-associated viral vector, lentiviral vector and adenoviral vector.
  • the viral vector is a recombinant lentiviral vector.
  • the recombinant lentiviral vector is selected from: human immunodeficiency virus (HIV-1 and HIV-2) vectors, simian immunodeficiency virus (SIV) vectors, feline immunodeficiency virus (FIV) vectors, Equine infectious anemia virus (EIAV) vector and caprine arthritic encephalitis virus (CAEV) vector.
  • the recombinant lentiviral vector is a recombinant simian immunodeficiency virus (SIV) vector.
  • the eighth aspect of the present invention also provides a recombinant virus particle.
  • the recombinant simian immunodeficiency virus vector is a recombinant simian immunodeficiency virus, also known as a recombinant simian immunodeficiency virus particle.
  • recombinant simian immunodeficiency virus or recombinant simian immunodeficiency virus particles are composed of recombinant simian immunodeficiency gene transfer vectors or shuttle plasmids (plasmids carrying foreign genes), packaging vectors, The rev expression vector and the VSV-G expression vector were prepared.
  • the recombinant monkey immunodeficiency gene transfer vector further comprises RRE, cPPT and WPRE elements.
  • the recombinant monkey immunodeficiency gene transfer vector comprises 5'LTR region, RRE, cPPT (central polypurine tract), the present invention in sequence from 5' to 3' Said expression cassette, WPRE (woodchuck hepatitis virus posttranscriptional regulatory element), 3' LTR region.
  • the packaging vector (refer to Chinese patent ZL200680012905.4 transformed from the original packaging vector sequence number: 27) in addition to gag, pol, also includes so-called modified genes vif, vpr and Known as tat and rev of regulatory genes.
  • the auxiliary genes (vif, vpr, tat) are removed from the packaging vector and rev are provided in the form of another expression vector, ie a rev expression vector.
  • VSV-G expression vector refers to a plasmid expressing VSV-G (Vesicular stomatitis virus; VSV) protein.
  • VSV-G Vesicular stomatitis virus
  • the recombinant SIV virus vector of the present invention can be pseudotyped with VSV-G.
  • VSV-G pseudotyping means that the envelope of the vector contains the surface glycoprotein VSV-G of vesicular stomatitis virus (VSV).
  • VSV-G expression vector Through the transfection of the VSV-G expression vector, the recombinant SIV virus particles coexist with the VSV-G protein during production, and pseudotyped recombinant SIV virus particles are obtained.
  • Recombinant lentiviruses such as SIV infect cells or living tissues to achieve long-term expression of foreign genes in cells or living tissues.
  • the ninth aspect of the present invention also provides a kit comprising the HIV-targeting RNA or a combination thereof, siRNA or a combination thereof, shRNA or a combination thereof, an shRNA coding sequence or a combination thereof, an expression cassette according to the present invention, or carrier.
  • the tenth aspect of the present invention also provides a cell comprising the HIV-targeting RNA or a combination thereof, siRNA or a combination thereof, shRNA or a combination thereof, shRNA coding sequence or a combination thereof according to the present invention, or comprising The expression cassette and vector of the present invention.
  • the cells of the present invention can be prepared and obtained by methods and techniques well known to those skilled in the art, as long as the final cells contain the aforementioned nucleic acid sequences or recombinant vectors. In a preferred embodiment of the present invention, the cells are obtained by infection with the recombinant viral vector according to the present invention.
  • said cells are derived from HIV recipient cells or peripheral blood mononuclear cells. In a further preferred embodiment of the invention said cells are derived from lymphocytes. In some embodiments, the cells are derived from T cells. In other embodiments, the cells are derived from naive T cells ( T), memory T cells, and effector T cells. In other embodiments, the cells are derived from cytotoxic T cells, helper T cells, and regulatory T cells. In other embodiments, the T cells are derived from CD4+ T cells, CD8+ T cells. In other embodiments, the cells are derived from NKT cells. In other embodiments, the cells are derived from ⁇ T cells. In other embodiments, the cells are derived from NK cells. In other embodiments, the cells are derived from antigen-presenting cells, such as macrophages, dendritic cells.
  • T naive T cells
  • the cells are derived from cytotoxic T cells, helper T cells, and regulatory T cells.
  • the T cells are derived from CD4+
  • the stem cells comprise hematopoietic stem cells (eg, CD34+ cells) or hematopoietic progenitor cells.
  • the stem cells include memory T stem cells (memory T stem cells), such as central memory T cells (central memory T cells), effector memory T cells (effector memory T cells) or stem cell-like Memory T cells (stem cell-like memory T cells).
  • the stem cells can be induced to differentiate into any T cells as described above.
  • the cells are derived from an HIV-infected patient. In some embodiments, the cells are derived from healthy humans.
  • the cells may be isolated (or ex vivo) cells, or cells in vivo.
  • the cells are derived from engineered cells, such as TCR-T cells or cells containing CAR molecules.
  • compositions comprising, pharmaceutical uses and methods of treatment
  • the eleventh aspect of the present invention also provides a pharmaceutical composition, which comprises the HIV-targeting RNA or its combination, siRNA or its combination according to the present invention, or it comprises the shRNA or its combination according to the present invention.
  • a pharmaceutical composition which comprises the HIV-targeting RNA or its combination, siRNA or its combination according to the present invention, or it comprises the shRNA or its combination according to the present invention.
  • Combination, shRNA coding sequence or combination thereof, or it comprises the expression cassette according to the present invention, or it comprises the vector according to the present invention, or it comprises the cell according to the present invention preferably also comprises pharmaceutically available Accepted carrier.
  • the pharmaceutical composition further includes other anti-HIV drugs known in the art.
  • the other anti-HIV drugs include but are not limited to nucleoside inhibitors, HIV vaccines, broad-spectrum neutralizing antibodies, CAR-T cells, etc.
  • the eleventh aspect of the present invention also provides HIV-targeting RNA or its combination, siRNA or its combination, shRNA or its combination, shRNA coding sequence or its combination, expression cassette, vector, cell or drug combination according to the present invention use of substances as medicines.
  • the eleventh aspect of the present invention also provides HIV-targeting RNA or its combination, siRNA or its combination, shRNA or its combination, shRNA coding sequence or its combination, expression cassette, vector, cell or drug combination according to the present invention Use of the drug in the preparation of medicines for treating and/or preventing HIV infection or AIDS.
  • the eleventh aspect of the present invention also provides a method for treating and/or preventing HIV infection or AIDS, which includes administering to a patient a therapeutically effective amount of the HIV-targeting RNA according to the present invention or a combination thereof, siRNA or a combination thereof, shRNA or a combination thereof, an shRNA coding sequence or a combination thereof, an expression cassette, a recombinant vector, a cell or a pharmaceutical composition.
  • the present invention also provides a method for treating HIV infection or AIDS using the above-mentioned treatment or prevention method in combination with other methods in the art.
  • the method for treating and/or preventing HIV infection or AIDS further comprises administering other anti-HIV drugs to the patient.
  • the other anti-HIV drugs include but are not limited to nucleoside inhibitors, HIV vaccines, broad-spectrum neutralizing antibodies, CAR-T cells, and the like.
  • therapeutically effective amount refers to HIV-targeting RNA or its combination, siRNA or its combination, shRNA or its combination, shRNA coding sequence or its combination, An expression cassette, vector, cell or pharmaceutical composition.
  • the twelfth aspect of the present invention also provides a drug administration method for treating AIDS, which includes the following steps:
  • PBMC peripheral blood mononuclear cells
  • step (1) Infecting the CD4 + T cells in step (1) with the recombinant virus vector of the present invention, and performing in vitro expansion of the infected CD4 + T cells;
  • CD3/CD28 antibody-coupled magnetic beads are used to activate and expand CD4 + T cells.
  • the multiplicity of infection of CD4 + T cells infected by the recombinant virus vector is 1-20, preferably 20.
  • HIV-targeting RNA or its combination, siRNA or its combination, shRNA or its combination, shRNA coding sequence or its combination, expression cassette, recombinant vector, cell or pharmaceutical composition of the present invention can significantly inhibit HIV virus replication, long-term inhibition Generation of escape mutations.
  • the anti-HIV drug of the present invention reduces the replication of the experimental virus strain HIV-1IIIB to below 20% of the virus control group;
  • the anti-HIV drug of the present invention reduces the replication of reverse transcriptase inhibitor strains and protease inhibitor drug-resistant strains to less than 10% of the virus control group;
  • the anti-HIV drugs provided by the present invention can inhibit the replication of HIV for a long time, and inhibit the generation of escape mutations to a large extent;
  • the anti-HIV drug provided by the present invention can effectively inhibit HIV virus replication in CD4 + T cells of HIV-positive patients, and significantly reduce the viral load.
  • Table 1 Exemplary siRNA sequences and corresponding target sequences on the HIV genome
  • Figure 1 is the vector map of pGTV-New.
  • Figure 2 shows the inhibitory effect of recombinant lentiviral single-target drugs on HIV.
  • Figure 3 is a graph showing the inhibition curves of recombinant lentiviruses SIV-Gag3 and SIV-G3L2 on HIV escape mutations.
  • Figure 4 is a graph showing the inhibition of HIV escape mutations by recombinant lentiviruses SIV-Gag2 and SIV-G2N.
  • HIV Human immunodeficiency virus
  • the experimental virus strain HIV-1IIIB is a laboratory standard virus strain of HIV-1, which has stable passage and replication ability in the human T lymphocyte line H9, and is widely used in the literature and practical application for the evaluation of HIV drug efficacy in vitro Model.
  • the HIV-1IIIB virus strain of the present invention comes from the British Medical Research Council AIDS Reagent Project, is multiplied and preserved by our laboratory, and its titer is 5 ⁇ 10 4 TCID50/ml, and is preserved below -70°C.
  • HIV human immunodeficiency virus
  • the first and second-line drugs for the treatment of HIV virus infection include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and fusion inhibitors, and drug resistance mutations to reverse transcriptase inhibitors and protease inhibitors are the main HIV drug resistance mutation, the present invention selects these two types of drug-resistant strains as experimental virus strains.
  • the HIV reverse transcriptase inhibitor-resistant strain MT2-010 and the HIV protease inhibitor-resistant strain MT2-884 used in the examples of the present invention were prepared according to the methods described in patent applications ZL201710172266.8 and ZL201210533551.5, respectively.
  • H9 cells and MT2 cells were purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd., and the specifications were 2 ⁇ 10 6 /cartridge. Resuscitated by our laboratory and subcultured.
  • Carrier treatment use EcoR I and NheI to cut pGTV-PEDF carrier (the preparation method of the carrier is detailed in 1-1 in Example 1 in patent document CN200680012905.4, which is incorporated by reference in its entirety Herein), recover about 5.5kb vector fragment;
  • fragment 1 amplification synthetic primer 1: ATCGAATTCC CGTTTGTGCT AG (SEQ ID NO.16), primer 2: AAGCTTGCGGGATCCCCGCG GCTCTAAAAT TTA (SEQ ID NO.17), using pGTV-PEDF as a template to amplify about 500bp fragment 1 , recycle fragment 1;
  • Fragment 2 amplification synthetic primer 3: AGCGCAGCGA GTCAGTGAGC G (SEQ ID NO.18), primer 4: GGATCC CGC AAGCTT AG ATCCGCACTT TTTAAAAG (SEQ ID NO.19), using pGTV-PEDF as a template, amplified about 500bp fragment 2, recovery fragment 2;
  • Overlap PCR Mix the recovered fragment 1 and fragment 2 as a template, amplify with primer 1 and primer 4 to obtain a fragment of about 1000 bp, after recovery, use EcoR I and NheI double enzyme digestion to recover the fragment;
  • H1-Gag1 sequence fragments H1-Gag2 sequence fragments, H1-Gag3 sequence fragments, U6-LTR2 sequence fragments, U6-Nef sequence fragments respectively (the synthetic Gag, LTR, Nef sequence parts mentioned in the vector construction process of the present invention are all Refers to the DNA sequence encoding the shRNA targeting the gene in the HIV genome), and HindIII and BamHI restriction sites were introduced on both sides during synthesis, wherein H1 and U6 are promoters.
  • the coding sequence of the shRNA targeting the Gag gene in the HIV genome is referred to as Gag1, Gag2, and Gag3 respectively
  • the coding sequence of the shRNA targeting the LTR gene in the HIV genome is referred to as LTR2
  • the coding sequence of the shRNA targeting the Nef gene in the HIV genome is referred to as is Nef, whose sequence is as follows:
  • ATAGTAAGAATGTATAGCTTCAAGAGAGGCTATACATTCTTACTAT (abbreviated as Gag3);
  • H1-Gag3, H1-Gag2, H1-Gag1, H1-LTR2, and H1-Nef sequence fragments synthesized above were respectively digested with HindIII and BamHI, and then ligated with the pGTV-new vector that had been digested with the same double enzymes to construct a A series of shuttle plasmids pGTV-Gag3, pGTV-Gag2, pGTV-Gag1, pGTV-LTR2 and pGTV-Nef carrying a single target in the HIV genome. Each shuttle plasmid was sequenced and verified to be correct.
  • H1-Gag3-U6-LTR2 sequence fragments H1-Gag2-U6-LTR2 sequence fragments, H1-Gag1-U6-LTR2 sequence fragments, H1-Gag2-U6-Nef sequence fragments respectively, and introduce HindIII and BamHI restriction site, where H1 and U6 are promoters, and there is a spacer between the expression box of H1 and the expression box of U6.
  • the H1-Gag3-U6-LTR2, H1-Gag2-U6-LTR2, H1-Gag1-U6-LTR2, and H1-Gag2-U6-Nef sequence fragments synthesized above were digested with HindIII and BamHI respectively, and the same double enzymes
  • the cut pGTV-new vector was connected, that is, a series of shuttle plasmids pGTV-Gag3-LTR2, pGTV-Gag2-LTR2, pGTV-Gag1-LTR2, and pGTV-Gag2-Nef carrying dual targets in the HIV genome were successfully constructed.
  • the above shuttle plasmid was sequenced and verified to be correct.
  • Cell line 293T cells (ATCC, CRL-11268) were inoculated according to each T225 culture flask (Coring, Cat#431082) 9 ⁇ 10 6 cells, in 20 ml of D-MEM medium containing 10% fetal bovine serum (Thermofisher, After culturing in Cat#11995-065) for 48 hours, the medium was replaced with 10 ml of preheated OPTI-MEM medium (Thermofisher, Cat#31985-070), which was used as cells to be transfected.
  • the solution was added to the above-mentioned culture flask inoculated with cells, cultivated in a 37°C, 5% CO 2 incubator for 3 hours, and then each bottle was supplemented with DMEM medium containing 20% FBS, and the final concentration of FBS was Adjust to 10%, and continue culturing overnight in a 37°C, 5% CO 2 incubator.
  • the genomic RNA of the virus is extracted, then reverse-transcribed, and the obtained cDNA is used as a template for real-time quantitative PCR.
  • For specific operation steps refer to the titer determination method described in 4-3 of Example 4 of Chinese Patent ZL201210078288.5 to measure the titer of recombinant lentivirus SIV genome (this patent document is incorporated herein by reference in its entirety).
  • the genome titers of the recombinant lentivirus SIV packaged in this example were all 3 ⁇ 10 10 Vg/ml.
  • the recombinant lentiviral vectors packaged by Gag2-Nef and packaging vectors, rev expression vectors, and VSV-G expression vectors are referred to as recombinant lentiviruses SIV-Gag3, SIV-Gag2, SIV-Gag1, SIV-LTR2, SIV-Nef, SIV-G3L2, SIV-G2L2, SIV-G1L2, SIV-G2N.
  • H9 cells were revived and subcultured with complete medium (RPMI-1640+10% FBS+PS). The cells were resuspended in OPTI-MEM medium, counted and adjusted to a concentration of 5 ⁇ 10 5 cells/ml, and inoculated in a 6-well plate with 2 ml per well. Set the experimental group SIV-Gag3, SIV-Gag2, SIV-Gag1, SIV-LTR2 or SIV-Nef, SIV-EGFP empty vector control group, and blank control group (without HIV-1IIIB infection, without lentivirus) on a 6-well plate carrier drug).
  • MOI infection index
  • HIV-1 P24 Antigen Detection Kit (96T)
  • 96T The "HIV-1 P24 Antigen Detection Kit (96T)" (Beijing Jin Boyi Biotechnology Co., Ltd.) was used for ELISA detection. Measure the OD value with a microplate reader, calculate the average value, calculate the relative concentration, and determine the antiviral activity of the recombinant lentivirus against HIV-1IIIB in H9 cells.
  • H9 cells were revived and subcultured with complete medium (RPMI-1640+10% FBS+PS). The cells were resuspended in OPTI-MEM medium, counted and adjusted to a concentration of 5 ⁇ 10 5 cells/ml, and inoculated in a 6-well plate with 2 ml per well.
  • recombinant lentivirus SIV-G3L2 Five doses of recombinant lentivirus SIV-G3L2 were added according to the infection index (MOI) of 125, 250, 500, 1000, and 2000, and the volumes added were 2.1 ⁇ l, 4.2 ⁇ l, 8.4 ⁇ l, 16.7 ⁇ l, and 33.3 ⁇ l, respectively. Dose one hole.
  • recombinant lentivirus SIV-EGFP with an MOI of 2000 was added as an empty vector control, with a volume of 33.3 ⁇ l. Mix well, culture at 37°C, 5% CO 2 for 24 hours, add 4ml of complete medium to each well, and culture at 37°C, 5% CO 2 for 72 hours.
  • HIV-1 P24 Antigen Detection Kit (96T)
  • 96T The "HIV-1 P24 Antigen Detection Kit (96T)" (Beijing Jin Boyi Biotechnology Co., Ltd.) was used for ELISA detection. Measure the OD value with a microplate reader, calculate the average value, calculate the relative concentration, and determine the antiviral activity of the recombinant lentivirus against HIV-1IIIB in H9 cells.
  • the recombinant lentiviruses SIV-G3L2, SIV-G2L2, SIV-G1L2 or SIV-G2N respectively reduced HIV replication to 10.94%, 12.32%, 15.01% of the control group without drug Or 13.28%, indicating that the recombinant lentivirus SIV-G3L2, SIV-G2L2, SIV-G1L2 or SIV-G2N can effectively inhibit the replication of HIV-1IIIB.
  • Example 4 the efficacy of recombinant lentiviral dual-target drugs on reverse transcriptase inhibitor-resistant strains
  • the experimental design is the same as that in Example 3, except that the tested cell line is MT2-010, a drug-resistant strain of reverse transcriptase inhibitors, and the infected cells are MT2 cells.
  • the first is cell culture and recombinant lentivirus infection: set the experimental group SIV-G3L2 or SIV-G2N, SIV-EGFP empty vector control group, and positive drug control group (plus efavirenz, EFV, Shanghai Disay) on a 6-well plate Connaught Biopharmaceutical Co., Ltd., concentration 1 ⁇ M), no drug control group (with MT2-010 infection, no recombinant lentiviral drug) and blank control group.
  • recombinant lentivirus Five doses of recombinant lentivirus were added according to the infection index (MOI) of 125, 250, 500, 1000 and 2000.
  • MOI infection index
  • a recombinant lentivirus SIV-EGFP with an MOI of 2000 was also added as an empty vector control. Mix well and incubate.
  • MT2 cells were diluted and seeded in 96-well plate, 1 ⁇ 10 4 cells/well. Dilute the reverse transcriptase inhibitor-resistant strain MT2-010 with complete medium, mix well, and add virus diluent to each well of cells except for the blank control group. Mix well and incubate. Samples were taken at the time described in Example 3, and ELISA detection was performed using the above kit. Measure the OD value with a microplate reader, calculate the average value, and calculate the relative concentration to determine the antiviral activity of the recombinant lentivirus against MT2-010 in MT2 cells.
  • the recombinant lentivirus SIV-G2N also showed a strong inhibitory effect on the replication of HIV reverse transcriptase inhibitor-resistant strains, and there was an obvious dose-effect relationship. Inhibition of HIV replication increased with increasing doses of recombinant lentivirus SIV-G2N.
  • MOI high-dose group
  • the recombinant lentivirus SIV-G2N reduced the replication of HIV to 11.31 ⁇ 1.34% of the control group without drug, which indicated that the recombinant lentivirus SIV-G2N could effectively inhibit reverse transcriptase inhibitors Replication of resistant strains.
  • Example 5 the efficacy of recombinant lentiviral drugs on drug-resistant strains of protease inhibitors
  • the experimental method is the same as step 1 of Example 3, except that the tested cell line is HIV protease inhibitor-resistant strain MT2-884, and the infected cells are MT2 cells.
  • the first is cell culture and recombinant lentivirus SIV-G3L2 or SIV-G2N infection: culture cells in 6-well plates, and set up experimental groups SIV-G3L2, SIV-G2N, SIV-EGFP empty vector control group, positive drug control group ( Add efavirenz, EFV, Shanghai Desano Biomedical Co., Ltd., concentration 1 ⁇ M), no drug control group (with MT2-884 infection, no recombinant lentiviral drug) and blank control group.
  • SIV-G3L2 or SIV-G2N infection culture cells in 6-well plates, and set up experimental groups SIV-G3L2, SIV-G2N, SIV-EGFP empty vector control group, positive drug control group ( Add efavirenz, EFV, Shanghai Desano Biomedical
  • recombinant lentivirus Five doses of recombinant lentivirus were added according to the infection index (MOI) of 125, 250, 500, 1000 and 2000.
  • MOI infection index
  • a recombinant lentivirus SIV-EGFP with an MOI of 2000 was also added as an empty vector control. Mix well and incubate.
  • MT2 cells were diluted and seeded in 96-well plate, 1 ⁇ 10 4 cells/well. Dilute HIV protease inhibitor drug-resistant strain MT2-884 with complete medium, and add virus diluent to each well of cells except for the blank control group. Mix well and incubate. Samples were taken at the time described in Example 3, and ELISA detection was performed using the above kit. Measure the OD value with a microplate reader, calculate the average value, and calculate the relative concentration to determine the antiviral activity of the recombinant lentivirus against MT2-884 in MT2 cells.
  • H9 cells were infected with the lentiviral vector drug SIV-G3L2 or SIV-G2N prepared in Example 1, and the control group was the recombinant lentiviral SIV-EGFP empty vector and the recombinant lentiviral single-target drug SIV-Gag3 or SIV-Gag2, respectively.
  • p24 in the supernatant was detected.
  • concentration of p24 in the supernatant increased significantly, indicating that the recombinant lentiviral drug failed to inhibit HIV, the culture of this cell line was stopped.
  • the detection results of different cell lines are shown in Table 10-11 and Figure 3-4.
  • the recombinant lentiviral SIV-eGFP empty vector had no inhibitory effect on the replication of HIV virus, and the obvious replication of HIV was detected when cultured for 3-6 days.
  • H9 cells After infection of H9 cells with the recombinant lentiviral single-target drug SIV-Gag3, H9 cells showed resistance to HIV, but the 4 monoclonal cell lines detected significant replication of HIV at day 18-21, which indicated that there was resistance to HIV at this time.
  • the recombinant lentiviral SIV-EGFP empty vector had no inhibitory effect on the replication of HIV virus, and the obvious replication of HIV was detected when cultured for 3-6 days.
  • H9 cells After infection of H9 cells with recombinant lentiviral single-target drug SIV-Gag2, H9 cells showed resistance to HIV, but all five monoclonal cell lines detected significant HIV replication at day 18-30, indicating that this Drug-resistant mutations to the recombinant lentiviral single-target drug SIV-Gag2 appeared.
  • the three monoclonal cell lines did not detect significant HIV replication within the experimental culture time range (30 days), and the two monoclonal cell lines did not detect HIV replication within the experimental culture time range (30 days). There was a small amount of replication in Tianshi, indicating that the recombinant lentiviral dual-target drug SIV-G2N not only has a significant inhibitory effect on HIV replication, but also can inhibit the generation of drug-resistant mutations to a large extent.
  • Example 7 Inhibitory effect of recombinant lentiviral drugs on HIV virus replication in CD4 + T cells of HIV patients
  • Ficoll-Paque PLUS (GE, Cat#17-1440-02) to separate EDTA anticoagulated whole blood according to the instructions of the kit (peripheral blood from 5 newly diagnosed HIV-positive patients, from the Key Laboratory of STD and AIDS in You’an Hospital, numbered respectively PBMC cells in 061302, 061903, 061904, 062003, 062004).
  • a high-speed refrigerated centrifuge (Thermo Scientific Sorvall ST40R)
  • use a horizontal rotor to centrifuge at 800g for 30min set the centrifugation speed up to 3, speed down to 0, and the temperature to 20°C. After centrifugation, gently aspirate the PBMC cell layer and place in a new centrifuge tube.
  • the PBMCs were washed with 3-5 times the volume of DPBS aspirated from the cell layer, and then centrifuged at 20° C. and 300 g for 10 min. After centrifugation, remove the supernatant, add pre-cooled erythrocyte lysate (Tianjin Haoyang NH4CL2009) to the cell pellet, and place at 4°C for 2 minutes to lyse the erythrocytes. Subsequently, pre-cooled DPBS was added to make the total volume 40-45ml, the cells were mixed evenly by pipetting up and down, and centrifuged at 300g at 4°C for 10min to obtain PBMC cells.
  • pre-cooled DPBS was added to make the total volume 40-45ml, the cells were mixed evenly by pipetting up and down, and centrifuged at 300g at 4°C for 10min to obtain PBMC cells.
  • Human CD4+ T cell isolation kit (Antibody Cocktail; Biotech-beads) was used to isolate CD4+ T cells. Specifically, according to the above PBMC cell count results, 40ul MACS Buffer resuspended cells were added per 1 ⁇ 10 7 cells (cell If it is less than 1.5 ⁇ 10 7 , it will be counted as 1 ⁇ 10 7 ; for cells >1.5 ⁇ 10 7 , it will be counted as 2 ⁇ 10 7 , and so on), then add 10ul Antibody Cocktail, mix well, and incubate at room temperature for 10 minutes in the dark. Then add 30ul (equal proportion) MACS Buffer and 20ul Biotech-beads, mix well, and incubate at room temperature in the dark for 10min.
  • CD4+ T cells were activated using magnetic beads coated with anti-CD3 antibody and anti-CD28 antibody.
  • kit Dynabeads Human T-Activator CD3/CD28 for T-Cell Expansion and Activation Thermofisher, 11131D.
  • CD4+ T cells were activated using magnetic beads coated with anti-CD3 antibody and anti-CD28 antibody.
  • kit Dynabeads Human T-Activator CD3/CD28 for T-Cell Expansion and Activation Thermofisher, 11131D.
  • mix the cells and the washed CD3/CD28 magnetic beads according to 1 ⁇ 10 6 cells corresponding to 25ul original concentration of magnetic beads, and supplement the final concentration of 30IU/ml IL-2 (R&D, 202-IL-010) and human serum albumin with a final concentration of 3-5%, or human AB serum were thoroughly mixed and incubated at 37°C for 48 hours.
  • the mixture of cells and magnetic beads was taken into a 15ml centrifuge tube, shaken at 2000rpm for 30s, and placed on a magnetic stand for 1 minute. Aspirate the supernatant into a new centrifuge tube, resuspend the cells with complete medium, centrifuge at 300g for 5 minutes, discard the supernatant after centrifugation, resuspend the cell pellet with fresh complete medium, and take a part for counting and flow detection.
  • the cells obtained above were centrifuged at 2000 rpm for 5 minutes to discard the supernatant, resuspended with 500 ⁇ l medium, and inoculated into each well, and cultured at 37° C., 5% CO 2 .
  • the above infection steps were repeated on days 2 and 3, respectively.
  • the culture supernatant was collected on the 7th day and the 14th day of culture, and the HIV-1 viral load was detected by HIV-1 nucleic acid quantitative detection kit (QPCR) (Daan Gene).
  • QPCR HIV-1 nucleic acid quantitative detection kit
  • the reaction program is 50°C, 15min;
  • the test results of the batches of samples are listed in Table 12.

Abstract

提供了一种靶向HIV的RNA、siRNA、shRNA、shRNA编码序列、RNA组合、siRNA组合、shRNA组合或shRNA编码序列的组合。还提供了包含上述序列或组合的表达盒、重组载体、重组病毒颗粒、试剂盒、细胞或药物组合物，以及它们在制备诊断、治疗和/或预防HIV感染或艾滋病的药物中的用途。

Description

靶向HIV的siRNA和shRNA及其相应的组合、表达盒、细胞及其应用 技术领域
本发明涉及生物医药领域，特别涉及靶向HIV的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒、载体、细胞、药物组合物及其应用。
背景技术
由HIV(Human Immunodeficiency virus)感染所致的艾滋病自1981年在美国首次发现以来，以异常迅猛的速度蔓延到世界各地。HIV属于逆转录病毒科，是一种RNA逆转录病毒，是由两条单链正链RNA分子(+ssRNA)组成的包膜病毒。HIV病毒侵入细胞后，病毒RNA在自身逆转录酶的作用下逆转录成单链cDNA，并在宿主细胞内合成双链cDNA，与宿主细胞的染色体整合，利用宿主细胞的转录和合成系统转录产生致病的mRNA/gRNA。HIV的主要目标是CD4阳性表达细胞，如CD4 +T细胞。目前世界上批准用于临床的抗艾滋病药物已有近二十种。但是，已有的任何一种药物都不能完全清除人体内的HIV病毒。几种药物组合使用的“鸡尾酒疗法”效果会优于单一药物治疗，但也不能完全清除HIV病毒。HIV在药物治疗后仍可在携带HIV基因组的CD4+细胞内再次激活扩增。
RNA干扰(RNAi)是一种抑制基因表达的机制，通过双链短干扰RNA(siRNA)诱导的特异性RNA降解过程，它发生在基因转录后，即mRNA水平上的对基因的调控，能够高效特异性阻断目的基因的表达。小干扰RNA(Small interfering RNA；siRNA)有时称为短干扰RNA(short interfering RNA)或沉默RNA(silencing RNA)，是一个长18到25个核苷酸的双股RNA，可以针对靶基因来设计并采用化学方法来合成，以瞬时转染细胞。或者是将针对靶基因的siRNA设计成发夹结构送入体内时，该发夹结构表达形成shRNA。shRNA是short hairpin RNA的缩写，翻译为“短发夹RNA”，shRNA包括两个短反向重复序列。目前已知shRNA主要参与RNA干扰(RNAi)现象，以带有专一性的方式调节基因的表达。shRNA进入细胞后，被宿主细胞内RNA解旋酶解旋为正义RNA链与反义RNA链，反义RNA链与体内的一些酶结合成沉默复合物RISC，识别含有其互补序列的mRNA，并与之结合。此时的RISC具有核酸酶的功能，能够切割降解mRNA，从而抑制相应基因表达。
到目前为止，艾滋病研究者们已经设计出了靶向HIV-1的编码序列以及非编码序列的siRNA(nef、tat、gag、vif、env、rev、LTR)(Boden等人，2004；Capodici等人，2002；Novina等人，2002；Han等人，2004；Jacque等人，2002；Lee等人，2002；Novina等人，2002)。然而，这种干扰方法只是在短期内有效。由于HIV病毒基因组的高突变率和siRNA的高度特异性，致使这些siRNA很容易失去作用，使得RNAi在长期抑制HIV复制方面变得困难(Das等人，2004；Westerhout等人，2005)。
因此，本领域中需要一种能够长期抑制HIV逃逸突变的药物和方法，以解决现有技术中存在的问题。
发明内容
因此，本发明提供了一种靶向HIV的RNA、siRNA、shRNA或shRNA编码序列，其能显著有效地抑制HIV病毒株、耐药突变病毒株的复制。特别地，本发明还提供了一种RNA组合、siRNA组合、shRNA组合或shRNA编码序列的组合，其不仅能够高效地抑制HIV病毒株或耐药突变病毒株的复制，还能够进一步长期有效抑制逃逸HIV突变株的产生，对HIV阳性患者具有很好的治疗效果。本发明所涉及的示例性的RNA、siRNA、shRNA及其编码序列示于表1和表2中。
一方面，本发明提供了一种靶向HIV的RNA序列，其靶向如SEQ ID NO：1-5中任一项所示序列。在另一方面，本发明提供的RNA序列如SEQ ID NO：6-10或20-24中任一项所示。
一方面，本发明提供了一种靶向HIV的siRNA，所述siRNA包括正义链和与之反向互补的反义链，由如下序列组成：
(i)正义链序列如SEQ ID NO：6所示，反义链的序列如SEQ ID NO：20所示；
(ii)正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示；
(iii)正义链序列如SEQ ID NO：8所示，反义链序列如SEQ ID NO：22所示；
(iv)正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；或
(v)正义链序列如SEQ ID NO：10所示，反义链序列如SEQ ID NO：24所示。
在另一方面，本发明提供了靶向HIV的shRNA或其编码序列，所述shRNA的序列包括：
(i)SEQ ID NO：6和SEQ ID NO：20所示序列；
(ii)SEQ ID NO：7和SEQ ID NO：21所示序列；
(iii)SEQ ID NO：8和SEQ ID NO：22所示序列；
(iv)SEQ ID NO：9和SEQ ID NO：23所示序列；或
(v)SEQ ID NO：10和SEQ ID NO：24所示序列。
在一个优选实施方式中，所述shRNA的序列如SEQ ID NO：25-29中任一项所示。
在一个优选实施方式中，所述shRNA的编码序列如SEQ ID NO：11-15中任一项所示。
在另一方面，本发明还提供了如上所述的RNA的组合。在一个优选实施方式中，所述RNA组合包括：
(i)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：6或20所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；
(ii)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：7或21所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；
(iii)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：8或22所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；或者
(iv)靶向Gag基因的RNA序列和靶向Nef基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：7或21所示，靶向Nef基因的RNA序列如SEQ ID NO：10或24所示。
在另一方面，本发明还提供了如上所述的siRNA的组合。在一个优选实施方式中，所述siRNA组合包括：
(i)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：6所示，反义链序列如SEQ ID NO：20所示，靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；
(ii)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示，靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；
(iii)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：8所示，反义链序列如SEQ ID NO：22所示，靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；或者
(iv)靶向Gag基因的siRNA和靶向Nef基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示，靶向Nef基因的siRNA的正义链序列如SEQ ID NO：10所示，反义链序列如SEQ ID NO：24所示。
在另一个方面，本发明还提供了靶向HIV的shRNA的组合。在一个优选实施方式中，所述shRNA组合包括：
(i)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中靶向Gag基因的shRNA的序列如SEQ ID NO：25所示，靶向LTR基因的shRNA的序列如SEQ ID NO：28所示；
(ii)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中靶向Gag基因的shRNA的序列如SEQ ID NO：26所示，靶向LTR基因的shRNA的序列如SEQ ID NO：28所示；
(iii)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中靶向Gag基因的shRNA的序列如SEQ ID NO：27所示；靶向LTR基因的shRNA的序列如SEQ ID NO：28所示；或者
(iv)靶向Gag基因的shRNA和靶向Nef基因的shRNA，其中靶向Gag基因的shRNA的序列如SEQ ID NO：26所示；靶向Nef基因的shRNA的序列如SEQ ID NO：29所示。
在另一方面，本发明还提供了如上所述的shRNA编码序列的组合。在一个优选实施方式中，所述shRNA编码序列的组合包括：
(i)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中所述的靶向Gag基因的shRNA的编码序列如SEQ ID NO：11所示，靶向LTR基因的shRNA的编码序列如SEQ ID NO：14所示，
(ii)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中所述的靶向Gag基因的shRNA的编码序列如SEQ ID NO：12所示，靶向LTR基因的shRNA的编码序列如SEQ ID NO：14所示，
(iii)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中所述的靶向Gag基因的shRNA的编码序列如SEQ ID NO：13所示，靶向LTR基因的shRNA的编码序列如SEQ ID NO：14所示，或者
(iv)靶向Gag基因的shRNA的编码序列和靶向Nef基因的shRNA的编码序列，其中所述的靶向Gag基因的shRNA的编码序列如SEQ ID NO：12所示，靶向Nef基因的shRNA的编码序列如SEQ ID NO：15所示。
在另一方面，本发明还提供了包含上述编码序列的表达盒。在另一方面，本发明还提供了包含上述编码序列，或上述表达盒的载体。在另一方面，本发明还提供了包含上述的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒或载体的细胞。在另一方面，本发明还提供了包含上述的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒、载体或细胞的药物组合物。其中，本发明所述的shRNA编码序列为DNA序列。
定义
除非另外指出，本发明的实践将采用本领域技术中的常规化学、生物化学、重组DNA技术和免疫学的方法。这样的技术在文献中有充分解释(参见例如FundamentalVirology，第二版，vol.I&II(B.N.Fields和D.M.Knipe编)；Handbook of ExperimentalImmunology，VoIs.I-FV(D.M.Weir和CC.Blackwell编，Blackwell ScientificPublications)；T.E.Creighton，Proteins:Structures and Molecular Properties(W.H.Freeman和Company，1993)；A.L.Lehninger，Biochemistry(Worth Publishers,Inc.，current addition)；Sambrook,等，Molecular Cloning:A Laboratory Manual(第2版，1989)；Methods In Enzymology(S.Colowick和N.Kaplan编，Academic Press,Inc.)。
为了便于理解本公开内容的各个实施方案，提供了特定术语的以下解释：
合成：通过人工手段在实验室产生，例如合成核酸可以在实验室化学合成。
分离的：“分离的”生物组分(例如，核酸分子、蛋白质、病毒或细胞)已经被从其中所述组分天然存在的生物体的细胞或组织中或者生物体本身中的其他生物组分(例如其他染色体和染色体外DNA和RNA、蛋白质和细胞)中基本上分离或纯化。已经“分离”的核酸分子和蛋白质包括通过标准纯化方法纯化的那些。该术语还包括通过在宿主细胞中重组表达制备的核酸分子和蛋白质，以及化学合成的核酸分子和蛋白质。
重组：重组核酸分子是指这样的核酸分子，其具有非天然存在的序列，或者具有通过两个序列片段的人为组合(否则地话，其将是分开的)制备的序列。这种人为组合可以通过化学合成或者通过分离的核酸分子片段的人为操作(如通过基因工程技术)实现。
同样地，重组病毒是包含非天然存在的序列或通过至少两个不同来源的序列的人为组合制备的序列的病毒。术语“重组”还包括仅通过天然核酸分子、蛋白质或病毒的一部分的添加、置换或缺失改变的核酸、蛋白质和病毒。本文使用的“重组SIV”是指其中包装有重组核酸分子(例如，编码shRNA重组核酸分子)的SIV颗粒。
序列同一性：两个或更多个核酸序列之间或者两个或更多个氨基酸序列之间的同一性或相似性是根据其序列之间的同一性或相似性表示。序列同一性可以根据百分比同一性测量；百分比越高，序列越相同。序列相似性可以根据百分比相似性测量(考虑保守性氨基酸置换)；百分比越高，序列越相似。当使用标准方法比对时，核酸或氨基酸序列的同源物或直系同源物具有相对高的序列同一性/相似性程度。与来自相关性更远的物种(例如，人和线虫(C.elegans)序列)相比，当直系同源蛋白质或cDNA来自更紧密相关的物种(例如，人和小鼠序列)时，这种同源性更显著。
序列同一性比较的长度可在基因组的全长上，基因编码序列的全长，或至少大约500至5000个核苷酸的片段是期望的。但是，在较小片段(例如具有至少大约9个核苷酸，通常至少大约20至24个核苷酸、至少大约28至32个核苷酸、至少大约36或更多个核苷酸)中的同一性也可以是期望的。通常，当提到两种不同序列之间的“同一性”、“同源性”或“相似性”时，参照“比对”序列确定“同一性”、“同源性”或“相似性”。“比对”序列或“比对”是指多个核酸序列或蛋白(氨基酸)序列，其通常含有与参考序列相比缺失或额外的碱基或氨基酸的校正。
使用任何公共或市售可得的多序列比对程序来进行比对。序列比对程序可用于氨基酸序列，例如“Clustal X”、“MAP”、“PIMA”、“MSA”、“BLOCKMAKER”、“MEME”和“Match-Box”程序。通常，这些程序中的任一种以默认设置使用，尽管本领域技术人员可以按需改变这些设置。或者，本领域技术人员可以采用至少提供如参考算法或程序所提供的同一性或比对水平的另一算法或计算机程序。参见例如J.D.Thomson等人,Nucl.Acids.Res.,“Acomprehensive comparison of multiple sequence alignments”,27(13):2682-2690(1999)。
多序列比对程序还可用于核酸序列。此类程序的实例包括“Clustal W”、“CAPSequence Assembly”、“BLAST”、 “MAP”和“MEME”，其可以通过互联网上的Web服务器来访问。此类程序的其它来源是本领域技术人员已知的。或者，还使用Vector NTI应用程序。还存在许多可用于测量核苷酸序列同一性的本领域已知的算法，包括在上述程序中包含的那些。作为另一实例，可以使用Fasta TM(GCG Version 6.1中的一个程序)来比较多核苷酸序列。Fasta TM提供了询问与搜索序列之间最佳重叠区域的比对和百分比序列同一性。例如，可以使用Fasta TM以其在GCG Version 6.1(经此引用并入本文)中提供的默认参数(字长为6，和用于打分矩阵的NOPAM因子)来确定核酸序列之间的百分比序列同一性。
可操作地连接：当第一核酸序列与第二核酸序列被放置为具有功能关系时，第一核酸序列与第二核酸序列可操作地连接。例如，如果启动子影响编码序列的转录或表达，则启动子与编码序列可操作地连接。通常，可操作地连接的DNA序列是连续的，并且当有必要连接两个蛋白质编码区时，其在相同阅读框中。
载体：载体是允许插入外源核酸而不破坏载体在宿主细胞中复制和/或整合的能力的核酸分子。载体可以包含允许其在宿主细胞中复制的核酸序列，例如复制起点。载体还可以包含一种或更多种选择性标记基因和其他遗传元件。表达载体是包含必要的调控序列以允许插入的基因转录和翻译的载体。在本文的一些实施方案中，载体是SIV载体。
“复制缺陷型病毒”或“病毒载体”是指合成或人工病毒粒子，其中含有相关基因的表达盒包装在病毒衣壳或包膜中，其中也包装在该病毒衣壳或包膜中的任何病毒基因组序列是复制缺陷的；即它们不能产生子代病毒体，但是保留了感染靶细胞的能力。在一个实施方案中，该病毒载体的基因组不包括编码复制所需的酶的基因(该基因组可以被工程化为“无内容”——仅含有侧接扩增和包装人工基因组所需信号的相关转基因)，但是这些基因可以在生产过程中提供。因此，其被认为对用于基因疗法是安全的，因为除了在复制所需病毒酶的存在下，复制和被子代病毒体感染不会发生。此类复制缺陷型病毒可以是腺相关病毒(AAV)、腺病毒、慢病毒(整合或非整合)或另一合适的病毒源。
“慢病毒”属于逆转录病毒亚属，以人类免疫缺陷病毒(HIV)为代表，可以感染分裂细胞并整合其基因组中，还具有感染包括神经元细胞、巨噬细胞、肝细胞、心肌细胞和干细胞在内的能力。“慢病毒载体”是以灵长类免疫缺陷病毒来源的一种病毒载体，已作为基因转移的有效工具，广泛应用于基因治疗的研究。携带外源基因的慢病毒载体通过感染细胞或活体组织，实现外源基因在细胞或活体组织中表达。示例性的慢病毒载体包括灵长类的重组慢病毒载体，例如人免疫缺陷病毒载体(human immunodeficiency virus,HIV)或猴免疫缺陷病毒载体(simian immunodeficiency virus，SIV)，或非灵长类的重组慢病毒载体。“猴免疫缺陷病毒(SIV)载体”是指在病毒颗粒中的核酸分子内，作为病毒载体功能所必需的序列为SIV基因由来序列的载体，所述必需的序列是指从5'端开始顺次为5'LTR的R区域，U5区域，包装信号(ф)、RRE、3'LTR的启动子区域以外的U3区域和R区域序列。。
启动子：指导/引起核酸(例如，基因)的转录的DNA区域。启动子包括转录起始位点附近的必要核酸序列。通常，启动子位于其转录的基因附近。启动子区域还任选地包括远端增强子或阻遏物元件，其可以位于距转录起始位点数千个碱基对远。
可药用载体：本公开内容中可以使用的可药用载体(溶剂(vehicle))是常规的。Remington’s Pharmaceutical Sciences,by E.W.Martin,MackPublishing Co.,Easton,PA,15th Edition(1975)描述了适合一种或更多种治疗性化合物、分子或试剂的药物递送的组合物和制剂。
预防、治疗或改善疾病：“预防”疾病(例如HIV感染)是指抑制疾病的全面发生。“治疗”是指在疾病开始发生后改善疾病或病理病症的体征或症状的治疗性介入。“改善”是指降低疾病的体征或症状的数目或严重性。
给药/给予：通过有效的途径向受试者提供或给予药剂，例如治疗剂(例如，重组SIV)。示例性给药途径包括但不限于注射(例如，皮下、肌内、真皮内、腹膜内和静脉内)、口服、导管内、舌下、直肠、经皮、鼻内、阴道和吸入途径。
治疗有效量：足以在用试剂治疗的受试者或者细胞中取得所需效果的特定药物或治疗试剂(例如重组SIV)的量。试剂的有效量取决于多种因素，包括但不限于被治疗的受试者或细胞，以及治疗性组合物的给药方式。
受试者：活的多细胞脊椎动物生物体，包括人和非人哺乳动物的类别。
靶向HIV的RNA序列
本发明的第一个方面提供了一种靶向HIV的RNA序列，所述RNA序列靶向SEQ ID NO：1-5中任一项所示序列。
在本发明的一个具体实施方式中，所述RNA序列如SEQ ID NO：6-10或20-24中任一项所示。
靶向HIV的siRNA
本发明的第二个方面提供了一种靶向HIV的siRNA，所述siRNA包括正义链和与之反向互补的反义链，其中，所述siRNA靶向HIV的Gag基因、LTR基因和/或Nef基因。
在本发明的一个具体实施方式中，所述siRNA靶向SEQ ID NO：1-5中任一项所示的序列。
在本发明的一个实施方式中，靶向HIV的Gag基因的siRNA正义链序列选自由SEQ ID NO：1-3中任一项所示序列中的连续19-23个核苷酸组成的序列；靶向HIV的LTR基因的siRNA正义链序列选自由SEQ ID NO：4中的连续19-26个核苷酸组成的序列；靶向HIV的Nef基因的siRNA正义链序列选自由SEQ ID NO：5中的连续19-25个核苷酸组成的序列。在本发明的另一个实施方式中，siRNA的反义链为与正义链反向互补的序列。
在本发明的一个实施方式中，所述靶向Gag基因的siRNA由如下序列组成：
(i)正义链序列如SEQ ID NO：6所示，反义链的序列如SEQ ID NO：20所示；
(ii)正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示；或
(iii)正义链序列如SEQ ID NO：8所示，反义链序列如SEQ ID NO：22所示。
在本发明的另一个实施方式中，所述靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示。
在本发明的另一个实施方式中，所述靶向Nef基因的siRNA的正义链序列如SEQ ID NO：10所示，反义链序列如SEQ ID NO：24所示。
靶向HIV的shRNA及其编码序列
本发明的第三个方面提供一种shRNA及其编码序列，所述shRNA靶向HIV的Gag基因、LTR基因和/或Nef基因。
在本发明的一个实施方式中，shRNA是由上述siRNA的正义链序列和与之反向互补的反义链序列通过中间非互补连接序列连接形成的shRNA，其中，正义链序列和反义链序列构成茎-环结构中的茎区，中间的非互补连接序列构成茎-环结构中的环区。
在本发明的一个实施方式中，所述shRNA在体内被加工后产生如前所述的siRNA。
在本发明的一个实施方式中，所述靶向Gag基因的shRNA的序列包括：
(i)SEQ ID NO：6和SEQ ID NO：20所示序列；
(ii)SEQ ID NO：7和SEQ ID NO：21所示序列；或者
(iii)SEQ ID NO：8和SEQ ID NO：22所示序列。
在本发明的另一个实施方式中，所述靶向LTR基因的shRNA的序列包括SEQ ID NO：9和SEQ ID NO：23所示序列。
在本发明的另一个实施方式中，所述靶向Nef基因的shRNA的序列包括SEQ ID NO：10和SEQ ID NO：24所示序列。
在本发明的优选实施方式中，所述shRNA的序列如SEQ ID NO：25-29中任一项所示；所述shRNA的编码序列如SEQ ID NO：11-15中任一项所示。
靶向HIV的RNA序列的组合
在本发明的第四个方面还提供了一种靶向HIV的RNA序列的组合。在本发明的一个具体实施方式中，所述组合包括如前所述的RNA序列中的至少一种。
在本发明的另一具体实施方式中，所述组合包括靶向Gag基因的RNA序列、靶向LTR基因的RNA序列或者靶向Nef基因的RNA序列中的至少两种。
在本发明的优选实施方案中，所述靶向HIV的RNA序列的组合包括：
(i)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，
(ii)靶向Gag基因的RNA序列和靶向Nef基因的RNA序列，或者
(iii)靶向LTR基因的RNA序列和靶向Nef基因的RNA序列。
进一步优选地，所述靶向HIV的Gag基因的RNA序列选自由SEQ ID NO：1-3中任一项所示序列中的连续19-23个核苷酸组成的序列或与之反向互补的序列，更优选地，如SEQ ID NO：6-8或20-22中任一项所示序列；靶向HIV的LTR基因的RNA序列选自SEQ ID NO：4中的连续19-26个核苷酸组成的序列或与之反向互补的序列，更优选地，如SEQ ID NO：9或23所示序列；靶向HIV的Nef基因的RNA序列选自SEQ ID NO：5中的连续19-25个核苷酸组成的序列或与之反向互补的序列，更优选地，如SEQ ID NO：10或24所示序列。
在本发明地一个优选实施方式中，所述靶向HIV的RNA序列包括：
(i)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：6或20所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；
(ii)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：7或21所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；
(iii)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：8或22所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；或者
(iv)靶向Gag基因的RNA序列和靶向Nef基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：7或21所示，靶向Nef基因的RNA序列如SEQ ID NO：10或24所示。
靶向HIV的siRNA的组合
本发明的第五个方面还提供了一种靶向HIV的siRNA组合。在本发明的一个具体实施方式中，所述组合包括如前所述的siRNA中的至少一种。
在本发明的另一个具体实施方式中，所述siRNA组合包括靶向Gag基因的siRNA、靶向LTR基因的siRNA 或者靶向Nef基因的siRNA中的至少两种。
在本发明的优选实施方案中，所述靶向HIV的siRNA包括：
(i)靶向Gag基因的siRNA和靶向LTR基因的siRNA，
(ii)靶向Gag基因的siRNA和靶向Nef基因的siRNA，或者
(iii)靶向LTR基因的siRNA和靶向Nef基因的siRNA。
进一步优选地，所述靶向Gag基因的siRNA正义链序列选自由SEQ ID NO：1-3中任一项所示序列中的连续19-23个核苷酸组成的序列，优选如SEQ ID NO：6-8中任一项所示序列，反义链序列为与正义链反向互补的序列；靶向LTR基因的siRNA正义链序列选自由SEQ ID NO：4中的连续19-26个核苷酸组成的序列，优选如SEQ ID NO：9所示序列，反义链序列为与正义链反向互补的序列；靶向Nef基因的siRNA正义链序列选自由SEQ ID NO：5中的连续19-25个核苷酸组成的序列，优选如SEQ ID NO：10所示序列，反义链序列为与正义链反向互补的序列。
在本发明的一个优选实施方式中，所述靶向HIV的siRNA组合包括：
(i)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：6所示，反义链序列如SEQ ID NO：20所示，靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；
(ii)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示，靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；
(iii)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：8所示，反义链序列如SEQ ID NO：22所示，靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；或
(iv)靶向Gag基因的siRNA和靶向Nef基因的siRNA的组合，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示，靶向Nef基因的siRNA的正义链序列如SEQ ID NO：10所示，反义链序列如SEQ ID NO：24所示。
靶向HIV的shRNA组合或shRNA编码序列的组合
本发明的第六个方面还提供了一种靶向HIV的shRNA组合或shRNA编码序列的组合。
在本发明的一个具体实施方式中，所述shRNA组合包括如前所述的shRNA中的至少一种。在本发明的另一个具体实施方式中，所述shRNA的编码序列的组合包括如前所述的shRNA的编码序列中的至少一种。
在本发明的另一个具体实施方式中，所述shRNA组合或shRNA编码序列的组合包括靶向Gag基因的shRNA或其编码序列、靶向LTR基因的shRNA或其编码序列，或者靶向Nef基因的shRNA或其编码序列中的至少两种。
在本发明的优选实施方案中，所述靶向HIV的shRNA组合或shRNA编码序列的组合包括：(i)靶向Gag基因的shRNA或其编码序列，和靶向LTR基因的shRNA或其编码序列，或者(ii)靶向Gag基因的shRNA或其编码序列，和靶向Nef基因的shRNA或其编码序列，或者(iii)靶向LTR基因的shRNA或其编码序列，和靶向Nef基因的shRNA或其编码序列。
优选地，所述靶向Gag基因的shRNA的序列包括：(i)SEQ ID NO：6和SEQ ID NO：20所示序列；(ii)SEQ ID NO：7和SEQ ID NO：21所示序列；或者(iii)SEQ ID NO：8和SEQ ID NO：22所示序列；所述靶向LTR基因的shRNA的序列包括SEQ ID NO：9和SEQ ID NO：23所示序列；所述靶向Nef基因的shRNA的序列包括SEQ ID NO：10和SEQ ID NO：24所示序列。
更进一步优选的，靶向Gag基因的shRNA的序列如SEQ ID NO：25-27中任一项所示；靶向LTR基因的shRNA的序列如SEQ ID NO：28所示；靶向Nef基因的shRNA的序列如SEQ ID NO：29所示。
更进一步优选的，所述靶向Gag基因的shRNA的编码序列如SEQ ID NO：11-13中任一项所示；靶向LTR基因的shRNA的编码序列如SEQ ID NO：14所示；靶向Nef基因的shRNA的编码序列如SEQ ID NO：15所示。
在本发明的一个优选实施方式中，所述靶向HIV的shRNA组合或shRNA编码序列的组合包括：
(i)靶向Gag基因的shRNA或其编码序列，和靶向LTR基因的shRNA或其编码序列，其中靶向Gag基因的shRNA包括SEQ ID NO：6和SEQ ID NO：20所示序列，靶向LTR基因的shRNA包括SEQ ID NO：9和SEQ ID NO：23所示序列；
(ii)靶向Gag基因的shRNA或其编码序列，和靶向LTR基因的shRNA或其编码序列，其中靶向Gag基因的shRNA包括SEQ ID NO：7和SEQ ID NO：21所示序列，靶向LTR基因的shRNA包括SEQ ID NO：9和SEQ ID NO：23所示序列；
(iii)靶向Gag基因的shRNA或其编码序列，和靶向LTR基因的shRNA或其编码序列，其中靶向Gag基因的shRNA包括SEQ ID NO：8和SEQ ID NO：22所示序列，靶向LTR基因的shRNA包括SEQ ID NO：9和SEQ ID NO：23所示序列；或
(iv)靶向Gag基因的shRNA或其编码序列，和靶向Nef基因的shRNA或其编码序列，其中靶向Gag基因的shRNA包括SEQ ID NO：7和SEQ ID NO：21所示序列，靶向Nef基因的shRNA包括SEQ ID NO：10和SEQ ID NO：24所示序列。
在本发明的一个优选实施方式中，所述靶向HIV的shRNA组合或shRNA编码序列的组合包括：
(i)靶向Gag基因的shRNA或其编码序列，和靶向LTR基因的shRNA或其编码序列，其中靶向Gag基因的shRNA的序列如SEQ ID NO：25所示，其编码序列如SEQ ID NO：11所示，靶向LTR基因的shRNA的序列如SEQ ID NO：28所示，其编码序列如SEQ ID NO：14所示；
(ii)靶向Gag基因的shRNA或其编码序列，和靶向LTR基因的shRNA或其编码序列，其中靶向Gag基因的shRNA的序列如SEQ ID NO：26所示，其编码序列如SEQ ID NO：12所示，靶向LTR基因的shRNA的序列如SEQ ID NO：28所示，其编码序列如SEQ ID NO：14所示；
(iii)靶向Gag基因的shRNA或其编码序列，和靶向LTR基因的shRNA或其编码序列，其中靶向Gag基因的shRNA的序列如SEQ ID NO：27所示，其编码序列如SEQ ID NO：13所示，靶向LTR基因的shRNA的序列如SEQ ID NO：28所示，其编码序列如SEQ ID NO：14所示；或
(iv)靶向Gag基因的shRNA或其编码序列，和靶向Nef基因的shRNA或其编码序列，其中靶向Gag基因的shRNA的序列如SEQ ID NO：26所示，其编码序列如SEQ ID NO：12所示，靶向Nef基因的shRNA的序列如SEQ ID NO：29所示，其编码序列如SEQ ID NO：15所示。
表达盒
本发明的第七个方面提供一种表达盒，其中，所述表达盒可表达根据如前所述的shRNA。
在本发明的一个实施方式中，所述表达盒包含如前所述的shRNA的编码序列或编码序列的组合。
在本发明的另一个优选实施方式中，所述表达盒包含shRNA的编码序列或其组合，所述shRNA包括：
(i)SEQ ID NO：6和SEQ ID NO：20所示序列；
(ii)SEQ ID NO：7和SEQ ID NO：21所示序列；
(iii)SEQ ID NO：8和SEQ ID NO：22所示序列；
(iv)SEQ ID NO：9和SEQ ID NO：23所示序列；或
(v)SEQ ID NO：10和SEQ ID NO：24所示序列。
在本发明的一个优选实施方式中，所述表达盒包含如SEQ ID NO：11-15中任一项所示序列或其组合。
在本发明中，所述表达盒还包含启动子。在本发明的一个优选实施方式中，启动子与上述编码序列可操作地连接。本发明使用的启动子可以是任何RNA聚合酶II启动子或RNA聚合酶III启动子；优选为RNA聚合酶III启动子。在本发明的一个更优选实施方式中，RNA聚合酶III启动子包括H1启动子、U6启动子、7SK启动子。在本 发明的另一优选实施方式中，所述RNA聚合酶II启动子或RNA聚合酶III启动子还包括任何合成的或改造的DNA片段。
在本发明的优选实施方案中，所述表达盒中包含的启动子序列选自H1和/或U6启动子。在本发明的一个优选实施方案中，靶向Gag的shRNA由H1启动子驱动表达。优选地，靶向Gag的shRNA的编码序列与H1启动子可操作地连接。在本发明的另一个优选实施方案中，靶向LTR或Nef的shRNA由U6启动子驱动表达。优选地，靶向LTR或Nef的shRNA的编码序列与U6启动子可操作地连接。
载体
本发明的第八个方面提供了一种载体，其包含根据本发明所述的RNA或其组合，siRNA或其组合，或者包含根据本发明所述的shRNA或其组合，或者shRNA编码序列或其组合；或者包含根据本发明所述的表达盒。
在本发明的一个实施方式中，所述载体包含本发明所述的RNA的组合，所述RNA的组合包含在同一个载体中。在本发明的另一个实施方式中，所述RNA的组合包含在不同的载体中。
在本发明的一个实施方式中，所述载体包含本发明所述的siRNA的组合，所述RNA的组合包含在同一个载体中。在本发明的另一个实施方式中，所述RNA的组合包含在不同的载体中。
在本发明的一个实施方式中，所述载体包含本发明所述的shRNA编码序列的组合，所述shRNA编码序列的组合包含在同一个载体中。在本发明的另一个实施方式中，所述shRNA编码序列的组合包含在不同的载体中。
在本发明的一个实施方式中，所述载体包含本发明所述的一个或多个表达盒。在本发明的一个优选实施方式中，所述多个表达盒包含在同一个载体中。在本发明的另一个优选实施方式中，所述多个表达盒包含在不同的载体中。
在本发明的优选实施方式中，所述载体包含：
(i)表达盒1，其包含H1启动子和靶向Gag的shRNA的编码序列，所述编码序列如SEQ ID NO：11-13中任一项所示；以及表达盒2，其包含U6启动子和靶向LTR的shRNA的编码序列，所述编码序列如SEQ ID NO：14所示；或者
(ii)表达盒1，其包含H1启动子和靶向Gag的shRNA的编码序列，所述编码序列如SEQ ID NO：12所示；以及表达盒2，其包含U6启动子和靶向Nef的shRNA的编码序列，所述编码序列如SEQ ID NO：15所示。
在本发明的一个实施方式中，载体可以为DNA载体、病毒载体、脂质体、纳米颗粒或外泌体。
在本发明的一个优选实施方式中，所述DNA载体选自由DNA质粒载体、结合其的分子耦联体和结合其的多 聚物所组成的组中。
在本发明的一个实施方式中，所述病毒载体选自由腺相关病毒载体、慢病毒载体和腺病毒载体所组成的组中。
本发明的优选实施方案中，所述病毒载体为重组慢病毒载体。
在本发明的优选实施方式，所述重组慢病毒载体选自：人类免疫缺陷病毒(HIV-1和HIV-2)载体、猴免疫缺陷病毒(SIV)载体、猫免疫缺陷病毒(FIV)载体、马感染性贫血病毒(EIAV)载体和山羊关节炎脑炎病毒(CAEV)载体。
在本发明的优选实施方案中，重组慢病毒载体为重组猴免疫缺陷病毒(SIV)载体。
本发明第八方面还提供了一种重组病毒颗粒，在本发明的优选一个实施例方案中，重组猴免疫缺陷病毒载体为重组猴免疫缺陷病毒，也称为重组猴免疫缺陷病毒颗粒。
在本发明的优选实施方案中，重组猴免疫缺陷病毒或重组猴免疫缺陷病毒颗粒由根据本发明所述的重组猴免疫缺陷基因转移载体或穿梭质粒(携带外源基因的质粒)、包装载体、rev表达载体和VSV-G表达载体制备获得。
在本发明的一个实施方案中，重组猴免疫缺陷基因转移载体还包含RRE、cPPT和WPRE元件。在本发明的一个优选实施方案中，所述重组猴免疫缺陷基因转移载体从5’至3’顺次包含5’LTR区域、RRE、cPPT(中心多嘌呤管道，central polypurine tract)、本发明所述的表达盒、WPRE(土拨鼠肝炎病毒转录后调控因子，woodchuck hepatitis virus posttranscriptional regulatory element)、3’LTR区域。
在本发明的一个实施方式中，包装载体(参见中国专利ZL200680012905.4由原始型包装载体序列号：27改造而来)中除了gag、pol之外，还包括被称作修饰基因vif、vpr和被称作调控基因的tat和rev。在本发明的另一个实施方式中，从包装载体中去除辅助的基因(vif、vpr、tat)，并将rev其以另外的表达载体的形式提供，即rev表达载体。
VSV-G表达载体，是指表达VSV-G(Vesicular stomatitis virus；VSV)蛋白的质粒。本发明的重组SIV病毒载体可以进行VSV-G假型化。VSV-G假型化是指，使载体的被膜包含水疱性口炎病毒(VSV)的表面糖蛋白VSV-G而言的。通过VSV-G表达载体的转染，使重组SIV病毒颗粒在产生时与VSV-G蛋白共存，得到假型化的重组SIV病毒颗粒。
重组慢病毒(例如SIV)通过感染细胞或活体组织，实现外源基因在细胞或活体组织中长期表达。
试剂盒
本发明的第九个方面还提供了一种试剂盒，其包含根据本发明的靶向HIV的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒，或者载体。
细胞
本发明的第十个方面还提供了一种细胞，其包含根据本发明的靶向HIV的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、或者其包含根据本发明所述的表达盒、载体。可通过本领域技术人员熟知的方法和技术来制备和获得本发明的细胞，只要最终的细胞包含如前所述的核酸序列或重组载体即可。在本发明的优选实施方案中，所述细胞由根据本发明的重组病毒载体感染获得。
在本发明的优选实施方案中，所述细胞来源于HIV受体细胞或外周血单核细胞。在本发明的进一步优选实施方案中，所述细胞来源于淋巴细胞。在一些实施方式中，所述细胞来源于T细胞。在另一些实施方式中，所述细胞来源于幼稚T细胞(
Figure PCTCN2022143442-appb-000001
T)、记忆性T细胞、效应性T细胞。在另一些实施方式中，所述细胞来源于细胞毒T细胞、辅助性T细胞、调节性T细胞。在另一些实施方式中，所述T细胞来源于CD4+T细胞，CD8+T细胞。在另一些实施方式中，所述细胞来源于NKT细胞。在另一些实施方案中，所述细胞来源于γδT细胞。在另一些实施方案中，所述细胞来源于NK细胞。在另一些实施方案中，所述细胞来源于抗原提呈细胞，例如巨噬细胞、树突状细胞。
在本发明的进一步优选实施方案中，所述细胞来源于干细胞。在本发明的一些实施方案中，所述干细胞包括造血干细胞(例如，CD34+细胞)或造血祖细胞。在本发明的另一些实施方案中，所述干细胞包括记忆性T干细胞(memory T stem cell)，例如中枢记忆T细胞(central memory T cell)、效应记忆T细胞(effector memory T cell)或干细胞样记忆T细胞(stem cell-like memory T cell)。在本发明的另一个实施方案中，所述干细胞经过诱导可分化为如前所述的任一种T细胞。
在一些实施方案中，所述细胞源自感染HIV的患者。在一些实施方案中，所述细胞源自健康人群。
在本发明的另一实施方案中，所述细胞可以是分离的(或离体的)细胞，或者是在体内的细胞。
在本发明的另一实施方案中，所述细胞来源于工程化细胞，例如TCR-T细胞或含有CAR分子的细胞。
药物组合物、制药用途和治疗方法
本发明的第十一方面还提供了一种药物组合物，其包含根据本发明所述的靶向HIV的RNA或其组合、siRNA或其组合、或者其包含根据本发明所述的shRNA或其组合、shRNA编码序列或其组合、或者其包含根据本发明所述的表达盒、或者其包含根据本发明所述的载体、或者其包含根据本发明所述的细胞，优选地还包含药学上可接受的载体。
在本发明的一个具体实施方式中，药物组合物中进一步包括本领域已知的其他的抗HIV药物。在本发明的另 一个具体实施方式中，所述其他的抗HIV药物包括但不限于核苷类抑制剂、HIV疫苗、广谱中和抗体、CAR-T细胞等。
本发明的第十一个方面还提供了根据本发明的靶向HIV的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒、载体、细胞或药物组合物作为药物的用途。
本发明的第十一个方面还提供了根据本发明的靶向HIV的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒、载体、细胞或药物组合物在制备治疗和/或预防HIV感染或艾滋病的药物中的用途。
本发明的第十一个方面还提供了一种治疗和/或预防HIV感染或艾滋病的方法，其包括向患者施用治疗有效量的根据本发明所述的靶向HIV的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒、重组载体、细胞或药物组合物。
在另一方面，本发明也提供了使用上述的治疗或预防方法与本领域中其他的方法联合治疗HIV感染或艾滋病的方法。在本发明的一个具体实施方式，治疗和/或预防HIV感染或艾滋病的方法进一步包括向患者施用其他的抗HIV药物。在本发明的另一个具体实施方式中，所述其他的抗HIV药物包括但不限于核苷类抑制剂、HIV疫苗、广谱中和抗体、CAR-T细胞等。
在本文中，治疗有效量是指有效地预防、缓解或改善疾病症状或延长患者存活时间的靶向HIV的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒、载体、细胞或药物组合物。
给药方法
本发明的第十二个方面还提供了一种用于治疗艾滋病的给药方法，其包括以下步骤：
(1)采集病人外周血，分离外周血单核细胞(PBMC)，纯化CD4 +T细胞；
(2)用本发明所述的重组病毒载体感染步骤(1)中的CD4 +T细胞，感染后的CD4 +T细胞进行体外扩增；和
(3)将扩增后的CD4 +T细胞回输到患者体内。
在本发明的一个实施方案中，采用CD3/CD28抗体偶联磁珠来激活和扩增CD4 +T细胞。
在本发明的一个优选实施方案中，重组病毒载体感染CD4 +T细胞的感染倍数为1-20，优选为20。
本发明的靶向HIV的RNA或其组合、siRNA或其组合、shRNA或其组合、shRNA编码序列或其组合、表达盒、重组载体、细胞或药物组合物能显著抑制HIV病毒的复制，长期抑制逃逸突变的产生。
本发明产生的有益效果如下：
(1)本发明抗HIV药物使实验病毒株HIV-1ⅢB的复制降低至病毒对照组的20％以下；
(2)本发明抗HIV药物使逆转录酶抑制剂株和蛋白酶抑制剂耐药株的复制分别降低至病毒对照组的10％以下；
(3)本发明提供的抗HIV药物能长期抑制HIV的复制，在很大程度上抑制逃逸突变的产生；和/或
(4)本发明提供的抗HIV药物能有效抑制HIV阳性患者CD4 +T细胞内HIV病毒复制，使病毒载量显著下降。
表1：示例性的siRNA的序列及对应于HIV基因组上的靶序列
Figure PCTCN2022143442-appb-000002
表2：示例性的shRNA及其编码序列
Figure PCTCN2022143442-appb-000003
Figure PCTCN2022143442-appb-000004
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为pGTV-New载体图谱。
图2为重组慢病毒单靶点药物对HIV的抑制作用。
图3为重组慢病毒SIV-Gag3和SIV-G3L2对HIV逃逸突变的抑制曲线图。
图4为重组慢病毒SIV-Gag2和SIV-G2N对HIV逃逸突变的抑制曲线图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。下述实施例中的实验方法，如无特殊说明，均为本领域的常规方法。
生物材料
1、人免疫缺陷病毒(HIV)实验室标准病毒株
实验病毒株HIV-1ⅢB是一种HIV-1的实验室标准病毒株，在人T淋巴细胞系H9上有稳定的传代和复制能力，是文献和实际应用中被广泛使用的HIV体外药效评价模型。本发明HIV-1ⅢB病毒株来自英国Medical  Research Council AIDS Reagent Project，由本实验室扩繁并保存，其滴度为5×10 4TCID50/ml，-70℃以下保存。
2、人免疫缺陷病毒(HIV)耐药株
治疗HIV病毒感染的一、二线药物包括逆转录酶抑制剂、蛋白酶抑制剂、整合酶抑制剂和融合抑制剂，对逆转录酶抑制剂和蛋白酶抑制剂的耐药突变是较为主要的HIV耐药突变，本发明选择这两类耐药株作为实验病毒株。本发明实施例所采用的HIV逆转录酶抑制剂耐药株MT2-010和HIV蛋白酶抑制剂耐药株MT2-884分别是按照专利申请ZL201710172266.8和ZL201210533551.5中所述方法制备获得。
3、H9细胞、MT2细胞均购自深圳市豪地华拓生物科技有限公司，规格均为2×10 6/支。由本实验室复苏，传代培养。
实施例1 携带靶向HIV基因组的shRNA的穿梭质粒的构建
1.中间载体pGTV-new构建：
(1)载体处理：采用EcoR I和NheI双酶切pGTV-PEDF载体(该载体的制备方法详见专利文献CN200680012905.4中实施例1中的1-1，该专利文献通过引用其全文并入本文中)，回收约5.5kb载体片段；
(2)片段1扩增：合成引物1：ATCGAATTCC CGTTTGTGCT AG(SEQ ID NO.16)，引物2：AAGCTTGCGGGATCCCCGCG GCTCTAAAAT TTA(SEQ ID NO.17)，以pGTV-PEDF为模板，扩增约500bp片段1，回收片段1；
(3)片段2扩增：合成引物3：AGCGCAGCGA GTCAGTGAGC G(SEQ ID NO.18)，引物4：GGATCC CGC AAGCTT AG ATCCGCACTT TTTAAAAG(SEQ ID NO.19)，以pGTV-PEDF为模板，扩增约500bp片段2，回收片段2；
(4)重叠PCR：将回收后的片段1和片段2混合作为模板，以引物1和引物4扩增得1000bp左右片段，回收后，采用EcoR I和NheI双酶切回收片段；
(5)将步骤(1)和(4)中经双酶切的载体和回收片段连接，构建成中间载体，命名为pGTV-New，载体图谱见图1。
2.携带靶向HIV基因组中单靶点的shRNA穿梭质粒的构建
分别合成H1-Gag1序列片段、H1-Gag2序列片段、H1-Gag3序列片段、U6-LTR2序列片段、U6-Nef序列片段(本发明载体构建过程中提到的合成Gag、LTR、Nef序列部分均指编码靶向HIV基因组中该基因的shRNA的DNA序列)，在合成时两侧分别引入HindIII和BamHI酶切位点，其中H1、U6为启动子。靶向HIV基因组中Gag基因的shRNA的编码序列分别简称为Gag1、Gag2、Gag3，靶向HIV基因组中LTR基因的shRNA 的编码序列简称为LTR2，靶向HIV基因组中Nef基因的shRNA的编码序列简称为Nef，其序列如下：
CAATGAGGAAGCTGCAGAATTCAAGAGATTCTGCAGCTTCCTCATTG(SEQ ID NO.11)(简称Gag1)；
GTGACATAGCAGGAACTACTTCAAGAGAGTAGTTCCTGCTATGTCAC(SEQ ID NO.12)(简称Gag2)；
ATAGTAAGAATGTATAGCCTTCAAGAGAGGCTATACATTCTTACTAT(SEQ ID NO.13)(简称Gag3)；
TGCTTCAAGCTAGTACCAGTTCAAGAGACTGGTACTAGCTTGAAGCA(SEQ ID NO.14)(简称LTR2)；
CACAAGGCTACTTCCCTGATTCAAGAGATCAGGGAAGTAGCCTTGTG(SEQ ID NO.15)(简称Nef)。
上述合成的H1-Gag3、H1-Gag2、H1-Gag1、H1-LTR2、H1-Nef序列片段分别经HindIII和BamHI双酶切，分别与经同样双酶切的pGTV-new载体连接，即构建成一系列的携带靶向HIV基因组中单靶点的穿梭质粒pGTV-Gag3、pGTV-Gag2、pGTV-Gag1、pGTV-LTR2和pGTV-Nef。各穿梭质粒经测序，验证正确。
3.携带靶向HIV基因组中双靶点的shRNA穿梭质粒的构建
分别合成H1-Gag3-U6-LTR2序列片段、H1-Gag2-U6-LTR2序列片段、H1-Gag1-U6-LTR2序列片段、H1-Gag2-U6-Nef序列片段，在合成时两侧引入HindIII和BamHI酶切位点，其中H1和U6是启动子，H1表达框与U6表达框之间有间隔序列spacer。
上述合成的H1-Gag3-U6-LTR2、H1-Gag2-U6-LTR2、H1-Gag1-U6-LTR2、H1-Gag2-U6-Nef序列片段分别经HindIII和BamHI双酶切，与经同样双酶切的pGTV-new载体连接，即一系列的携带靶向HIV基因组中双靶点的穿梭质粒pGTV-Gag3-LTR2、pGTV-Gag2-LTR2、pGTV-Gag1-LTR2、pGTV-Gag2-Nef构建成功。上述穿梭质粒经测序，验证正确。
4、重组慢病毒SIV的制备
(1)重组慢病毒SIV的包装和纯化
包装载体、rev表达载体、VSV-G表达载体的构建方法详见专利ZL200680012905.4实施例1中的方法。
将细胞株293T细胞(ATCC,CRL-11268)按照每一个T225培养瓶(Coring，Cat#431082)9×10 6细胞进行接种，在20ml含有10％胎牛血清的D-MEM培养基(Thermofisher，Cat#11995-065)中培养48小时后，将培养基置换为10ml已预热的OPTI-MEM培养基(Thermofisher,Cat#31985-070)，作为待转染细胞备用。
为每一个T225培养瓶分别配置转染体系。首先，将实施例1中获得的穿梭质粒60μg与包装载体30μg、rev表达载体12μg、及VSV-G表达载体12μg共同溶解于2.25ml的HBSS缓冲液中，形成质粒混合物。然后，配制167nM的CaCl 2溶液，将上述2.25ml质粒混合物加入2.25ml的上述CaCl 2溶液中，立即涡旋5s混匀，并 在室温条件下孵育10～15min，获得约4.5ml含有质粒DNA-CaCl 2复合物的溶液。随后，将所述溶液加入到上述接种有细胞的培养瓶中，在37℃、5％CO 2培养箱中培养3小时，然后每瓶补加含20％FBS的DMEM培养基，将FBS终浓度调整为10％，继续在37℃、5％CO 2培养箱中培养过夜。
转染后20小时,将每个T225培养瓶中的培养基置换为20ml新鲜的已预热的DMEM培养基。转染后48小时，回收上清，并用0.45μm的过滤器过滤上清以获得初滤液，随后利用高速离心机进行浓缩操作。具体而言，将初滤液置于高速离心管中，于4℃，40000g条件下离心2小时。将离心管内的上清液完全移除后，向每个离心管内加入DPBS以覆盖沉淀，随后，重悬沉淀，获得重组慢病毒SIV浓缩液。上述病毒浓缩液可直接使用或者分装后于-80℃冻存备用。
(2)重组慢病毒SIV基因组滴度测定
抽提病毒的基因组RNA，然后逆转录，再利用得到的cDNA为模板进行实时定量PCR。具体操作步骤参见中国专利ZL201210078288.5的实施例4中4-3记载的滴度测定方法进行重组慢病毒SIV基因组滴度测定(该专利文献通过引用其全文并入本文中)。经检测本实施例中包装出的重组慢病毒SIV的基因组滴度均为3×10 10Vg/ml。
在本申请上下文中，分别由穿梭质粒pGTV-Gag3、pGTV-Gag2、pGTV-Gag1、pGTV-LTR2和pGTV-Nef、pGTV-Gag3-LTR2、pGTV-Gag2-LTR2、pGTV-Gag1-LTR2或pGTV-Gag2-Nef和包装载体、rev表达载体、VSV-G表达载体包装出来的重组慢病毒载体，分别简称为重组慢病毒SIV-Gag3、SIV-Gag2、SIV-Gag1、SIV-LTR2、SIV-Nef、SIV-G3L2、SIV-G2L2、SIV-G1L2、SIV-G2N。
实施例2 重组慢病毒单靶点药物对HIV的抑制作用
(1)H9细胞给药：
复苏H9细胞，用完全培养基(RPMI-1640+10％FBS+PS)传代培养。将细胞重悬于OPTI-MEM培养基，计数并调整浓度为5×10 5个/ml，接种于6孔板，每孔2ml。在6孔板上设置实验组SIV-Gag3、SIV-Gag2、SIV-Gag1、SIV-LTR2或SIV-Nef、SIV-EGFP空载体对照组、和空白对照组(无HIV-1IIIB感染，无慢病毒载体药物)。按感染指数(MOI)为2000分别加入上述重组慢病毒，混匀，于37℃、5％CO 2中培养24小时，每孔补加完全培养基4ml，于37℃5％CO 2中培养72小时。
(2)攻毒
将6孔板中每孔吸弃5ml上清，另加5ml新的完全培养基。将细胞稀释并接种于96孔板，1×10 4个/孔。用5.75ml完全培养基稀释0.5ml HIV-1IIIB，混匀，除空白对照组外，每孔细胞中加入100μl病毒稀释液。混匀，于37℃、5％CO 2培养。分别在HIV-1IIIB感染后的第2天和第4天半量换液，感染后第7天每孔取样150μl，密封后56℃加热30分钟，将HIV病毒热灭活后，-20℃存储，待ELISA检测。
(3)样品ELISA检测
采用的“HIV-1P24抗原检测试剂盒(96T)”(北京金博义生物技术有限公司)进行ELISA检测。用酶标仪测定OD值，求平均值，计算相对浓度，确定重组慢病毒在H9细胞中对HIV-1IIIB的抗病毒活性。
结果如图2所示，5个重组慢病毒单靶点药物在H9细胞上对HIV IIIB实验株的复制都有显著的抑制作用。
实施例3、重组慢病毒双靶点药物对实验株的药效实验
(1)H9细胞给药：
复苏H9细胞，用完全培养基(RPMI-1640+10％FBS+PS)传代培养。将细胞重悬于OPTI-MEM培养基，计数并调整浓度为5×10 5个/ml，接种于6孔板，每孔2ml。在6孔板上设置实验组SIV-G3L2、SIV-G2L2、SIV-G1L2、或SIV-G2N、SIV-EGFP空载体对照组、阳性药物对照组(加依非韦伦，EFV，上海迪赛诺生物医药有限公司，浓度1μM)、不加药对照组(有HIV-1IIIB感染，无重组慢病毒药物)和空白对照组(无HIV-1IIIB感染，无慢病毒载体药物)。按感染指数(MOI)为125、250、500、1000、2000五个剂量分别加入重组慢病毒SIV-G3L2，加入的体积分别为2.1μl、4.2μl、8.4μl、16.7μl、33.3μl，每个剂量一孔。另加入MOI为2000的重组慢病毒SIV-EGFP作为空载体对照，体积为33.3μl。混匀，于37℃、5％CO 2中培养24小时，每孔补加完全培养基4ml，于37℃5％CO 2中培养72小时。
(2)攻毒
将6孔板中每孔吸弃5ml上清，另加5ml新的完全培养基。将细胞稀释并接种于96孔板，1×10 4个/孔。用5.75ml完全培养基稀释0.5ml HIV-1IIIB，混匀，除空白对照组外，每孔细胞中加入100μl病毒稀释液。混匀，于37℃、5％CO 2培养。分别在HIV-1IIIB感染后的第2天和第4天半量换液，感染后第7天每孔取样150μl，密封后56℃加热30分钟，将HIV病毒热灭活后，-20℃存储，待ELISA检测。
(3)样品ELISA检测
采用的“HIV-1P24抗原检测试剂盒(96T)”(北京金博义生物技术有限公司)进行ELISA检测。用酶标仪测定OD值，求平均值，计算相对浓度，确定重组慢病毒在H9细胞中对HIV-1IIIB的抗病毒活性。
表3 受试药物SIV-G3L2在H9细胞中对HIV-1IIIB的抗病毒活性
Figure PCTCN2022143442-appb-000005
Figure PCTCN2022143442-appb-000006
表4 受试药物SIV-G2L2在H9细胞中对HIV-1IIIB的抗病毒活性
Figure PCTCN2022143442-appb-000007
表5 受试药物SIV-G1L2在H9细胞中对HIV-1IIIB的抗病毒活性
Figure PCTCN2022143442-appb-000008
Figure PCTCN2022143442-appb-000009
表6 受试药物SIV-G2N在H9细胞中对HIV-1IIIB的抗病毒活性
Figure PCTCN2022143442-appb-000010
结果如表3-表6所示，在H9细胞中重组慢病毒SIV-G3L2、SIV-G2L2、SIV-G1L2或SIV-G2N对HIV病毒的复制表现出抑制作用，并且具有明显的量效关系，随着重组慢病毒SIV-G3L2、SIV-G2L2、SIV-G1L2或SIV-G2N剂量的增加，对HIV复制的抑制作用增加。在高剂量组(MOI＝2000)中，重组慢病毒SIV-G3L2、SIV-G2L2、SIV-G1L2或SIV-G2N分别将HIV的复制降低至不加药对照组的10.94％、12.32％、15.01％或13.28％，说明重组慢病毒SIV-G3L2、SIV-G2L2、SIV-G1L2或SIV-G2N能有效抑制HIV-1IIIB的复制。
实施例4、重组慢病毒双靶点药物对逆转录酶抑制剂耐药株的药效
实验设计同实施例3，不同点在于受试细胞株为逆转录酶抑制剂的耐药株MT2-010，感染细胞为MT2细胞。首先是细胞培养及重组慢病毒感染：在6孔板上设置实验组SIV-G3L2或SIV-G2N、SIV-EGFP空载体对照组、阳性药物对照组(加依非韦伦，EFV，上海迪赛诺生物医药有限公司，浓度1μM)、不加药对照组(有MT2-010感染，无重组慢病毒药物)和空白对照组。按感染指数(MOI)为125、250、500、1000、2000五个剂量分别加入重组慢病毒。另加入MOI为2000的重组慢病毒SIV-EGFP作为空载体对照。混匀，培养。
然后是攻毒：将MT2细胞稀释并接种于96孔板，1×10 4个/孔。用完全培养基稀释逆转录酶抑制剂的耐药株MT2-010，混匀，除空白对照组外，每孔细胞中加入病毒稀释液。混匀，培养。如实施例3中所述时间取样，利用上述试剂盒进行ELISA检测。用酶标仪测定OD值，求平均值，计算相对浓度，确定重组慢病毒在MT2细胞中对MT2-010的抗病毒活性。
表7 重组慢病毒SIV-G3L2对逆转录酶抑制剂耐药株MT2-010的抗病毒活性
Figure PCTCN2022143442-appb-000011
结果如表7所示，在MT2细胞中，重组慢病毒SIV-G3L2对HIV逆转录酶抑制剂耐药株的复制表现出抑制作用，且具有明显量效关系。随着重组慢病毒SIV-G3L2剂量的增加，对HIV复制的抑制作用增加。在高剂量组(MOI＝2000)中，重组慢病毒SIV-G3L2将HIV的复制降低至不加药对照组的9.14±2.09％，这说明重组慢病毒SIV-G3L2能有效抑制逆转录酶抑制剂耐药株的复制。
表8 重组慢病毒SIV-G2N对逆转录酶抑制剂耐药株MT2-010的抗病毒活性
Figure PCTCN2022143442-appb-000012
如表8所示，在MT2细胞中，重组慢病毒SIV-G2N对HIV逆转录酶抑制剂耐药株的复制同样表现出较强的抑制作用，且具有明显量效关系。随着重组慢病毒SIV-G2N剂量的增加，对HIV复制的抑制作用增加。在高剂量组(MOI＝2000)中，重组慢病毒SIV-G2N将HIV的复制降低至不加药对照组的11.31±1.34％，这说明重组慢病毒SIV-G2N能有效抑制逆转录酶抑制剂耐药株的复制。
实施例5、重组慢病毒药物对蛋白酶抑制剂耐药株的药效
实验方法同实施例3的步骤1，不同点在于受试细胞株为HIV蛋白酶酶抑制剂的耐药株MT2-884，感染细胞为MT2细胞。首先是细胞培养及重组慢病毒SIV-G3L2或SIV-G2N感染：在6孔板中培养细胞，并设置实验组SIV-G3L2、SIV-G2N、SIV-EGFP空载体对照组、阳性药物对照组(加依非韦伦，EFV，上海迪赛诺生物医药有限公司，浓度1μM)、不加药对照组(有MT2-884感染，无重组慢病毒药物)和空白对照组。按感染指数(MOI)为125、250、500、1000、2000五个剂量分别加入重组慢病毒。另加入MOI为2000的重组慢病毒SIV-EGFP作为空载体对照。混匀，培养。
然后是攻毒：将MT2细胞稀释并接种于96孔板，1×10 4个/孔。用完全培养基稀释HIV蛋白酶酶抑制剂的耐药株MT2-884，除空白对照组外，每孔细胞中加入病毒稀释液。混匀，培养。如实施例3中所述时间取样，利用上述试剂盒进行ELISA检测。用酶标仪测定OD值，求平均值，计算相对浓度，确定重组慢病毒在MT2细胞中对MT2-884的抗病毒活性。
表9 重组慢病毒SIV-G3L2对HIV蛋白酶抑制剂耐药株MT2-884的抗病毒活性
Figure PCTCN2022143442-appb-000013
结果如表9所示，在MT2细胞中，重组慢病毒SIV-G3L2对HIV蛋白酶抑制剂耐药株的复制表现出抑制作用，且具出明显的量效关系。随着重组慢病毒SIV-G3L2给药剂量的增加，对HIV复制的抑制作用增加。在高剂量组中(MOI＝2000)，重组慢病毒SIV-G3L2将HIV的复制降低至不加药对照组的9.72±0.70％，这说明重组慢病毒SIV-G3L2能有效抑制HIV蛋白酶抑制剂耐药株的复制。
在MT2细胞中，重组慢病毒SIV-G2N对HIV蛋白酶抑制剂耐药株的复制表现出抑制作用，且具出明显的量效关系。随着重组慢病毒SIV-G2N给药剂量的增加，对HIV复制的抑制作用增加。在高剂量组中(MOI＝2000)，重组慢病毒SIV-G2N将HIV的复制降低至不加药对照组的9.02±0.70％，这说明重组慢病毒SIV-G2N能有效抑制HIV蛋白酶抑制剂耐药株的复制(结果未显示)。
实施例6、重组慢病毒药物对HIV逃逸突变的抑制
用实施例1制备的慢病毒载体药物SIV-G3L2或SIV-G2N感染H9细胞，对照组分别为重组慢病毒SIV-EGFP空载体和重组慢病毒单靶点药物SIV-Gag3或SIV-Gag2，病毒滴度均为3×10 10Vg/ml。六孔板中接种H9细胞，1×10 6个/孔。同时每孔分别加入33.3μl上述的重组慢病毒，即MOI＝2000。感染后24小时，每孔补2ml完全培养基，继续培养48小时后将细胞稀释为单克隆培养。
提取重组慢病毒感染后的H9细胞的DNA，通过PCR扩增条带大小检测病毒基因整合情况。三种病毒感染的样品各随机挑选5个单克隆细胞，进行HIV逃逸突变实验。感染后的细胞株分别命名为H9-EGFP-1、H9-EGFP-2、H9-EGFP-3、H9-EGFP-4、H9-EGFP-5，H9-Gag3-1、H9-Gag3-2、H9-Gag3-3、H9-Gag3-4、H9-Gag3-5，H9-G3L2-1、H9-G3L2-2、H9-G3L2-3、H9-G3L2-4、H9-G3L2-5。
上述15个单细胞株的培养体系中，按MOI＝0.002TCID50攻毒HIV-1IIIB，共培养30天，每3天进行一次细胞传代，并检测上清p24。当上清中p24浓度明显上升，说明重组慢病毒药物对HIV抑制失败，则停止这一细胞株培养。不同细胞株的检测结果见表10-11和图3-4。
表10 SIV-G3L2双靶点药物对HIV逃逸突变的抑制
Figure PCTCN2022143442-appb-000014
Figure PCTCN2022143442-appb-000015
如表10和图3所示，重组慢病毒SIV-eGFP空载体对HIV病毒的复制没有抑制效果，培养3-6天时检测到HIV的明显复制。重组慢病毒单靶点药物SIV-Gag3感染H9细胞后，H9细胞表现出对HIV的抗性，但4个单克隆细胞株在第18-21天检测到HIV的明显复制，说明此时出现对重组慢病毒单靶点药物SIV-Gag3的耐药突变。重组慢病毒双靶点药物SIV-G3L2感染H9细胞后，5个单克隆细胞株在实验培养时间范围内(30天)都没有检测到HIV的明显复制，说明重组慢病毒双靶点药物SIV-G3L2不仅对HIV的复制有明显抑制作用，而且可以在很大程度上抑制耐药突变的产生。
表11 SIV-G2N双靶点药物对HIV逃逸突变的抑制
Figure PCTCN2022143442-appb-000016
如表11和图4所示，重组慢病毒SIV-EGFP空载体对HIV病毒的复制没有抑制效果，培养3-6天时检测到HIV的明显复制。重组慢病毒单靶点药物SIV-Gag2感染H9细胞后，H9细胞表现出对HIV的抗性，但所有的5个单克隆细胞株在第18-30天均检测到HIV的明显复制，说明此时出现对重组慢病毒单靶点药物SIV-Gag2的耐药突变。重组慢病毒双靶点药物SIV-G2N感染H9细胞后，3个单克隆细胞株在实验培养时间范围内(30天)都没有检测到HIV的明显复制，2个单克隆细胞株在27-30天时有少量复制，说明重组慢病毒双靶点药物SIV-G2N不仅对HIV的复制有明显抑制作用，而且可以在很大程度上抑制耐药突变的产生。
实施例7、重组慢病毒药物对HIV患者CD4 +T细胞内HIV病毒复制的抑制作用
1、PBMC细胞分离
利用Ficoll-Paque PLUS(GE，Cat#17-1440-02)，按照试剂盒说明书分离EDTA抗凝全血(5例初次确诊HIV阳性患者外周血，来自佑安医院性病艾滋病重点实验室，分别编号为061302、061903、061904、062003、062004)中的PBMC细胞。具体地，利用高速冷冻离心机(Thermo Scientific Sorvall ST40R)，使用水平转子以800g离心30min，设置离心升速为3，降速为0，温度为20℃。离心结束后，轻轻地吸出PBMC细胞层，并置于新的离心管中。使用所吸出细胞层的3-5倍体积的DPBS清洗PBMC，随后，在20℃，300g条件下，离心10min。离心结束后移除上清，向细胞沉淀中加入预冷的红细胞裂解液(天津灏洋NH4CL2009)，4℃放置2分钟以裂解红细胞。随后，加入预冷的DPBS，使总体积为40-45ml，上下吹打使细胞混匀，在300g，4℃条件下离心10min，从而获得PBMC细胞。
2、CD4 +T细胞分离与培养
采用人CD4+T细胞分离试剂盒(Antibody Cocktail；Biotech-beads)进行CD4+T细胞分离，具体地，根据上述PBMC细胞计数结果，按每1×10 7细胞加入40ul MACS Buffer重悬细胞(细胞不足1.5×10 7，按1×10 7计；细胞＞1.5×10 7，按2×10 7计算，以此类推)，再加入10ul Antibody Cocktail，充分混匀，室温避光孵育10min。然后再加入30ul(等比例)MACS Buffer，和20ul Biotech-beads，混匀，室温避光孵育10min。同时将LS分选柱置于分悬架上，并在分选柱上加放筛网，并加入2ml MACS Buffer润洗两次。孵育好的细胞悬液，加入1-2ml MACS Buffer，混匀，加至已润洗好的细胞分选柱上，收集流出后的悬液。细胞悬液流尽后，加入2ml MACS Buffer洗两遍柱子，并收集。收集的细胞悬液，充分混匀后，取300ul细胞悬液，部分用于计数。每2×10 6细胞加1ml T细胞完全培养基(rHuIL2200IU/ml，1X GlutaMAX)重悬细胞。
利用包被了抗CD3抗体和抗CD28抗体的磁珠来激活CD4+T细胞。按照试剂盒Dynabeads Human T-Activator CD3/CD28for T-Cell Expansion and Activation(Thermofisher，11131D)的说明书来操作。首先根据获得的CD4+T细胞的计数结果，将细胞和已清洗好的CD3/CD28磁珠按照1×10 6个细胞对应25ul原始浓度的磁珠进行混合，并补充终浓度为30IU/ml的IL-2(R&D，202-IL-010)和终浓度为3-5％的人血清白蛋白，或人AB血清，充分混匀后，37℃培养48小时。将细胞与磁珠混合物取至15ml离心管中，2000rpm振荡30s后，放置于磁力架上1分钟。吸取上清至新的离心管中，利用完全培养基重悬细胞，300g离心5分钟，离心后弃上清，用新鲜完全培养基重悬细胞沉淀，并取部分进行计数及流式检测。
3、CD4 +T细胞感染与HIV病毒载量检测
Recombinant Human Fibronectin Fragment
Figure PCTCN2022143442-appb-000017
以10μg/cm 2的浓度包被表面未处理的24孔板，37℃孵育1小时。用DPBS洗2次，加入500μl 1％PBS-BSA，37℃封闭1小时。用DPBS洗3次，加入500μl完全培养基。将重组慢病毒SIV-G3L2，SIV-G2N或SIV-GFP，按照MOI＝20加入各条件组，37℃孵育1小时。 1小时后，每孔弃250μl培养液。将上述获得的细胞2000rpm，离心5分钟弃上清，用500μl培养基重悬，并接种至各孔，37℃、5％CO 2培养。分别在2天和3天重复以上感染步骤。在培养第7天、第14天收集培养上清，用HIV-1核酸定量检测试剂盒(QPCR)(达安基因)检测HIV-1病毒载量。参照试剂盒说明书，提取病毒核酸，配制QPCR体系，反应程序为50℃，15min；95℃15min；(94℃15sec，55℃45sec)×45cycles；采用FAM/TAMRA和VIC/NONE双荧光检测，多个批次的样本检测结果列在表12中。
表12 SIV-G3L2和SIV-G2N双靶点药物对HIV患者CD4+T细胞内HIV病毒复制的抑制作用
Figure PCTCN2022143442-appb-000018
由表12可知，重组慢病毒药物SIV-G3L2或SIV-G2N均可以有效抑制HIV阳性患者CD4 +T细胞内HIV病 毒复制，经过7天的体外培养，几例患者CD4 +T细胞培养上清液中病毒载量显著下降。
以上所述仅为本发明的较佳实施例而已，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均包含在本发明的保护范围内。

Claims (48)

  1. 一种用于靶向HIV的RNA序列，其序列如SEQ ID NO：6-10或20-24中任一项所示。
  2. 一种靶向HIV的siRNA，其包括正义链和与之互补的反义链，所述siRNA由如下序列组成：
    (i)正义链序列如SEQ ID NO：6所示，反义链的序列如SEQ ID NO：20所示；
    (ii)正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示；
    (iii)正义链序列如SEQ ID NO：8所示，反义链序列如SEQ ID NO：22所示；
    (iv)正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；或
    (v)正义链序列如SEQ ID NO：10所示，反义链序列如SEQ ID NO：24所示。
  3. 一种靶向HIV的shRNA，所述shRNA包括权利要求1或2中所述的任一序列。
  4. 一种如权利要求3中所述的shRNA，其序列如SEQ ID NO：25-29中任一项所示。
  5. 一种编码如权利要求3或4中所述的shRNA的序列。
  6. 一种如权利要求5中所述的shRNA的编码序列，其序列如SEQ ID NO：11-15中任一项所示。
  7. 一种靶向HIV的RNA序列的组合，所述组合包括如权利要求1中所述的RNA序列中的至少一种。
  8. 一种靶向HIV的siRNA组合，所述组合包括如权利要求2中所述的siRNA中的至少一种。
  9. 一种靶向HIV的shRNA组合，所述组合包括如权利要求3或4中所述的shRNA中的至少一种。
  10. 一种靶向HIV的shRNA的编码序列的组合，所述组合包括如权利要求5或6中所述的shRNA的编码序列中的至少一种。
  11. 一种靶向HIV的RNA序列的组合，所述组合包括靶向Gag基因的RNA序列、靶向LTR基因的RNA序列或者靶向Nef基因的RNA序列中的至少两种。
  12. 一种如权利要求11中所述的RNA序列的组合，所述组合包括：
    (i)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，
    (ii)靶向Gag基因的RNA序列和靶向Nef基因的RNA序列，或者
    (iii)靶向LTR基因的RNA序列和靶向Nef基因的RNA序列。
  13. 一种如权利要求11或12中所述的RNA序列的组合，其中：
    靶向HIV的Gag基因的RNA序列选自由SEQ ID NO：1-3中任一项所示序列中的连续19-23个核苷酸组成的序列或与之反向互补的序列，优选如SEQ ID NO：6-8或20-22中任一项所示序列；
    靶向HIV的LTR基因的RNA序列选自由SEQ ID NO：4中的连续19-26个核苷酸组成的序列或与之反向互补的序列，优选如SEQ ID NO：9或23所示序列；
    靶向HIV的Nef基因的RNA序列选自由SEQ ID NO：5中的连续19-25个核苷酸组成的序列或与之反向 互补的序列，优选如SEQ ID NO：10或24所示序列。
  14. 一种如权利要求11-13中任一项所述的RNA序列的组合，所述组合包括：
    (i)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：6或20所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；
    (ii)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：7或21所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；
    (iii)靶向Gag基因的RNA序列和靶向LTR基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：8或22所示，靶向LTR基因的RNA序列如SEQ ID NO：9或23所示；或者
    (iv)靶向Gag基因的RNA序列和靶向Nef基因的RNA序列，其中靶向Gag基因的RNA序列如SEQ ID NO：7或21所示，靶向Nef基因的RNA序列如SEQ ID NO：10或24所示。
  15. 一种靶向HIV的siRNA组合，所述组合包括靶向Gag基因的siRNA、靶向LTR基因的siRNA或者靶向Nef基因的siRNA中的至少两种。
  16. 一种如权利要求15中所述的siRNA组合，所述组合包括：
    (i)靶向Gag基因的siRNA和靶向LTR基因的siRNA，
    (ii)靶向Gag基因的siRNA和靶向Nef基因的siRNA，或者
    (iii)靶向LTR基因的siRNA和靶向Nef基因的siRNA。
  17. 一种如权利要求15或16中所述的siRNA组合，其中：
    靶向HIV的Gag基因的siRNA正义链序列选自由SEQ ID NO：1-3中任一项所示序列中的连续19-23个核苷酸组成的序列，优选如SEQ ID NO：6-8中任一项所示序列，反义链序列为与正义链反向互补的序列；
    靶向HIV的LTR基因的siRNA正义链序列选自由SEQ ID NO：4中的连续19-26个核苷酸组成的序列，优选如SEQ ID NO：9所示序列，反义链序列为与正义链反向互补的序列；
    靶向HIV的Nef基因的siRNA正义链序列选自由SEQ ID NO：5中的连续19-25个核苷酸组成的序列，优选如SEQ ID NO：10所示序列，反义链序列为与正义链反向互补的序列。
  18. 一种如权利要求15-17中任一项所述的siRNA组合，所述组合包括：
    (i)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：6所示，反义链序列如SEQ ID NO：20所示，靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；
    (ii)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示，靶向LTR基因的siRNA的正义链序列如SEQ  ID NO：9所示，反义链序列如SEQ ID NO：23所示；
    (iii)靶向Gag基因的siRNA和靶向LTR基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：8所示，反义链序列如SEQ ID NO：22所示，靶向LTR基因的siRNA的正义链序列如SEQ ID NO：9所示，反义链序列如SEQ ID NO：23所示；或者
    (iv)靶向Gag基因的siRNA和靶向Nef基因的siRNA，其中靶向Gag基因的siRNA的正义链序列如SEQ ID NO：7所示，反义链序列如SEQ ID NO：21所示，靶向Nef基因的siRNA的正义链序列如SEQ ID NO：10所示，反义链序列如SEQ ID NO：24所示。
  19. 一种靶向HIV的shRNA的组合，所述组合包括靶向Gag基因的shRNA、靶向LTR基因的shRNA或者靶向Nef基因的shRNA中的至少两种。
  20. 一种如权利要求19中所述的靶向HIV的shRNA组合，所述组合包括：
    (i)靶向Gag基因的shRNA和靶向LTR基因的shRNA，
    (ii)靶向Gag基因的shRNA和靶向Nef基因的shRNA，或者
    (iii)靶向LTR基因的shRNA和靶向Nef基因的shRNA。
  21. 一种如权利要求19或20中所述的靶向HIV的shRNA组合，其中：
    所述靶向Gag基因的shRNA的序列包括：(i)SEQ ID NO：6和SEQ ID NO：20所示序列；(ii)SEQ ID NO：7和SEQ ID NO：21所示序列；或者(iii)SEQ ID NO：8和SEQ ID NO：22所示序列；
    所述靶向LTR基因的shRNA的序列包括SEQ ID NO：9和SEQ ID NO：23所示序列；
    所述靶向Nef基因的shRNA的序列包括SEQ ID NO：10和SEQ ID NO：24所示序列。
  22. 一种如权利要求19-21任一项中所述的靶向HIV的shRNA组合，其中所述组合包括：
    (i)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中靶向Gag基因的shRNA的序列包括SEQ ID NO：6和SEQ ID NO：20所示序列，靶向LTR基因的shRNA的序列包括SEQ ID NO：9和SEQ ID NO：23所示序列；
    (ii)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中靶向Gag基因的shRNA的序列包括SEQ ID NO：7和SEQ ID NO：21所示序列，靶向LTR基因的shRNA的序列包括SEQ ID NO：9和SEQ ID NO：23所示序列；
    (iii)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中所述靶向Gag基因的shRNA的序列包括SEQ ID NO：8和SEQ ID NO：22所示序列；所述靶向LTR基因的shRNA的序列包括SEQ ID NO：9和SEQ ID NO：23所示序列；或者
    (iv)靶向Gag基因的shRNA和靶向Nef基因的shRNA，其中所述靶向Gag基因的shRNA的序列包括 SEQ ID NO：7和SEQ ID NO：21所示序列，所述靶向Nef基因的shRNA的序列包括SEQ ID NO：10和SEQ ID NO：24所示序列。
  23. 一种如权利要求19或20中所述的靶向HIV的shRNA组合，其中：
    靶向Gag基因的shRNA选自SEQ ID NO：25-27中任一项所示序列；
    靶向LTR基因的shRNA为SEQ ID NO：28所示序列；
    靶向Nef基因的shRNA为SEQ ID NO：29所示序列。
  24. 一种如权利要求19-23中任一项所述的靶向HIV的shRNA组合，所述组合包括：
    (i)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中靶向Gag基因的shRNA的序列如SEQ ID NO：25所示，靶向LTR基因的shRNA的序列如SEQ ID NO：28所示；
    (ii)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中靶向Gag基因的shRNA的序列如SEQ ID NO：26所示，靶向LTR基因的shRNA的序列如SEQ ID NO：28所示；
    (iii)靶向Gag基因的shRNA和靶向LTR基因的shRNA，其中靶向Gag基因的shRNA的序列如SEQ ID NO：27所示；靶向LTR基因的shRNA的序列如SEQ ID NO：28所示；或者
    (iv)靶向Gag基因的shRNA和靶向Nef基因的shRNA的，其中靶向Gag基因的shRNA的序列如SEQ ID NO：26所示；靶向Nef基因的shRNA如SEQ ID NO：29所示。
  25. 一种靶向HIV的shRNA的编码序列的组合，所述组合包括靶向Gag基因的shRNA的编码序列、靶向LTR基因的shRNA的编码序列或者靶向Nef基因的shRNA的编码序列中的至少两种。
  26. 一种如权利要求25中所述的靶向HIV的shRNA编码序列组合，所述组合包括：
    (i)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，
    (ii)靶向Gag基因的shRNA的编码序列和靶向Nef基因的shRNA的编码序列，或者
    (iii)靶向LTR基因的shRNA的编码序列和靶向Nef基因的shRNA的编码序列。
  27. 一种如权利要求25或26中所述的靶向HIV的shRNA的编码序列组合，其中：
    所述靶向Gag基因的shRNA包括：(i)SEQ ID NO：6和SEQ ID NO：20所示序列；(ii)SEQ ID NO：7和SEQ ID NO：21所示序列；或者(iii)SEQ ID NO：8和SEQ ID NO：22所示序列；
    所述靶向LTR基因的shRNA包括SEQ ID NO：9和SEQ ID NO：23所示序列；
    所述靶向Nef基因的shRNA包括SEQ ID NO：10和SEQ ID NO：24所示序列。
  28. 一种如权利要求25-27任一项中所述的靶向HIV的shRNA编码序列组合，其中所述组合包括：
    (i)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中靶向Gag基因的 shRNA包括SEQ ID NO：6和SEQ ID NO：20所示序列，靶向LTR基因的shRNA包括SEQ ID NO：9和SEQ ID NO：23所示序列；
    (ii)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中靶向Gag基因的shRNA包括SEQ ID NO：7和SEQ ID NO：21所示序列，靶向LTR基因的shRNA包括SEQ ID NO：9和SEQ ID NO：23所示序列；
    (iii)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中靶向Gag基因的shRNA包括(iii)SEQ ID NO：8和SEQ ID NO：22所示序列；靶向LTR基因的shRNA包括SEQ ID NO：9和SEQ ID NO：23所示序列；或者
    (iv)靶向Gag基因的shRNA的编码序列和靶向Nef基因的shRNA的编码序列，其中所述靶向Gag基因的shRNA包括SEQ ID NO：7和SEQ ID NO：21所示序列，所述靶向Nef基因的shRNA包括SEQ ID NO：10和SEQ ID NO：24所示序列。
  29. 一种如权利要求25或26中的所述靶向HIV的shRNA编码序列组合，其中：
    靶向Gag基因的shRNA的编码序列选自SEQ ID NO：11-13中任一项所示序列；
    靶向LTR基因的shRNA的编码序列为SEQ ID NO：14所示序列；
    靶向Nef基因的shRNA的编码序列为SEQ ID NO：15所示序列。
  30. 一种如权利要求25-29中任一项所述的靶向HIV的shRNA编码序列组合，所述组合包括：
    (i)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中所述的靶向Gag基因的shRNA的编码序列如SEQ ID NO：11所示，靶向LTR基因的shRNA的编码序列如SEQ ID NO：14所示，
    (ii)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中所述的靶向Gag基因的shRNA的编码序列如SEQ ID NO：12所示，靶向LTR基因的shRNA的编码序列如SEQ ID NO：14所示，
    (iii)靶向Gag基因的shRNA的编码序列和靶向LTR基因的shRNA的编码序列，其中所述的靶向Gag基因的shRNA的编码序列如SEQ ID NO：13所示，靶向LTR基因的shRNA的编码序列如SEQ ID NO：14所示，或者
    (iv)靶向Gag基因的shRNA的编码序列和靶向Nef基因的shRNA的编码序列，其中所述的靶向Gag基因的shRNA的编码序列如SEQ ID NO：12所示，靶向Nef基因的shRNA的编码序列如SEQ ID NO：15所示。
  31. 一种表达盒，其包含权利要求5、6中任一项所述的shRNA编码序列，或权利要求10或25-30中任一 项所述的shRNA编码序列的组合。
  32. 一种如权利要求31中所述的表达盒，其进一步包含启动子，其中所述启动子可以是任何RNA聚合酶II启动子或RNA聚合酶III启动子；优选为RNA聚合酶III启动子，所述RNA聚合酶II启动子或RNA聚合酶III启动子还包括任何合成的或改造的DNA片段。
  33. 一种如权利要求32中所述的表达盒，其中RNA聚合酶III启动子为H1启动子、U6启动子、7SK启动子。
  34. 一种如权利要求31-33中任一项所述的表达盒，其中靶向Gag基因的shRNA由H1启动子驱动，靶向LTR或Nef的shRNA由U6启动子驱动表达，或者靶向Gag基因的shRNA由U6启动子驱动表达，靶向LTR或Nef的shRNA由H1启动子驱动表达。
  35. 一种载体，其包含如权利要求1所述的RNA、权利要求2所述的siRNA、权利要求3或4中所述的shRNA、权利要求5或6中所述的shRNA的编码序列、权利要求7或11-14中任一项所述的RNA序列的组合、权利要求8或15-18中任一项所述的siRNA组合、权利要求9或19-24中任一项所述的shRNA组合、或者权利要求10或25-30中任一项所述的shRNA编码序列的组合、或如权利要求31-34中任一项所述的表达盒。
  36. 一种如权利要求35中所述的载体，其包含两个或两个以上的表达盒，其中所述两个或两个以上的表达盒包含在同一个载体中，或者所述两个或两个以上的表达盒分别包含在不同的载体中。
  37. 一种如权利要求35或36中所述的载体，所述载体选自质粒载体、病毒载体、脂质体、纳米颗粒、或外泌体；其中所述病毒载体选自腺相关病毒载体、慢病毒载体或腺病毒载体。
  38. 一种如权利要求37所述的载体，其中所述慢病毒载体选自人类免疫缺陷病毒(HIV-1和HIV-2)载体、猴免疫缺陷病毒(SIV)载体、猫免疫缺陷病毒(FIV)载体、马感染性贫血病毒(EIAV)载体和山羊关节炎脑炎病毒(CAEV)载体。
  39. 一种细胞，其包含权利要求1中所述的RNA序列、权利要求2所述的siRNA、权利要求3或4中所述的shRNA、权利要求5或6中所述的shRNA的编码序列、权利要求7或11-14中任一项所述的RNA序列的组合、权利要求8或15-18中任一项所述的siRNA组合、权利要求9或19-24中任一项所述的shRNA组合、权利要求10或25-30中任一项所述的shRNA的编码序列组合、权利要求31-34中任一项所述的表达盒、或权利要求35-38中任一项所述的载体。
  40. 一种如权利要求39中所述的细胞，所述细胞来源于HIV受体细胞、外周血单核细胞或淋巴细胞；优选的，所述细胞来源于T细胞(例如，幼稚T细胞、记忆性T细胞、效应性T细胞、细胞毒T细胞、辅助性T细胞、调节性T细胞、CD4+T细胞、CD8+T细胞、NKT细胞、γδT细胞等)、NK细胞、抗原提呈细胞(例如，巨噬细胞、树突状细胞等)。
  41. 一种如权利要求39所述的细胞，其中所述细胞来源于干细胞，包括但不限于造血干细胞、造血祖细胞、记忆性T干细胞(例如中枢记忆T细胞、效应记忆T细胞或干细胞样记忆T细胞)。
  42. 一种如权利要求39-41中任一项所述的细胞，所述细胞是来源于哺乳动物，灵长类或人体的细胞。
  43. 一种如权利要求39中所述的细胞，所述细胞来源于工程化细胞。
  44. 一种药物组合物，其包含权利要求1中所述的RNA序列、权利要求2所述的siRNA、权利要求3或4中所述的shRNA、权利要求5或6中所述的shRNA的编码序列、权利要求7或11-14中任一项所述的RNA序列的组合、权利要求8或15-18中任一项所述的siRNA组合、权利要求9或19-24中任一项所述的shRNA组合、权利要求10或25-30中任一项所述的shRNA的编码序列组合、权利要求31-34中任一项所述的表达盒、或权利要求35-38中任一项所述的载体、或权利要求39-43中任一项所述的细胞，以及药学上可接受的载体。
  45. 如权利要求44所述的药物组合物，所述药物组合物中进一步包括其他的抗HIV药物，包括但不限于核苷类抑制剂、HIV疫苗、广谱中和抗体、CAR-T细胞等。
  46. 权利要求1中所述的RNA序列、权利要求2所述的siRNA、权利要求3或4中所述的shRNA、权利要求5或6中所述的shRNA的编码序列、权利要求7或11-14中任一项所述的RNA序列的组合、权利要求8或15-18中任一项所述的siRNA组合、权利要求9或19-24中任一项所述的shRNA组合、权利要求10或25-30中任一项所述的shRNA的编码序列组合、权利要求31-34中任一项所述的表达盒、或权利要求35-38中任一项所述的载体、或权利要求39-43中任一项所述的细胞、或权利要求44或45所述的药物组合物在制备用于治疗HIV感染或艾滋病的药物中的应用。
  47. 一种预防和/或治疗HIV感染或艾滋病的方法，包括向患者施用权利要求1中所述的RNA序列，权利要求2所述的siRNA、权利要求3或4中所述的shRNA、权利要求5或6中所述的shRNA的编码序列、权利要求7或11-14中任一项所述的RNA序列的组合，权利要求8或15-18中任一项所述的siRNA组合、权利要求9或19-24中任一项所述的shRNA组合、权利要求10或25-30中任一项所述的shRNA的编码序列组合、权利要求31-34中任一项所述的表达盒、或权利要求35-38中任一项所述的重组载体、或权利要求39-43中任一项所述的细胞、或权利要求44或45中所述的药物组合物。
  48. 如权利要求47中所述的方法，该方法进一步包括向患者施用其他的抗HIV药物，包括但不限于核苷类抑制剂、HIV疫苗、广谱中和抗体、CAR-T细胞等。
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