MULTIPLEXED SHRNAS AND USES THEREOF

Abstract

Nucleic acid molecules such as shRNA dusters and artificial miRNA dusters are disclosed. Also disclosed are methods of use, compositions, cells, viral particles, and kits relating to the nucleic acid molecules disclosed herein.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the continuation of U.S. patent application Ser. No. 15/519,359, filed on Apr. 14, 2017, which is a U.S. National Phase of International Application No. PCT/US2015/055537, filed on Oct. 14, 2015 under the PCT (Patent Cooperation Treaty), and claims priority to U.S. Provisional Patent Application Nos. 62/063,583, filed Oct. 14, 2014, and 62/240,709, filed on Oct. 13, 2015. The entire contents of these applications are incorporated herein by reference.

FIELD OF THE INVENTION

The disclosure relates to multiplexed shRNAs and uses thereof.

BACKGROUND

Following successful clinical trials, there has been a resurgence of interest in using ribonucleic acid interference (RNAi) to treat many diseases including cancer and HIV-1 infection (Davidson and McCray (2011). Nat Rev Genet 12: 329-340). Although short hairpin RNAs (shRNA) can be used to inhibit human immunodeficiency virus (HIV-1) replication (Lee et al. (2005). Blood 106: 818-826; Lee et al. (2002). Nat Biotechnol 20: 500-505; DiGiusto et al. (2010). Sci Transl Med 2: 36ra43), one potential problem of shRNAs expressed under the commonly used Pol III promoters is that the overexpressed shRNA can cause toxicities by competing with endogenous microRNAs (miRNAs) for cytoplasmic transport (Grimm et al. (2006). Nature 441: 537-541; McBride et al. (2008). Proc Natl Acad Sci USA 105: 5868-5873; Boudreau et al. (2008) RNA 14: 1834-1844). Moreover, shRNAs in the cytoplasm that are processed by the Dicer enzyme can also bind all Ago proteins, further compromising endogenous miRNA function (Grimm et al. (2010). J Clin Invest 120: 3106-3119).

A major concern in using traditional shRNA for HIV-1 therapy is the rapid emergence of escape mutations since HIV-1 evolves very quickly due to highly error-prone reverse transcription (Boden et at (2003). J Virol 77: 11531-11535; Das et at (2004). J Virol 78: 2601-2605). Therefore, to minimize this problem, it is important to be able to express multiple shRNAs in a single vector to target host factors and viral genes. However, attempts to express multiple shRNAs using tandem repeats of the same promoter were unsuccessful because the cassettes were prone for deletion due to homologous recombination (Brake et at (2008). Mol Ther 16: 557-564). Similarly, expression of multiple shRNAs using the same miRNA backbone is also likely to lead to deletion by homologous recombination. Alternatively, multiple shRNAs may be expressed using the naturally occurring polycistronic miRNA clusters (Liu et at (2008), Nucleic Acids Res 36: 2811-2824; Aagaard et al. (2008). Gene Ther 15: 1536-1549; Chung et al. (2012). Hum Gene Ther 23: 1200-1208). However, currently the maximum number of shRNAs that can be expressed in a polycistronic miRNA backbone is limited (Liu et at (2008). Nucleic Acids Res 36: 2811-2824). Although mathematical modeling suggests that a combination of four shRNAs may be sufficient to overcome escape, this requires all four shRNAs to be matched to each of the circulating 100's of viral variants and the viral quasispecies present in patients (McIntyre et al. (2011). AIDS Res Ther 8: 1).

MiRNAs are transcribed as long (up to several kb) primary miRNAs (pri-miRNAs) containing the miRNA duplex with long 5′ and 3′ flanking sequences (Lee et al. (2004). EMBO J 23: 4051-4060; Cai et at (2004). RNA 10: 1957-1966). Pri-miRNAs are processed in the nucleus by Drosha/DGCR8 complex to generate pre-miRNAs, which are then exported to the cytoplasm for further processing by Dicer to generate mature miRNAs (Lee et at (2003). Nature 425: 415-419; Han et at (2004). Genes Dev 18: 3016-3027). Generally, long stretches of flanking sequences are used to design shRNAs in a endogenous miRNA backbone (shRNA-miRs) in the hope that it will lead to processing by Drosha/DGCR8, just like primary miRNAs (Chang et al. (2006). Nat Methods 3: 707-714). However, multiplexing shRNAs with different miRNA backbones by this method is impractical because of the long stretches of flanking sequences.

Delivery of shRNA to the relevant (HIV-1 susceptible) target cells in vivo remains a great challenge. It is a common strategy to deliver shRNA via a VSV-G pseudotyped lentiviral vector because the VSV receptor is ubiquitously expressed in most cell types. VSV-G pseudotyping allows transduction of many different cell types. However, CD4 T cells, the major targets of HIV-1, can be transduced by VSV-G pseudotyped lentivirus only after activation, which could impact their repertoire and long-term survival (Agosto et al. (2009). Viral 83: 8153-8162; Yu et al, (2009). PLoS Pathog 5: e1000633).

In spite of current therapies, the need exists for developing novel therapies that treat and prevent HIV and AIDS.

SUMMARY

The disclosure provides, at least in part, nucleic acid molecules such as shRNA clusters encoding shRNA-like molecules and artificial miRNA clusters encoding modified pri-miRNA-like molecules. The shRNA clusters and artificial miRNA clusters disclosed herein can be used, for example, to produce artificial RNA molecules, e.g., RNAi molecules. Cells, viral particles, compositions (e.g., pharmaceutical compositions), kits, and methods relating to the nucleic acid molecules, e.g., shRNA clusters and artificial miRNA clusters, are also disclosed. The nucleic acid molecules (e.g., shRNA clusters and artificial miRNA clusters), artificial RNA molecules (e.g., RNAi molecules), cells, viral particles, compositions (e.g., pharmaceutical compositions), and kits and methods disclosed herein can be used to treat or prevent a disease, e.g., HIV infection and/or AIDS.

In an aspect, the disclosure features a nucleic acid molecule, e.g., an shRNA cluster, encoding a plurality of shRNA-like molecules, wherein each of the plurality of shRNA-like molecules comprises: a stem region comprising an artificial RNA molecule comprising a guide strand and a passenger strand, wherein the guide strand is substantially complementary to a target mRNA; and a backbone region comprising a 5′ flanking region, a terminal loop region, and a 3′ flanking region.

In an embodiment, the backbone region of at least one shRNA-like molecule is not repeated among the plurality of shRNA-like molecules, e.g., the plurality of shRNA-like molecules do not include backbone regions that are identical or substantially identical. In an embodiment, the backbone regions of the plurality of shRNA-like molecules are not identical or substantially identical to the backbone regions of pri-miRNAs encoded by a single naturally-occurring miRNA cluster, e.g., at least two of the plurality of shRNA-like molecules are derived from pri-miRNAs encoded by different naturally-occurring miRNA clusters. In an embodiment, the backbone region of at least one shRNA-like molecule is not repeated among the plurality of shRNA-like molecules, and the backbone regions of the plurality of shRNA-like molecules are not identical or substantially identical to the backbone regions of pri-miRNAs encoded by a single naturally-occurring miRNA cluster.

In an embodiment, the plurality of shRNA-like molecules comprise two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) shRNA-like molecules. In another embodiment, the plurality of shRNA-like molecules comprise four or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) shRNA-like molecules. In yet another embodiment, the plurality of shRNA-like molecules comprise seven or more (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) shRNA-like molecules. In still another embodiment, the plurality of shRNA-like molecules comprise ten or more (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) shRNA-like molecules.

In an embodiment, the shRNA cluster is an artificial miRNA cluster, e.g., an artificial miRNA cluster described herein. In an embodiment, the shRNA cluster encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) miRNA-based shRNA molecules (shRNA-miR molecules). In an embodiment, the shRNA cluster encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) shRNA molecules that are not derived from naturally-occurring pri-miRNAs.

In an embodiment, the shRNA cluster comprises a first nucleotide sequence encoding a first shRNA-like molecule and a second nucleotide sequence encoding a second shRNA-like molecule. In an embodiment, the first shRNA-like molecule is derived from a naturally-occurring pri-miRNA for a first miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a first miRNA). In another embodiment, the first shRNA-like molecule is not derived from a naturally-occurring pri-miRNA. In an embodiment, the second shRNA-like molecule is derived from a naturally-occurring pri-miRNA for a second miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a second miRNA). In another embodiment, the second shRNA-like molecule is not derived from a naturally-occurring pri-miRNA.

In an embodiment, the backbone region has one, two, or three of the following properties:

1) the terminal loop is about 3 to about 50 nucleotides, e.g., about 5 to about 40 nucleotides, about 8 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 12 to about 18 nucleotides, in length;

2) the 5′ flanking region is about 5 to about 300 nucleotides, e.g., about 10 to about 200 nucleotides, about 15 to about 150 nucleotides, about 20 to about 100 nucleotides, about 30 to about 50 nucleotides, or about 15 to about 30 nucleotides, e.g., about 15, 20, or 30 nucleotides, in length; or

3) the 3′ flanking region is about 5 to about 300 nucleotides, e.g., about 10 to about 200 nucleotides, about 15 to about 150 nucleotides, about 20 to about 100 nucleotides, about 30 to about 50 nucleotides, or about 15 to about 30 nucleotides, e.g., about 15, 20, or 30 nucleotides, in length.

Without being bound by theory, it is believed that in an embodiment, the 5′ flanking region and the 3′ flanking region can form a duplex region, e.g., a partial duplex region. In an embodiment, the shRNA cluster further comprises a third nucleotide sequence encoding a third shRNA molecule. In an embodiment, the third shRNA-like molecule is derived from a naturally-occurring pri-miRNA for a third miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a third miRNA). In an embodiment, the third miRNA is naturally expressed from a transcript different than the transcript from which the first, second, or both miRNAs is expressed, e.g., not encoded by the same naturally-occurring miRNA cluster as the first, second, or both miRNAs. In another embodiment, the third shRNA-like molecule is not derived from a naturally-occurring pri-miRNA.

In an embodiment, the shRNA cluster further comprises a fourth nucleotide sequence encoding a fourth shRNA molecule. In an embodiment, the fourth shRNA-like molecule is derived from a naturally-occurring pri-miRNA fora fourth miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a fourth miRNA). In an embodiment, the fourth miRNA is naturally expressed from a transcript different than the transcript from which the first, second, or third miRNA is expressed, e.g., not encoded by the same naturally-occurring miRNA cluster as the first, second, or third miRNA. In another embodiment, the fourth shRNA-like molecule is not derived from a naturally-occurring pri-miRNA.

In an embodiment, the shRNA cluster further comprises a fifth nucleotide sequence encoding a fifth shRNA molecule. In an embodiment, the fifth shRNA-like molecule is derived from a naturally-occurring pri-miRNA for a fifth miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a fifth miRNA). In an embodiment, the fifth miRNA is naturally expressed from a transcript different than the transcript from which the first, second, third, or fourth miRNA is expressed, e.g., not encoded by the same naturally-occurring miRNA cluster as the first, second, third, or fourth miRNA. In another embodiment, the fifth shRNA-like molecule is not derived from a naturally-occurring pri-miRNA.

In an embodiment, the shRNA cluster further comprises a sixth nucleotide sequence encoding a sixth shRNA molecule. In an embodiment, the sixth shRNA-like molecule is derived from a naturally-occurring pri-miRNA for a sixth miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a sixth miRNA). In an embodiment, the sixth miRNA is naturally expressed from a transcript different than the transcript from which the first, second, third, fourth, or fifth miRNA is expressed, e.g., not encoded by the same naturally-occurring miRNA cluster as the first, second, third, fourth, or fifth miRNA. In another embodiment, the sixth shRNA-like molecule is not derived from a naturally-occurring pri-miRNA.

In an embodiment, the shRNA cluster further comprises a seventh nucleotide sequence encoding a seventh shRNA molecule. In an embodiment, the seventh shRNA-like molecule is derived from a naturally-occurring pri-miRNA for a seventh miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a seventh miRNA). In an embodiment, the seventh miRNA is naturally expressed from a transcript different than the transcript from which the first, second, third, fourth, fifth, or sixth miRNA is expressed, e.g., not encoded by the same naturally-occurring miRNA cluster as the first, second, third, fourth, fifth, or sixth miRNA. In another embodiment, the seventh shRNA-like molecule is not derived from a naturally-occurring pri-miRNA.

In an embodiment, the shRNA cluster further comprises an 8^th, 9^th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th nucleotide sequence encoding an 8^th, 9^th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th shRNA-like molecule. In an embodiment, the 8^th, 9^th, 10^th, 11^th, 12^th, 13^th 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th shRNA-like molecule is derived from a naturally-occurring pri-miRNA for an 8^th, 9^th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises an 8^th, 9^th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th, miRNA). In an embodiment, the 8^th, 9^th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th, shRNA-like molecule is not derived from a naturally-occurring pri-miRNA.

In an embodiment, the shRNA cluster has one, two, or all of the following properties:

1) the terminal loop region is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a terminal loop region of a naturally-occurring pri-miRNA;

2) the 5′ flanking region that is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a 5′ flanking region of a naturally-occurring pri-miRNA molecule; or

3) the 3′ flanking region that is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a 3′ flanking region of a naturally-occurring pri-miRNA.

In an embodiment, the shRNA cluster encodes a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of artificial RNA molecules, e.g., produced by the plurality of shRNA-like molecules. In an embodiment, the artificial RNA molecule is an RNAi molecule. In an embodiment, the artificial RNA molecule is an siRNA. In an embodiment, the artificial RNA molecule does not comprise a naturally-occurring miRNA.

In an embodiment, the artificial RNA molecules produced by (e.g., processed from) the first and second shRNA-like molecules, target different mRNAs, e.g., mRNAs transcribed from different genes.

In an embodiment, each of the artificial RNA molecules produced by (e.g., processed from) the plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of shRNA-like molecules, targets a different mRNA, e.g., mRNAs transcribed from different genes. In an embodiment, at least two (e.g., 2, 3, 4, 5, or more) of the artificial RNA molecules produced by (e.g., processed from) the plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of shRNA-like molecules, target the same mRNA or different mRNAs transcribed from a same gene.

In an embodiment, the artificial RNA molecule, when expressed in a cell, substantially inhibits expression (e.g., reduces the expression level) of the target mRNA (e.g., by cleavage of the target mRNA, inhibiting translation of the target mRNA, or both). In an embodiment, the cell is a T cell, e.g., CD4+ T cell, a stem cell, e.g., a hematopoietic stem cell, or a CD34+ cell. In an embodiment, the cell is a resting T cell. In another embodiment, the cell is an activated T cell. In an embodiment, the cell is a peripheral blood mononuclear cell (PBMC) or is isolated from PBMCs.

In an embodiment, the mRNA is encoded by a mammalian (e.g., human) gene, e.g., a gene encoding a receptor or co-receptor for a virus, e.g., an HIV co-receptor, e.g., CCR5 or CXCR4. In an embodiment, the target mRNA is encoded by a viral gene, e.g., an HIV gene (e.g., HIV-1 gene), e.g., Gag, Env, Tat, Pol2, Pol1, or Vif.

In an embodiment, the plurality of artificial RNA molecules, when expressed in a cell, substantially inhibit the expression of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) viral genes, e.g., one or more (e.g., 2, 3, 4, 5, or all) HIV genes chosen from Gag, Env, Tat, Pol2, Pol1, or Vif. In an embodiment, the plurality of artificial RNA molecules, when expressed in a cell, substantially inhibit the expression of HIV Gag, Env, Tat, Pol2, Pol1, and Vif genes.

In an embodiment, the guide strand is substantially complementary (e.g., at least 80%, 85%, 90%, 95% or 100% complementary) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene. In an embodiment, the guide strand is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In an embodiment, the shRNA cluster encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene; and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In another embodiment, the shRNA cluster encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene; and four or more (e.g., 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In yet another embodiment, the shRNA cluster encodes one artificial RNA molecule comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene, and six artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In an embodiment, the passenger strand is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene.

In an embodiment, the passenger strand is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In an embodiment, the shRNA cluster encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene; and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In another embodiment, the shRNA cluster encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene; and four or more (e.g., 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In yet another embodiment, the shRNA cluster encodes one artificial RNA molecule comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene, and six artificial RNA molecules each comprising a guide strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In an embodiment, the virus is an RNA virus. In an embodiment, the virus is a fast-evolving virus. In an embodiment, the viral gene is from an RNA virus. In an embodiment, the virus is from a fast-evolving virus.

In an embodiment, the shRNA-like molecule is derived from a pri-miRNA for an miRNA described herein, e.g., miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150. In an embodiment, the plurality of shRNA-like molecules are derived from two or more (e.g., three, four, five, six, or all) pri-miRNAs for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150. In an embodiment, the plurality of shRNA-like molecules are derived from pri-miRNAs for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, and miR-150.

In an embodiment, the terminal loop region is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a terminal loop region of a pri-miRNA for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150.

In an embodiment, the 5′ flanking region is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a 5′ flanking of a pri-miRNA for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150.

In an embodiment, the 3′ flanking region is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a 3′ flanking region of a pri-miRNA for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150.

In an embodiment, the shRNA cluster comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) nucleotide sequences, each of which is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a nucleotide sequence of a modified pri-miRNA-like molecule disclosed in Tables 4-5. In an embodiment, the shRNA cluster comprises the nucleotide sequence(s) of one or more (e.g., 2, 3, 4, 5, 6, or 7) of the modified pri-miRNA-like molecules disclosed in Tables 4-5. In an embodiment, the shRNA cluster comprises the nucleotide sequences of the seven modified pri-miRNA-like molecule disclosed in Tables 4-5.

In an embodiment, the shRNA cluster comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) nucleotide sequences, each of which is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a guide strand nucleotide sequence disclosed in Tables 4-5.

In an embodiment, the shRNA cluster comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) guide strand nucleotide sequences disclosed in Tables 4-5.

In an embodiment, the shRNA cluster comprises the seven guide strand nucleotide sequences disclosed in Tables 4-5.

In an embodiment, the shRNA cluster comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) nucleotide sequences, each of which is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a passenger strand nucleotide sequence disclosed in Tables 4-5.

In an embodiment, the shRNA cluster comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) passenger strand nucleotide sequences disclosed in Tables 4-5. In an embodiment, the shRNA cluster comprises the seven passenger strand nucleotide sequences disclosed in Tables 4-5.

In an embodiment, the shRNA cluster comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) nucleotide sequences, each of which is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a terminal loop region, 5′ flanking region, or 3′ flanking region nucleotide sequence disclosed in Tables 4-5.

In an embodiment, the shRNA cluster comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) terminal loop region, 5′ flanking region, or 3′ flanking region nucleotide sequences disclosed in Tables 4-5. In an embodiment, the shRNA cluster comprises the seven terminal loop region, 5′ flanking region, or 3′ flanking region nucleotide sequences disclosed in Tables 4-5.

In an embodiment, the shRNA cluster further comprises a plurality of spacer sequences (e.g., about 0 to about 250 nucleotides, about 1 to about 200 nucleotides, about 10 to about 150 nucleotides, about 20 to about 100 nucleotides, or about 30 to about 50 nucleotides, in length), wherein the spacer sequence is between the sequences encoding two shRNA-like molecules.

In an embodiment, the shRNA cluster is efficiently processed in the nucleus, e.g., by Drosha/DGCR8. In another embodiment, the shRNA cluster preserves the secondary structure of at least a portion of a native pri-miRNA, e.g., a pri-miRNA for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150.

In an embodiment, the stem region does not include a bulge or a mismatch between the guide strand and the passenger strand. In another embodiment, the stem region comprises a bulge or a mismatch between the guide strand and the passenger strand.

In an embodiment, the guide strand is about 15 to about 30 nucleotides, e.g., about 18 to about 25 nucleotides or about 19 to about 21 nucleotides, in length. In another embodiment, the passenger strand is about 15 to about 30 nucleotides, e.g., about 18 to about 25 nucleotides or about 19 to about 21 nucleotides, in length.

In an embodiment, shRNA cluster is an expression cassette.

In an embodiment, the shRNA cluster further comprises a promoter, e.g., a Pol II promoter or a Pol III promoter, e.g., operably linked to an shRNA-like molecule (e.g., the plurality of shRNA-like molecules).

In an embodiment, the promoter is a constitutive promoter, an inducible promoter, a ubiquitous promoter, a tissue-specific promoter, a cell-type-specific promoter, and/or a developmental stage-specific promoter. In an embodiment, the promoter is an EF-1α-derived promoter. In another embodiment, the promoter is a CMV-derived promoter.

In an embodiment, the shRNA cluster further comprises one or more (e.g., two or three) selectable markers. In an embodiment, the shRNA cluster further comprises a reporter gene.

In an embodiment, the shRNA cluster further comprises a polyadenylation signal. In an embodiment, the shRNA cluster further comprises one or more (e.g., two) long terminal repeats (LTRs). In an embodiment, the shRNA cluster further comprises an internal ribosome entry site (IRES).

In an embodiment, the shRNA cluster is in a viral vector, e.g., a retroviral or lentiviral vector, e.g., a self-inactivating (SIN) retroviral or lentiviral vector.

In one aspect, the disclosure features a nucleic acid molecule, e.g., an artificial miRNA cluster, encoding a plurality of modified pri-miRNA-like molecules, e.g., miRNA-based shRNA molecules (shRNA-miR molecules), the nucleic acid molecule comprising: a first nucleotide sequence encoding a first modified pri-miRNA-like molecule (e.g., shRNA-miR molecule) derived from a naturally-occurring pri-miRNA for a first miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a first miRNA); and a second nucleotide sequence encoding a second modified pri-miRNA-like molecule (e.g., shRNA-miR molecule) derived from a naturally-occurring pri-miRNA for a second miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a second miRNA), wherein the first and second miRNAs are naturally expressed from different transcripts or are not in the same naturally-occurring miRNA cluster.

In an embodiment, each of the plurality of modified pri-miRNA-like molecules (e.g., shRNA-miR molecules) comprises: a stem region comprising an artificial RNA molecule comprising a guide strand and a passenger strand, wherein the guide strand is substantially complementary to a target mRNA, a terminal loop region, a 5′ flanking region, and a 3′ flanking region.

In an embodiment, the modified pri-miRNA-like molecule (e.g., shRNA-miR molecule) has one or more (e.g., 2 or all) of the following:

1) a terminal loop region that is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a terminal loop region of a naturally-occurring pri-miRNA;

2) a 5′ flanking region that is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a 5′ flanking region of a naturally-occurring pri-miRNA; or

3) a 3′ flanking region that is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a 3′ flanking region of a naturally-occurring pri-miRNA.

In an embodiment, the terminal loop is about 3 to about 50 nucleotides, e.g., about 5 to about 40 nucleotides, about 8 to about 30 nucleotides, about 10 to about 20 nucleotides, or about 12 to about 18 nucleotides, in length.

In an embodiment, the 5′ flanking region is about 5 to about 300 nucleotides, e.g., about 10 to about 200 nucleotides, about 15 to about 150 nucleotides, about 20 to about 100 nucleotides, about 30 to about 50 nucleotides, or about 15 to about 30 nucleotides, e.g., about 15, 20, or 30 nucleotides, in length.

In an embodiment, the 3′ flanking region is about 5 to about 300 nucleotides, e.g., about 10 to about 200 nucleotides, about 15 to about 150 nucleotides, about 20 to about 100 nucleotides, about 30 to about 50 nucleotides, or about 15 to about 30 nucleotides, e.g., about 15, 20, or 30 nucleotides, in length.

Without being bound by theory, it is believed that in an embodiment, the 5′ flanking region and the 3′ flanking region can form a duplex region, e.g., a partial duplex region. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a third nucleotide sequence encoding a third modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a third miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a third miRNA).

In an embodiment, the third miRNA is naturally expressed from a transcript different than the transcript from which the first, second, or both miRNAs is expressed, e.g., not in the same naturally-occurring miRNA cluster as the first, second, or both miRNAs.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a fourth nucleotide sequence encoding a fourth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a fourth miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a fourth miRNA).

In an embodiment, the fourth miRNA is naturally expressed from a transcript different than the transcript from which the first, second, or third miRNA is expressed, e.g., not in the same naturally-occurring miRNA cluster as the first, second, or third miRNA.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a fifth nucleotide sequence encoding a fifth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a fifth miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a fifth miRNA).

In an embodiment, the fifth miRNA is naturally expressed from a transcript different than the transcript from which the first, second, third, or fourth miRNA is expressed, e.g., not in the same naturally-occurring miRNA cluster as the first, second, third, or fourth miRNA.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a sixth nucleotide sequence encoding a sixth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a sixth miRNA molecule (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a sixth miRNA).

In an embodiment, the sixth miRNA molecule is naturally expressed from a transcript different than the transcript from which the first, second, third, fourth, or fifth miRNA is expressed, e.g., not in the same naturally-occurring miRNA cluster as the first, second, third, fourth, or fifth miRNA.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a seventh nucleotide sequence encoding a seventh modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a seventh miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises a seventh miRNA).

In an embodiment, the seventh miRNA is naturally expressed from a transcript different than the transcript from which the first, second, third, fourth, fifth, or sixth miRNA is expressed, e.g., not in the same naturally-occurring miRNA cluster as the first, second, third, fourth, fifth, or sixth miRNA.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises an 8^th, 9^th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th nucleotide sequence encoding an 8^th, 9^th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for an 8^th, 9th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17^th, 18^th, 19^th, or 20^th, miRNA (e.g., a naturally-occurring pri-miRNA that gives rise to or comprises an 8^th, 9^th, 10^th, 11^th, 12^th, 13^th, 14^th, 15^th, 16^th, 17th, 18^th, 19^th, or 20^th miRNA).

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) modified pri-miRNA-like molecules derived from naturally-occurring pri-miRNAs. In an embodiment, at least two (e.g., 2, 3, 4, 5, or more) of the modified pri-miRNA-like molecules in the plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) are derived from the same naturally-occurring pri-miRNA. In an embodiment, each of the plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of the modified pri-miRNA-like molecules is derived from a different naturally-occurring pri-miRNA.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes seven modified pri-miRNA-like molecules derived from naturally-occurring pri-miRNAs. In an embodiment, each of the seven modified pri-miRNA-like molecules is derived from a different naturally-occurring pri-miRNA. In an embodiment, the seven different naturally-occurring pri-miRNAs are not in the same miRNA cluster.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further encodes at least one (e.g., 1, 2, 3, 4, 5, or more) shRNA molecule that is not derived from a naturally-occurring pri-miRNA.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of artificial RNA molecules. In an embodiment, the artificial RNA molecule is an RNAi molecule. In an embodiment, the artificial RNA molecule is an siRNA. In an embodiment, the artificial RNA molecule does not comprise a naturally-occurring miRNA.

In an embodiment, the artificial RNA molecules produced by the first and second modified pri-miRNA-like molecules, target different mRNAs, e.g., mRNAs transcribed from different genes.

In an embodiment, each of the artificial RNA molecules encoded by the plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of modified pri-miRNA-like molecules, targets a different mRNA, e.g., mRNAs transcribed from different genes. In an embodiment, at least two (e.g., 2, 3, 4, 5, or more) of the artificial RNA molecules produced by (e.g., processed from) the plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of modified pri-miRNA-like molecules, target a same mRNA or different mRNAs transcribed from the same gene.

In an embodiment, the artificial RNA molecule, when expressed in a cell, substantially inhibits expression (e.g., reduces the expression level) of the target mRNA (e.g., by cleavage of the target mRNA, inhibit translation of the target mRNA, or both). In an embodiment, the cell is a T cell, e.g., CD4+ T cell, a stem cell, e.g., a hematopoietic stem cell, or a CD34+ cell. In an embodiment, the cell is a resting T cell. In another embodiment, the cell is an activated T cell. In an embodiment, the cell is a peripheral blood mononuclear cell (PBMC) or is isolated from PBMCs.

In an embodiment, the mRNA is encoded by a mammalian (e.g., human) gene, e.g., a gene encoding a receptor or co-receptor for a virus, e.g., an HIV co-receptor, e.g., CCR5 or CXCR4. In an embodiment, the target mRNA is encoded by a viral gene, e.g., an HIV gene (e.g., HIV-1 gene), e.g., Gag, Env, Tat, Pol2, Pol1, or Vif.

In an embodiment, the plurality of artificial RNA molecules, when expressed in a cell, substantially inhibit the expression of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) viral genes, e.g., one or more (e.g., 2, 3, 4, 5, or all) HIV genes chosen from Gag, Env, Tat, Pol2, Pol1, or Vif. In an embodiment, the plurality of artificial RNA molecules, when expressed in a cell, substantially inhibit the expression of HIV Gag, Env, Tat, Pol2, Pol1, and Vif genes.

In an embodiment, the guide strand is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene. In an embodiment, the guide strand is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene; and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In another embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene; and four or more (e.g., 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In yet another embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes one artificial RNA molecule comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene, and six artificial RNA molecules, each comprising a guide strand that is substantially complementary (e.g., at least 80%, 85%, 90%, 95%, or 100% complementary) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In an embodiment, the passenger strand is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene.

In an embodiment, the passenger strand is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene; and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In another embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene; and four or more (e.g., 5, 6, 7, 8, 9, 10, or more) artificial RNA molecules, each comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In yet another embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) encodes one artificial RNA molecule comprising a passenger strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a mammalian gene, e.g., a human gene, and six artificial RNA molecules each comprising a guide strand that is substantially identical (e.g., at least 80%, 85%, 90%, 95%, or 100% identical) to a sequence in a target mRNA encoded by a viral gene, e.g., an HIV gene.

In an embodiment, the virus is an RNA virus. In an embodiment, the virus is a fast-evolving virus. In an embodiment, the viral gene is from an RNA virus. In an embodiment, the virus is from a fast-evolving virus.

In an embodiment, the modified pri-miRNA-like molecule is derived from a pri-miRNA for an miRNA described herein, e.g., miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150. In an embodiment, the plurality of modified pri-miRNA-like molecules are derived from two or more (e.g., three, four, five, six, or all) pri-miRNAs for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150. In an embodiment, the plurality of modified pri-miRNA-like molecules are derived from pri-miRNAs for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, and miR-150.

In an embodiment, the terminal loop region is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a terminal loop region of a pri-miRNA for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150.

In an embodiment, the 5′ flanking region is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a 5′ flanking of a pri-miRNA for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150.

In an embodiment, the 3′ flanking region is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a 3′ flanking region of a pri-miRNA for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) nucleotide sequences, each of which is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a nucleotide sequence of a modified pri-miRNA-like molecule disclosed in Tables 4-5. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises the nucleotide sequence(s) of one or more (e.g., 2, 3, 4, 5, 6, or 7) modified pri-miRNA-like molecules disclosed in Tables 4-5. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises the nucleotide sequences of the seven modified pri-miRNA-like molecule disclosed in Tables 4-5.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) nucleotide sequences, each of which is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a guide strand nucleotide sequence disclosed in Tables 4-5. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) guide strand nucleotide sequences disclosed in Tables 4-5. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises the seven guide strand nucleotide sequences disclosed in Tables 4-5.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) nucleotide sequences, each of which is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a passenger strand nucleotide sequence disclosed in Tables 4-5. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) passenger strand nucleotide sequences disclosed in Tables 4-5. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises the seven passenger strand nucleotide sequences disclosed in Tables 4-5.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) nucleotide sequences, each of which is substantially homologous to (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from) a terminal loop region, 5′ flanking region, or 3′ flanking region nucleotide sequence disclosed in Tables 4-5. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises one or more (e.g., 2, 3, 4, 5, 6, or 7) terminal loop region, 5′ flanking region, or 3′ flanking region nucleotide sequences disclosed in Tables 4-5. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) comprises the seven terminal loop region, 5′ flanking region, or 3′ flanking region nucleotide sequences disclosed in Tables 4-5.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a plurality of spacer sequences (e.g., about 0 to about 250 nucleotides, about 1 to about 200 nucleotides, about 10 to about 150 nucleotides, about 20 to about 100 nucleotides, or about 30 to about 50 nucleotides, in length), wherein the spacer sequence is between the sequences encoding two modified pri-miRNA-like molecules.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) is efficiently processed in the nucleus, e.g., by Drosha/DGCR8. In another embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) preserves the secondary structure of at least a portion of a naturally-occurring pri-miRNA, e.g., a pri-miRNA for miR-30a, miR-21, miR-20a, miR-16-1, miR-122, miR-185, or miR-150.

In an embodiment, the stem region does not include a bulge or a mismatch between the guide strand and the passenger strand. In another embodiment, the stem region comprises a bulge or a mismatch between the guide strand and the passenger strand.

In an embodiment, the guide strand is about 15 to about 30 nucleotides, e.g., about 18 to about 25 nucleotides or about 19 to about 21 nucleotides, in length. In another embodiment, the passenger strand is about 15 to about 30 nucleotides, e.g., about 18 to about 25 nucleotides or about 19 to about 21 nucleotides, in length.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) is an expression cassette. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a promoter, e.g., a Pol II promoter or a Pol III promoter, e.g., operably linked to a modified pri-miRNA-like molecule (e.g., the plurality of modified pri-miRNA-like molecules).

In an embodiment, the promoter is a constitutive promoter, an inducible promoter, a ubiquitous promoter, a tissue-specific promoter, a cell-type-specific promoter, and/or a developmental stage-specific promoter. In an embodiment, the promoter is an EF-1α-derived promoter. In another embodiment, the promoter is a CMV-derived promoter.

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises one or more (e.g., two or three) selectable markers. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a reporter gene. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises a polyadenylation signal. In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises one or more (e.g., two) long terminal repeats (LTRs). In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) further comprises an internal ribosome entry site (IRES).

In an embodiment, the nucleic acid molecule (e.g., artificial miRNA cluster) is in a viral vector, e.g., a retroviral or lentiviral vector, e.g., a self-inactivating (SIN) retroviral or lentiviral vector.

In another aspect, the disclosure features a nucleic acid molecule (e.g., artificial miRNA cluster) encoding seven modified pri-miRNA-like molecules (e.g., shRNA-miR molecules), the nucleic acid molecule comprising: a first nucleotide sequence encoding a first modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA that gives rise to miR-30a, a second nucleotide sequence encoding a second modified pri-miRNA-like molecule derived from a naturally occurring pri-miRNA that gives rise to miR-21, a third nucleotide sequence encoding a third modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA that gives rise to miR-20a, a fourth nucleotide sequence encoding a fourth modified pri-miRNA-like molecule derived from a naturally occurring pri-miRNA that gives rise to miR-16-1, a fifth nucleotide sequence encoding a fifth modified pri-miRNA-like molecule derived from a naturally occurring pri-miRNA that gives rise to miR-122, a sixth nucleotide sequence encoding a sixth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA that gives rise to miR-185, a seventh nucleotide sequence encoding a seventh modified pri-miRNA-like molecule derived from a naturally occurring pri-miRNA that gives rise to miR-150, wherein each of the seven modified pri-miRNA-like molecules comprises: a stem region comprising an artificial RNA molecule comprising a guide strand and a passenger strand, wherein the guide strand is substantially complementary to a target mRNA encoded by a gene selected from a group consisting of human CCR5 gene and HIV (e.g., HIV-1) Gag, Env, Tat, Pol2, Pol1, and Vif genes; a terminal loop region, e.g., a terminal loop region described herein; a 5′ flanking region, e.g., a 5′ flanking region described herein; and a 3′ flanking region, e.g., a 3′ flanking region described herein.

In an aspect, the disclosure features a transcript, e.g., a primary RNA transcript or a processed RNA transcript, encoded by a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein.

In an aspect, the disclosure features a plurality of shRNA-like molecules encoded by a nucleic acid molecule (e.g., shRNA cluster) described herein.

In another aspect, the disclosure features a plurality of modified pri-miRNA-like molecules (e.g., shRNA-miR molecules) encoded by a nucleic acid molecule (e.g., artificial miRNA cluster) described herein.

In yet another aspect, the disclosure features a plurality of artificial RNA molecules encoded by a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein.

In an aspect, the disclosure features a cell comprising a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein.

In an embodiment, the cell is infected by a virus, e.g., an RNA virus, e.g., an HIV (e.g., HIV-1). In an embodiment, the virus is a fast-evolving virus. In an embodiment, the cell is isolated or derived from a patient, e.g., a patient having an HIV (e.g., HIV-1) infection or AIDS.

In another aspect, the disclosure features a cell comprising a plurality of shRNA-like molecules described herein or a plurality of modified pri-miRNA-like molecules described herein.

In an embodiment, the cell is infected by a virus, e.g., an RNA virus, e.g., an HIV (e.g., HIV-1). In an embodiment, the virus is a fast-evolving virus. In an embodiment, the cell is isolated or derived from a patient, e.g., a patient having an HIV (e.g., HIV-1) infection or AIDS.

In an aspect, the disclosure features a viral particle comprising a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein.

In an embodiment, the viral particle is a retrovirus or a lentivirus.

In another aspect, the disclosure features a viral particle comprising a plurality of shRNA-like molecules described herein or modified pri-miRNA-like molecules described herein.

In an embodiment, the viral particle is a retrovirus or a lentivirus.

In yet another aspect, the disclosure features a viral particle comprising a plurality of artificial RNA molecules (e.g., RNAi molecules) described herein.

In an embodiment, the viral particle is a retrovirus or a lentivirus.

In an aspect, the disclosure features a method for inhibiting the expression of a gene (e.g., a gene described herein) in a cell (e.g., a cell described herein), the method comprising contacting a cell with a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein, under conditions that allow expression of artificial RNA molecules, e.g., the artificial RNA molecules described herein.

In an embodiment, the cell is infected by a virus, e.g., an RNA virus, e.g., an HIV (e.g., HIV-1). In an embodiment, the virus is a fast-evolving virus. In an embodiment, the cell is isolated or derived from a patient, e.g., a patient having an HIV (e.g., HIV-1) infection or AIDS.

In an embodiment, the gene is a mammalian cellular gene, e.g., a gene encoding an HIV co-receptor, e.g., CCR5 or CXCR4. In an embodiment, the gene is a viral gene, e.g., an HIV (e.g., HIV-1) gene, e.g., Gag, Env, Tat, Pol2, Pol1, or Vif.

In an embodiment, the cell is contacted with the nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) in vivo. In an embodiment, the cell is contacted with the nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) ex vivo. In an embodiment, the cell is contacted with the nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) in vitro.

In another aspect, the disclosure features a method for inhibiting the expression of a gene (e.g., a gene described herein) in a cell (e.g., a cell described herein), the method comprising contacting a cell with a viral particle described herein, under conditions that allow expression of artificial RNA molecules, e.g., the artificial RNA molecules described herein.

In an embodiment, the cell is infected by a virus, e.g., an RNA virus, e.g., an HIV (e.g., HIV-1). In an embodiment, the virus is a fast-evolving virus. In an embodiment, the cell is isolated or derived from a patient, e.g., a patient having an HIV (e.g., HIV-1) infection or AIDS. In an embodiment, the gene is a mammalian cellular gene, e.g., a gene encoding an HIV co-receptor, e.g., CCR5 or CXCR4. In an embodiment, the gene is a viral gene, e.g., an HIV (e.g., HIV-1) gene, e.g., Gag, Env, Tat, Pol2, Pol1, or Vif.

In an embodiment, the cell is contacted with the nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) in vivo. In an embodiment, the cell is contacted with the nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) ex vivo. In an embodiment, the cell is contacted with the nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) in vitro.

In an aspect, the disclosure features a method for treating or preventing a disorder, e.g., an HIV (e.g., HIV-1) infection or AIDS, the method comprising administering a cell (e.g., a cell described herein) to a subject having an HIV infection or AIDS, or is at risk of having an HIV infection or AIDS, thereby treating or preventing the disorder, e.g., the HIV infection or AIDS.

In another aspect, the disclosure features a method for treating or preventing a disorder, e.g., an HIV (e.g., HIV-1) infection or AIDS, the method comprising: isolating a cell from a subject having an HIV infection or AIDS, or is at risk of having an HIV infection or AIDS; contacting the isolated cell with a viral particle described herein; and administering the cell contacted with the viral particle to the subject, thereby treating or preventing the disorder, e.g., the HIV infection or AIDS.

In yet another aspect, the disclosure features a method for treating or preventing a disorder, e.g., an HIV (e.g., HIV-1) infection or AIDS, the method comprising administering a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein to a subject having an HIV infection or AIDS, or at risk of having an HIV infection or AIDS, thereby treating or preventing the disorder, e.g., the HIV infection or AIDS.

In still another aspect, the disclosure features a method for treating or preventing a disorder, e.g., an HIV (e.g., HIV-1) infection or AIDS, the method comprising administering a viral particle described herein to a subject having an HIV infection or AIDS, or at risk of having an HIV infection or AIDS, thereby treating or preventing the disorder, e.g., the HIV infection or AIDS.

In an aspect, the disclosure features a cell described herein for use in treating or preventing an HIV infection or AIDS in a subject.

In another aspect, the disclosure features an shRNA cluster described herein or a nucleic acid molecule described herein for use in treating or preventing a disorder, e.g., an HIV infection or AIDS in a subject.

In yet another aspect, the disclosure features viral particle described herein for use in treating or preventing a disorder, e.g., an HIV infection or AIDS in a subject.

In an aspect, the disclosure features use of a cell described herein in the manufacture of a medicament for the treatment or prevention of a disorder, e.g., an HIV (e.g., HIV-1) infection or AIDS, in a subject.

In another aspect, the disclosure features use of a nucleic acid molecule (e.g., an shRNA cluster or artificial miRNA cluster) described herein in the manufacture of a medicament for the treatment or prevention of a disorder, e.g., an HIV (e.g., HIV-1) infection or AIDS, in a subject.

In yet another aspect, the disclosure features use of a viral particle described herein in the manufacture of a medicament for the treatment or prevention of a disorder, e.g., an HIV (e.g., HIV-1) infection or AIDS, in a subject.

In an aspect, the disclosure features a method for producing a cell that expresses a plurality of artificial RNA molecules described herein, the method comprising: contacting a cell (e.g., a cell described herein) with a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein, and optionally, culturing the cells under conditions that allow expression of the artificial RNA molecules, thereby producing the cell.

In an embodiment, the method comprises contacting the cell with a viral particle described herein. In an embodiment, the method further comprises isolating a cell from a subject, e.g., a subject having an HIV (e.g., HIV-1) infection or AIDS, prior to contacting the cell with the nucleic acid molecule.

In another aspect, the disclosure features a method for producing a cell that expresses a plurality of artificial RNA molecules (e.g., RNAi molecules) described herein, the method comprising: contacting a cell (e.g., a cell described herein) with a viral particle described herein, and optionally, culturing the cells under conditions that allow expression of the artificial RNA molecules, thereby producing the cell.

In an embodiment, the method further comprises isolating a cell from a subject, e.g., a subject having an HIV infection or AIDS, prior to contacting the cell with the nucleic acid molecule.

In an aspect, the disclosure features a method of designing a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein, the method comprising altering a backbone region of a naturally-occurring pri-miRNA, wherein the backbone region comprises a 5′ flanking region, a terminal loop region, and a 3′ flanking region.

In an embodiment, the method comprises one, two, or all of the following:

1) adding one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region;

2) deleting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region; or

3) substituting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region.

In an aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising a nucleic acid molecule (e.g., an shRNA cluster or artificial miRNA cluster) described herein and a pharmaceutically acceptable carrier.

In another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising a cell described herein and a pharmaceutically acceptable carrier.

In yet another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising a viral particle described herein and a pharmaceutically acceptable carrier.

In an aspect, the disclosure features a kit comprising a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA molecule) described herein and instructions for use of the nucleic acid molecule.

In another aspect, the disclosure features a kit comprising a cell described herein and instructions for use of the cell.

In yet another aspect, the disclosure features a kit comprising a viral particle described herein and instructions for use of the viral particle.

In an aspect, the disclosure features an artificial miRNA cluster to prevent infection of cells by HIV-1 comprising multiplexed miRNA-based shRNA. In an embodiment, said multiplexed miRNA-based shRNA comprises seven shRNAs. In another embodiment, the artificial miRNA cluster is also effective in controlling replication of cells infected with HIV-1.

In another aspect, the disclosure features a method of treatment for patients infected with HIV-1 comprising administering an effective amount of multiplexed miRNA-based shRNAs to a patient. In an embodiment, said multiplexed miRNA-based shRNA comprises seven shRNAs.

In yet another aspect, the disclosure features a method of preventing infection by HIV-1 comprising administering an effective amount of multiplexed miRNA-based shRNAs to a patient. In an embodiment, said multiplexed miRNA-based shRNA comprises seven shRNAs.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, in which Ike reference numerals refer to identical or functionally-similar elements throughout the separate views and which are incorporated in and form a part of the specification, further illustrate the embodiments and, together with the detailed description, serve to explain the embodiments disclosed herein.

FIGS. 1A-1D illustrate optimized designs of multiplexed shRNA-miRs, in accordance with the disclosed embodiments. Plasmids encoding (FIG. 1A) pri-miR-30 or (FIG. 1B) pri-miR-150 containing indicated lengths of flanking sequence (10, 50, and 100 ng) were co-transfected with psiCHECK-2 vector (100 ng) harboring the target sequence in the 3′ UTR of the Renilla luciferase gene and gene silencing was tested by dual luciferase assay 24 hours later. Bar graphs represent mean+SD of triplicates. (FIG. 1C) Schematic of single and multiplexed shRNA-miR designed to express indicated 1, 2, 4, and 7 shRNA-miRs. “X” represents restriction enzyme sites. (FIG. 1D) Schematic showing the position of shRNA-miR target sites in the HIV-1 genome.

FIGS. 2A-2C illustrate competency of shRNA-miR expressed from multiplexed shRNA-miRs contruct, in accordance with the disclosed embodiments. (FIG. 2A) Plasmids encoding indicated individual or multiplexed shRNA-miRs (2 shRNA=CCR5 and Vit 4 shRNAs=CCR5, Gag, Pol 1 and Vif; 7 shRNAs=CCR5, Gag, Env, Tat, Pol 1, Pol 2, and Vif) were co-transfected (10, 50, and 100 ng) with psiCHECK vector (100 ng) containing shRNA-miR target sequences in the 3′ UTR of R-luc into 293 FT cells. Gene silencing was determined by dual luciferase assay performed 24 hours after co-transfection. The ratio of renilla luciferase (Rluc, reporter) to firefly luciferase (Floc, internal control), normalized to the negative control mCherry vector expressing no shRNA-miR transfection is shown. The experiments were performed in triplicate. (FIG. 2B) Jurkat cells were transfected with single, dual, or multiple shRNA-miR encoding constructs and assayed for apoptosis by Annexin V staining. Staurosporine was used as positive control. (FIG. 2C) Indicated shRNAmiR expression vector-transduced PBMCs were subjected to MTS assay 2 and 4 days after transduction. Bar graphs represent mean+SD of triplicates.

FIGS. 3A-3C illustrate the effect of multiplexed shRNA-miR on CCR5, in accordance with disclosed embodiments. (FIG. 3A) TZM-bl cells were transduced with lentivirus encoding CCR5 shRNA-miR only or multiplexed shRNA-miRs and CCR5 expression assayed 48 hours later by flow cytometry. (FIG. 3B) 293 T cells were co-transfected with pNL4-3 plasmid together with plasmids encoding no shRNA-miR (mock), CCR5 shRNA-miR only, or multiplexed shRNA-miRs. Supernatants collected 48 hours later were used to infect TZM-bl cells and after another 48 hours, cell lysates were analyzed for luciferase activity. (FIG. 3C) TZM-bl cells were transduced with HIV-1 env pseudotyped lentivirus encoding indicated shRNA-miRs and infected with HIV-1_NL4-3 (left) and HIV-1_BaL (right) at an MOI of 0.01. p24 antigen level in culture supernatants obtained 9 days after infection was measured by ELISA.

FIGS. 4A-4B illustrate transduction and protection of resting and activated T cells by HIV-1 env-pseudotyped lentiviral vector expressing multiplexed shRNA-miRs, in accordance with disclosed embodiments. (FIG. 4A) Activated and (FIG. 4B) resting T cells were transduced with HIV-1 env-pseudotyped lentivirus encoding no shRNA-miR (mock), CCR5 only shRNA-miR, or 7 shRNA-miRs (transduction efficiency in terms of GFP expression is shown below the schematic of treatment), infected with HIV-1NL4-3 (left panels) or HIV-1Bal (right panels) and p24 antigen levels in culture supernatants were measured by ELISA on days 0, 3, 9, and 15 after infection (top panels). Cells were analyzed by flow cytometry for GFP expression on days 0, 3, 9, and 15 after infection (bottom panels).

FIGS. 5A-5C illustrate inhibition of HIV-1 replication in HIV-seropositive donor PBMCs transduced with HIV-1 env-pseudotyped lentiviral vector expressing multiplexed shRNA-miRs, in accordance with disclosed embodiment. (FIG. 5A) Time course of treatment and testing. (FIG. 5B) PBMCs from four different HIV-1 seropositive donors were transduced with HIV-1 env-pseudotyped lentivirus encoding no shRNA-miR (mock), Tat shRNA-miR, or 7 shRNA-miRs and p24 antigen level in culture supernatants measured by ELISA on indicated days after infection. (FIG. 5C) Culture supernatants from Tat shRNA-miR transduced cells in FIG. 5B obtained on day 35 were sequenced for Tat shRNA-miR target region in the HIV-1 genome.

FIGS. 6A-6C illustrate prevention of CD4 T cell loss and endogenous HIV-1 replication in 7 shRNA-miR treated HIV seropositive PBMC transplanted Hu-PBL mice, in accordance with disclosed embodiments. (FIG. 6A) Time course of treatment and analysis.

FIGS. 6B-6C NOD/SCID/IL2-Rγc−/− mice were transplanted with PBMCs form two seropositive donors (FIG. 6B on ART) and (FIG. 6C treatment naive) that were transduced ex vivo with HIV-1 env-pseudotyped lentivirus encoding either no shRNA-miR (mock), only Tat shRNA-miR, or 7 shRNA-miRs and monitored for CD4 T cell counts on days 8, 29, and 43 and serum p24 levels on day 43. A representative dot plots of CD4 and CD8 T cell reconstitution on CD3 gated cells (left) and cumulative data from four mice on CD4 T cell levels (middle) and plasma p24 levels (right) is shown. P values between groups for p24 levels are indicated.

FIG. 7 shows exemplary shRNA-miRNA structures and sequences. Left panel shows each shRNA-miR hairpin. The predicted siRNA sequence is underlined. Arrows indicate the Drosha cleavage sites. Right panel represents dominant small RNA sequences obtained by deep sequencing of individual or multiplexed shRNA-miR constructs.

FIG. 8 depicts the frequency of individual siRNA reads generated from 7 shRNA-miRs. 293 T cells were transfected with indicated single or 7 shRNA-miRs and 48 h later small RNAs obtained from cell lysates cloned and deep sequenced. Small RNA reads were normalized by dividing the read numbers by the endogenous miR-16 (Table 3). Relative expression was calculated using the formula: (small RNA reads for indicated shRNA-miRs in 7 shRNA-miR transfected cell/small RNA reads for endogenous miR-16)/(small RNA read number for individual shRNA-miRs in single shRNA-miR transfected cell/small RNA reads for endogenous miR-16)×100.

FIG. 9 is a graphical representation of data showing mCherry (vector alone, single, and multiplex shRNA-miRs) expression in vector transduced PBMCs over time.

FIG. 10 depicts that activated (A CD4) and resting CD4 T (R CD4) cells were transduced with VSV-G or HIV env-pseudotyped lentivirus and mCherry expression determined 48 hours later by flow cytometry.

FIG. 11 depicts that activated and resting CD4 T cells transduced with HIV-1 env-pseudotyped lentivirus encoding 7 shRNA-miRs in FIG. 4 were monitored for GFP expression on days 0, 3, 9, and 15 after infection to determine enrichment of HIV-resistant cells.

FIG. 12 is schematic of multiplexed shRNA-miRs. Synthesized Ultramer oligos were cloned into pLVX vector with indicated restriction enzyme sites. The inserted sequences can be found in Table 3.

DETAILED DESCRIPTION

The embodiments will now be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. The embodiments disclosed herein can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Unnecessary detail of known functions and operations may be omitted from the current description so as not to obscure the present invention and further discussion of the embodiments can be found in examples. Like numbers refer to like elements throughout. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Definitions

As used herein, the articles “a” and “an” refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

As used herein, the term “or” is used herein to mean, and is used interchangeably with, the term “and/or”, unless context clearly indicates otherwise.

As used herein, the term “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

As used herein, the terms “nucleic acid”, “nucleic acid sequence”, “nucleotide sequence” or “polynucleotide sequence”, and “polynucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may be either single-stranded or double-stranded, and if single-stranded may be the coding strand or non-coding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components, A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid may be a recombinant polynucleotide or a polynucleotide of genomic, cDNA, enzymatically processed, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

As used herein, the term “gene” refers to a nucleic acid, e.g., a locus or region of DNA that encodes a function RNA (e.g., a coding or non-coding RNA, e.g., an mRNA or miRNA) or polypeptide. A gene can include both exon and (optionally) intron sequences. The nucleic acid can also optionally include non-coding sequences such as promoter and/or enhancer sequences.

As used herein, the term “mRNA” refers to a nucleic acid transcribed from a gene from which a polypeptide is translated, and may include non-translated regions such as a 5′ UTR and/or a 3′ UTR. It will be understood that shRNA-like molecules, modified pri-miRNA-like molecules (e.g., shRNA-miR molecules), or artificial RNA molecules (e.g., RNAi molecules) described herein may comprise a nucleotide sequence that is complementary to any sequence of an mRNA molecule, including translated regions, the 5′ UTR, the 3′ UTR, and sequences that include both a translated region and a portion of either 5′ UTR or 3′ UTR. An shRNA-Ike molecule, modified pri-miRNA-like molecule (e.g., shRNA-miR molecule), or artificial RNA molecule (e.g., RNAi molecule) described herein may comprise a nucleotide sequence that is complementary to a region of an mRNA molecule spanning the start codon or the stop codon.

As used herein, a sequence “encoding” a particular molecule is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of mRNA) into an RNA or polypeptide, in vitro or in vivo, when operably linked to an appropriate regulatory sequence.

As used herein, the term “operably linked” means that a sequence is linked to a regulatory sequence in a manner which allows expression of the sequence. Regulatory sequences include, e.g., promoters, enhancers, and other expression control elements that are art-recognized and are selected to direct expression of the sequence.

As used herein, the term “recombinant” refers to a material by recombinant DNA techniques, e.g., produced from cells transformed by an exogenous DNA construct encoding the desired RNA.

As used herein, the term “vector” refers to a vehicle for introducing a nucleic acid into a cell. Vectors include, but are not limited to, plasmids, phagemids, viruses, bacteria, and vehicles derived from viral or bacterial sources. Vectors can also include aptamers, where the aptamer either forms part of or is conjugated to the RNAi molecule (Dassie et al., Nature Biotechnology 27, 839-846 (2009), Thou and Rossi, Silence, 1:4 (2010), McNamera et al., Nature Biotechnology 24, 1005-1015 (2006)). As used herein, a “plasmid” is a circular, double-stranded DNA molecule. A useful type of vector for use in the present invention is a viral vector, wherein heterologous DNA sequences are inserted into a viral genome that can be modified to delete one or more viral genes or parts thereof. Certain vectors are capable of autonomous replication in a host cell (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be stably integrated into the genome of a host cell and are thereby replicated along with the host genome.

As used herein, the term “transfected cell” or “transduced cell” refers to a cell that has been genetically modified. Genetic modification can be stable or transient. Methods of transfection or transduction (i.e., introducing vectors or constructs into cells) include, but are not limited to, liposome fusion (transposomes), viral infection, and routine nucleic acid transfection methods such as electroporation, calcium phosphate precipitation, and microinjection. Successful transfection or transduction will have an intended effect in the transduced cell, such as gene expression, gene silencing, enhancing a gene target, or triggering target physiological event.

As used herein, the term “isolated” refers to material that is removed from its original or native environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature.

As used herein, the term “derived from” means originating from or taken from a specified source. In the case of a sequence fragment, the term means that a partial (e.g., about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more) or complete sequence is present in the specified source and has been cloned or copied from the source.

As used herein, the term “synthetic” refers to a material prepared by chemical synthesis.

As used herein, the term “artificial” refers to a material that is not naturally occurring or differs from a material isolated from a natural source.

The compositions and methods disclosed herein encompass, at least in part, nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 80%, 85%, 90%, 95% identical or higher to the sequence specified.

In the context of nucleotide sequence, the term “substantially homologous” or “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a nucleic acid or polypeptide having common functional activity, or encode a common structural nucleic acid or polypeptide domain or a common functional nucleic acid or polypeptide activity. For example, nucleotide sequences can have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence, e.g., a sequence provided herein.

Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a typical embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, 50%, or 60%, e.g., at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com) using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One suitable set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

The nucleic acid sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid as described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. See www.ncbi.nlm.nih.gov.

The compositions and methods disclosed herein also encompass, at least in part, nucleic acids having sequences substantially complementary or complementary to a reference sequence.

As used herein, the term “substantially complementary” refers to sequences of nucleotides where a majority (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) or all of the bases in the sequence are complementary, or one or more (e.g., no more than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%) bases are non-complementary, or mismatched. A complementary sequence can be a reverse complement of the sequence allowing for Watson-Crick base pairing, wobble base pairing, or both, whereby G pairs with either C or U, and A pairs with either U or T. A sequence may be complementary to the entire length of another sequence or it may be complementary to a specified portion or length of another sequence. One skilled in the art will recognize that the U may be present in RNA, and that T may be present in DNA. Therefore, a U within an RNA sequence may pair with A or G in either an RNA sequence or a DNA sequence, while an A within either of an RNA or DNA sequence may pair with a U in a RNA sequence or T in a DNA sequence. Two sequences that are substantially complementary may hybridize to each other, e.g., under low stringency, medium stringency, high stringency, or very high stringency conditions.

As used herein, the term “wobble base pairing” with regard to two complementary nucleic acid sequences refers to the base pairing of G to uracil U rather than C, when one or both of the nucleic acid strands contains the ribonucleobase U.

As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions 4) are suitable conditions and the ones that should be used unless otherwise specified.

As used herein, the term “treat,” “treating”, or “treatment” means that a subject (e.g., a human) who has a disorder and/or experiences a symptom of a disorder, will, in an embodiment, suffer a less severe symptom and/or recover faster when a therapeutic agent is administered than if the therapeutic agent were never administered. In an embodiment, when an HIV infection or AIDS is treated, an assay to detect HIV in the subject will detect fewer HIV after effective treatment for the HIV infection or AIDS. For example, a diagnostic assay, such as PCR (e.g., VCR) will detect fewer or no HIV in a biological sample of a subject after administration of a therapeutic agent for the effective treatment of the HIV infection or AIDS. Treatment can, e.g., partially or completely alleviate, ameliorate, relieve, inhibit, or reduce the severity of and/or reduce incidence and optionally delay onset of one or more manifestations of the effects or symptoms, features, and/or causes of a particular infection, disease, disorder, and/or condition (e.g., an HIV infection). In an embodiment, treatment is of a subject who does not exhibit certain signs of the relevant infection, disease, disorder, and/or condition, and/or of a subject who exhibits only early signs of the infection, disease, disorder, and/or condition. In an embodiment, treatment is of a subject who exhibits one or more established signs of the relevant infection, disease, disorder, and/or condition. In an embodiment, treatment is of a subject diagnosed as suffering from an HIV infection or AIDS.

As used herein, the term “prevent,” “preventing”, or “prevention” means that a subject (e.g., a human) is less likely to have a disorder (e.g., an HIV infection or AIDS) if the subject receives a therapeutic agent, e.g., prior to (e.g., 1 day, 2 days, 1 week, 2 weeks, 3 weeks, or 1 month or more) being exposed to an agent that causes the disorder, e.g., HIV.

As used herein, a “subject” or “patient” can be a human or non-human animal.

Design of shRNA Clusters and Modified Pri-miRNA Clusters

As used herein, an “shRNA duster” refers to a group of nucleotide sequences encoding shRNA-like molecules located within a short distance (e.g., within about 10 Kb, e.g.; within about 5 kb, 4 kb, 3 kb, 2 kb, or 1 kb) on a same nucleic acid. In an embodiment, expression of the shRNA-like molecules encoded by an shRNA duster is under the control of a common regulatory sequence (e.g., a promoter). In an embodiment, the shRNA-like molecules are transcribed as a single transcript.

As used herein, an “miRNA duster” refers to a group of nucleotide sequences encoding miRNA-like molecules located within a short distance (e.g., within about 10 Kb, e.g., within about 5 kb, 4 kb, 3 kb, 2 kb, or 1 kb) on a same nucleic acid, e.g., a same chromosome. In an embodiment, expression of the modified pri-miRNA-like molecules encoded by an miRNA duster is under the control of a common regulatory sequence (e.g., a promoter). In an embodiment, the modified pri-miRNA-like molecules are transcribed as a single transcript.

Exemplary approaches to define and analyze miRNA dusters are described, e.g., in Chan et al. (2012) Genomics 100(3): 141-148, which is incorporated by reference herein in its entirety.

The nucleic acid molecules (e.g., shRNA dusters or artificial miRNA dusters) described herein can encode artificial RNA molecules, e.g.; interfering RNA or small inhibitory RNA molecules (RNAi molecules), RNAi molecules include, but are not limited to, short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and micro-RNAs (mRNAs) in all stages of processing. These molecules can have different origins: siRNAs can be synthetic or processed from double-stranded precursors (dsRNAs) with two distinct strands of base-paired RNA; siRNAs that are derived from repetitive sequences in the genome are called rasiRNAs; and miRNAs are derived from a single transcript that forms base-paired hairpins. Base pairing of siRNAs and miRNAs can be perfect (i.e., fully complementary) or imperfect, including bulges in the duplex region. Mechanisms of utilizing RNAi molecules in mammalian cell are described in more detail below.

RNAi can be used a powerful tool for in vitro, ex vivo, or in vivo studies of gene function in mammalian cells and for therapy in both human and veterinary contexts and as a tool for in vitro and in vivo studies of gene function. Inhibition of a target gene via RNAi is generally sequence-specific.

While not wishing to be bound by theory, it is believed that there are at least three mechanisms of utilizing RNAi in mammalian cells. The first is cytoplasmic delivery of siRNA molecules, which are either chemically synthesized or enzymatically processed dsRNA. These siRNAs are introduced into cells using standard transfection methods. The siRNAs enter the RISC to silence target mRNA expression.

The second mechanism is nuclear delivery, e.g., via viral vectors, of gene expression cassettes expressing a short hairpin RNA (shRNA). The shRNA is modeled on micro interfering RNA (miRNA); an endogenous trigger of the RNAi pathway (Lu et al., 2005, Advances in Genetics 54: 117-142). Conventional shRNAs, which mimic pre-miRNA, are transcribed by RNA Polymerase II or III as single-stranded molecules that form stem-loop structures. Once produced, they exit the nucleus, are cleaved by DICER, and enter the RISC as siRNAs.

The third mechanism is similar to the second mechanism except that the shRNA is modeled on primary miRNA (e.g.; shRNA-miR) rather than pre-miRNA transcripts (Sewell et al., 2006, Drug Discovery Today 11: 975-982), The use of this transcript produces a more physiological shRNA that reduces toxic effects. The shRNA-miR is first cleaved to produce shRNA and then cleaved again by DICER to produce siRNA. The siRNA is then incorporated into the RISC for target mRNA degradation.

For mRNA degradation, translational repression, or deadenylation, mature miRNAs or siRNAs are loaded into the RNA Induced Silencing Complex (RISC) by the RISC-loading complex (RLC). Subsequently, the guide strand leads the RISC to cognate target mRNAs in a sequence-specific manner and the Slicer component of RISC hydrolyses the phosphodiester bound coupling the target mRNA nucleotides paired to nucleotide 10 and 11 of the RNA guide strand. Slicer forms together with distinct classes of small RNAs the RNAi effector complex, which is the core of RISC. Therefore, in an embodiment, the “guide strand” is that portion of the double-stranded RNA that associates with RISC as opposed to the “passenger strand”, which is not associated with RISC.

As used herein, the term “modified pri-miRNA-like molecule” or “shRNA-miR molecule” refers to micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide and passenger strands of an siRNA duplex are incorporated into an existing (or natural) pri-miRNA or into a modified or synthetic (designed) pri-miRNA, When transcribed, a modified pri-miRNA-like molecule or shRNA-miRNA molecule forms a structure identical or similar to a natural pri-miRNA. The modified pri-miRNA-like molecule or shRNA-miR molecule can be subsequently processed by Drosha and its co-factors into modified pre-miRNA or shRNA. The modified pri-miRNA-like molecule or shRNA-miR molecule can have a 5′ flanking region, terminal loop region, and/or a 3′ flanking region that is substantially identical to the 5′ flanking region, terminal loop region, and/or a 3′ flanking region of a naturally occurring pri-miRNA. In some embodiments, one or more insertions, deletions, and substitutions can be introduced into the 5′ flanking region, terminal loop region, and/or the 3′ flanking region of a naturally-occurring pri-miRNA sequence to produce the backbone of a pri-miRNA-like molecule or shRNA-miR molecule. While not wishing to be bound by theory, it is believed that any naturally-occurring pri-miRNA can provide a backbone for designing modified pri-miRNA-like molecules or shRNA-miR molecules. For example, any miRNAs described in miRBase can be used. miRBase Release 21: June 2014 is publically available at mirbase.org/pub/mirbase, which is incorporated by reference herein in its entirety (Kozomara and Griffiths-Jones NAR 2014 42:D68-D73; Kozomara and Griffiths-Jones NAR 2011 39:D152-D157; Griffiths-Jones et at NAR 2008 36:D154-D158; Griffiths-Jones et at NAR 2006 34:D140-D144; Griffiths-Jones NAR 2004 32:D109-D111).

The typical structural features of primary miRNAs and mRNA precursors, and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design are known in the art, e.g. as described in Krol et at (2004) The Journal of Biological Chemistry, 279, 42230-42239.

Without wishing to be bound by theory, it is believed that primary transcripts of the miRNA genes, pri-miRNAs are processed in the nucleus to pre-miRNAs by the ribonuclease Drosha (Lee et at (2003) Nature 425, 415-419) and exported from the nucleus by Exportin-5 (Lund et at (2003) Science 303, 95-98). The 60-90-nt miRNA precursors form the stem-loop structures and the cytoplasmic ribonuclease class III enzyme Dicer (Hutvágner et at (2002) Science 297; 2056-2060) excises miRNAs from the pre-miRNA hairpin stem. Dicer, either alone or with the help of Drosha, cleaves both strands of the precursor to form a double-stranded miRNA/miRNA* duplex (Lee et at (2003) Nature 425, 415-419), and typically only one strand (e.g., a guide strand as opposed to a passenger strand) accumulates which enters the RNAi-induced silencing complex (RISC) (Khvorova et at (2003) Cell 115, 209-216).

As used herein, a “stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion or stem region) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion or terminal loop region). The terms “hairpin” and “fold-back” structures can also be used to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches. The stem region can contain an artificial RNA molecule (e.g., RNAi molecule) as described herein.

As used herein, a terminal loop region refers to a region flanked by a guide strand and a passenger strand of an RNAi molecule (e.g., a double-stranded siRNA or miRNA/miRNA*).

As described herein, a stem region can be a region formed by a guide strand and a passenger strand of an RNAi molecule (e.g., a double-stranded siRNA or miRNA/miRNA*) and connected to a terminal loop region. The guide strand can be either 5′ or 3′ to the terminal loop region. Likewise, the passenger strand can be either 3′ or 5′ to the terminal loop region. The stem region can form a duplex with or without one or more mismatches or bulges.

As used herein, a 5′ flanking region refers to a region immediately adjacent to a strand (either a guide strand or a passenger strand) that is 5′ to the terminal loop region. As used herein, a 3′ flanking region refers to a region immediately adjacent to a strand (either a guide strand or a passenger strand) that is 3′ to the terminal loop region. The 5′ flanking region or 3′ flanking region described herein, as well as the structure formed by the 5′ and 3′ flanking regions, may have one or more structural features of a corresponding region of a naturally-occurring pri-miRNA. While not wishing to be bound by theory, it is believed that in an embodiment, the presence of the 5′ flanking region and/or 3′ flanking region may improve production of an RNAi molecule, e.g., an siRNA and miRNA, embedded in an shRNA-like molecule or modified pri-miRNA-like molecule described herein. The length of the 5′ flanking region or 3′ flanking region may vary. Exemplary approaches for selecting a 5′ flanking region or 3′ flanking region (e.g., determining the optimal length of the 5′ flanking region or 3′ flanking region) are described herein in the examples.

One can design and express shRNA-like molecules, modified pri-miRNA-like molecules (e.g., shRNA-miR molecules), or artificial RNA molecules (e.g., RNAi molecules) based on the features of the native gene encoding the pri-miRNA. In particular, the pri-miRNA architecture can be used to express shRNA-like molecules, modified pri-miRNA-like molecules (e.g., shRNA-miR molecules), or artificial RNA molecules (e.g.; RNAi molecules) from pol H promoter-based expression plasmids by using a variety of RNA pol II-based expression vectors or even from poi III promoter-based expression plasmids using pol III-dependent promoters. In certain embodiments, expression vectors may employ use of expression cassettes comprising the sequence encoding an shRNA-like molecule, modified pri-miRNA-like molecule (e.g., shRNA-miR molecule), or artificial RNA molecule (e.g., RNAi molecule). In certain embodiments, expression vectors encoding shRNA-like molecules; modified pri-miRNA-like molecules (e.g., shRNA-miR molecules), or artificial RNA molecules (e.g., RNAi molecules) may be based on self-inactivating lentivirus (SIN) vector backbones. In certain embodiments, expression vectors encoding shRNA-like molecules, modified pri-miRNA-like molecules (e.g., shRNA-miR molecules), or artificial RNA molecules (e.g., RNAi molecules) may be based on CMV-based or MSCV-based vector backbones. Generally; appropriate vector backbones include vector backbones used in construction of expression vectors for conventional shRNAs. Exemplary use of expression cassettes in construction of shRNA expression vectors also useful in the construction of expression cassettes encoding the modified pri-miRNA molecules or shRNA-miR molecules of the disclosure can be found, e.g.; in Gottwein E. and Cullen B. Meth. Enzymol. 427:229-243, 2007, Dickens et al. Nature Genetics, 39:914-921; 2007, Chen et al, Science 303: 83-86, 2004; Zeng and Cullen, RNA 9: 112-123; 2003, the contents of which are specifically incorporated herein by reference.

Exemplary guidelines for designing RNAi molecules, e.g., siRNAs or shRNAs, are known in the art, e.g., as described in Elbashir, et al, (2001) EMBO J 20: 6877-6888, Brown et al. (2002) Ambion TechNotes 9(1): 3-5; Sui, et al. (2002) Proc. Natl. Acad. Sci. US A 99(8): 5515-5520; Lee et al. Nature Biotechnology 20: 500-505; Yu et al. (2002) Proc. Natl. Acad. Sci. USA 99(9): 6047-6052; Paul et al. (2002) Nature Biotechnology 20: 505-508; Brummelkamp et al. (2002) Science 296: 550-553; Jacque, et al., (2002) Nature 418: 435-438; Miyagishi et al. (2002) Nature Biotechnology 20; 497-500; Paddison et al. (2002) Genes Devel. 16: 948-958; the contents of which are specifically incorporated herein by reference.

The nucleic acid molecules described herein can be modified, e.g., to enhance stability in vivo. Modified nucleic acids include molecules containing nucleotide analogues, including those molecules having additions, deletions, and/or substitutions in the nucleobase, sugar, or backbone; and molecules that are cross-linked or otherwise chemically modified. The modified nucleotide(s) may be within portions of the nucleic acid molecule, or throughout it. For instance, the shRNA-like molecule may be modified, or contain modified nucleic acids in regions at its 5′ end, its 3′ end, or both, and/or within the guide strand, passenger strand, or both, and/or within nucleotides that overhang the 5′ end, the 3′ end, or both. See U.S. Pat. Nos. 6,107,094 and 5,898,031; U.S. Publication Nos. 2008/0249039, 2007/0191294, 2008/0213891, 2007/0135372, and 2005/0020521; all of which are hereby incorporated by reference.

Vectors

The disclosure provides vectors that include a nucleic acid molecule (e.g., shRNA duster or artificial miRNA duster) described herein, e.g., for expression of an shRNA-like molecule, modified pri-miRNA-like molecule (e.g., shRNA-miR molecule), or artificial RNA molecule (e.g., RNAi molecule) described herein.

In certain embodiments, expression vectors encoding shRNA-like molecules, modified pri-miRNA-like molecules (e.g., shRNA-miR molecules), or artificial RNA molecules (e.g., RNAi molecules) as described herein, may be based on self-inactivating lentivirus (SIN) vector backbones. In certain embodiments, expression vectors encoding the shRNA-like molecules, modified pri-miRNA-like molecules (e.g., shRNA-miR molecules), or artificial RNA molecules (e.g., RNAi molecules) described herein may be based on CMV-based or MSCV-based vector backbones. Exemplary vector backbones and methodologies for construction of expression vectors suitable for use with the shRNA clusters or nucleic acid molecules (e.g., artificial miRNA dusters) described herein, and methods for introducing such expression vectors into various mammalian cells can be found, e.g., in Premsrurit P K. et al., Cell, 145(1):145-158, 2011, Gottwein E. and Cullen B. Meth. Enzymol, 427:229-243, 2007, Dickens et at, Nature Genetics, 39:914-921, 2007, Chen et at, Science 303: 83-86, 2004; Zeng and Cullen, RNA 9: 112-123, 2003, the contents of which are specifically incorporated herein by reference.

The vectors can be targeting vectors, such as those using flp recombination into the colA locus allowing single copy integration. Other targeting sites in the mouse genome include, but are not limited to ROSA26 and HPRT. Additionally, transposase may be used to introduce mimics into the genome of an animal or the cell of an animal. See Premsrurit P K. et al., Cell, 145(1):145-158, (2011), the contents of which are specifically incorporated herein by reference.

General principles of vector construction and expression of sequences from vector constructs, as well as methods of making and using the vectors, are described, e.g., in International Publication No. WO 09/055,724, which is incorporated herein by reference.

Artificial RNA molecules (e.g., RNAi molecules) described herein can be expressed from vectors to provide sustained silencing and high yield delivery into almost any cell type. In a certain embodiment, the vector is a viral vector. Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral, and avian viral vectors. The use of viral vector-based RNAi delivery not only allows for stable single-copy genomic integrations, but also avoids the non-sequence specific response via cell-surface toll-like receptor 3 (TLR3).

Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP-FE-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. A producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles can then be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.

In certain embodiments, cells can be engineered using a lentivirus and lentivirus based vectors. Such an approach is advantageous in that it allows for tissue-specific expression in animals through use of cell type-specific poi H promoters, efficient transduction of a broad range of cell types, including nondividing cells and cells that are hard to infect by retroviruses, and inducible and reversible gene knockdown by use of tet-responsive and other inducible promoters. Methods for expressing artificial RNA molecules (e.g., RNAi molecules) by producing and using lentivirus engineered cells are known in the art. For exemplary descriptions of such methods, see, for example, Stegmeier F. et al., Proc Acad Sci USA 2005, 102(37):13212-13217, Klinghoffer et al., RNA 2010, 16:879-884, the contents of which are specifically incorporated herein. Efficient production of replication-incompetent recombinant lentivirus may be achieved, for example, by co-transfection of expression vectors and packaging plasmids using commercially available packaging cell lines, such as TLA-HEK293™, and packaging plasmids, available from Thermo Scientific/Open Biosystems, Huntsville, Ala.

In certain embodiments, cells can be engineered using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that can integrate its DNA into nondividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, International Publication No. WO 2015/031686, U.S. Pat. Nos. 7,927,585, 7,906,111, 7,261,544, 6,221,646, 5,589,377, 5,478,745, 5,474,935, 5,436,146, 5,436,146, 5,173,414, and 5,139,941, and. For example, an AAV vector can include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The recombinant AAV vector can be transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells.

Typically any method for introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct. A viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA. Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like. Thus the shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.

Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters. It has been shown that shRNAs are also most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al., 2005, Nat. Genet, 39: 914-921). Therefore, in a certain embodiment, the coding sequence of the RNAi molecule is controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type H promoters. Examples of useful promoters in the context of the invention are tetracycline-inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline transactivator (rtTA) systems. Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types. A certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Publication No. WO 2004/029219, European Publication No. EP 2166107, and Fewell et al., 2006, Drug Discovery Today 11: 975-982, for an exemplary description of inducible shRNA.

To facilitate the monitoring of the target gene knockdown, cells harboring the RNAi-expressing construct can additionally comprise a marker or reporter construct, such as a fluorescent construct. The reporter construct can express a marker, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, yellow fluorescent protein (YFP), such as VENUS, enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and citrine and red fluorescent protein from discosoma (dsRED). Other suitable detectable markers include chloramphenicol acetyltransferase (CAT), luminescent proteins such as luciferase lacZ (β-galactosidase) and horseradish peroxidase (HRP), nopaline synthase (NOS), octopine synthase (OCS), and alkaline phosphatase. The marker gene can be separately introduced into the cell harboring the shRNA construct (e.g., co-transfected, etc.). Alternatively, the marker gene can be on the shRNA construct and the marker gene expression can be controlled by the same or a separate translation unit, for example, by an IRES (internal ribosomal entry site). In one aspect of the invention, marker genes can be incorporated into “sensor” expression vectors for use in high throughput methods for determining the knockdown efficiency against particular genes and for identifying the most potent target sequences for a particular target gene. Such methods, including the design and use of plasmids and reporter constructs for testing the potency of particular shRNA molecules are described, e.g., in International Publication No. WO20091055724, the contents of which are herein specifically incorporated by reference in its entirety.

Reporters can also be those that confer resistance to a drug, such as neomycin, ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, doxycycline, and tetracyclin. Reporters can also be lethal genes, such as herpes simplex virus-thymidine kinase (HSV-TK) sequences, as well as sequences encoding various toxins including the diphtheria toxin, the tetanus toxin, the cholera toxin, and the pertussis toxin. A further negative selection marker is the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene for negative selection in 6-thioguanine.

To facilitate the quantification of specific shRNAs in a complex population of cells infected with a library of shRNAs, each shRNA construct can additionally comprise a barcode. A barcode is a unique nucleotide sequence (generally a 19-mer) linked to each shRNA. The barcode can be used to monitor the abundance of each shRNA via micoarray hybridization (Fewell et al., 2006, Drug Discovery Today 11: 975-982). In a certain embodiment, each shRNA construct also comprises a unique barcode. For more information on the use of barcodes in shRNA pooled analyses, see WO 04/029219, Bernards et al., 2006, Nature Methods 3: 701-706, and Chang et al., 2006, Nature Methods 3: 707-714.

Exemplary Target Genes

The nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein can be used to express artificial RNA molecules (e.g., RNAi molecules) that target any genes. For example, the target genes can include mammalian genes (e.g., human genes) or viral genes. In an embodiment, the target gene is a mammalian gene encoding a viral receptor or co-receptor, e.g., an HIV co-receptor, e.g., CCR5 or CXCR4. In another embodiment, the target gene is a viral gene, e.g., an HIV gene, e.g., Gag, Env, Tat, Pol2, Pol1, or Vif. Artificial RNA molecules (e.g., RNAi molecules) can be designed based on the gene sequences provided herein or known in the art.

Exemplary CCR and HIV gene sequences are provided as follows:

Homo sapiens CC chemokine receptor 5 (CCR5)
CTGTTTAAAGACAAAAAGGCCCCAAAAAGGAGGGATGGCACGAAACACCCT
CCAATATGGGCATGGAGTCTAGAGTGACAAAGTGATCAAAAGTTCATTTCCTATGGGG
TGTCCGAATGTACTTAATAATAAAAAGAGAACAAGAGCCATGCAAACTGAGAGGGACA
AAGTAGAAAGAGTAGCAGACACCAAGCAACTAAGTCACAGCATGATAAGCTGCTAGCT
TGTTGTCATTATTGTATCCAGAACAACATTTCATTTAAATGCTGAAGAATTTCCCATGGG
TCCCCACTTTCTTGTGAATCCTTGGGCTGAACCCCCCCGTCCTGAGTGGTTACTAGAA
CACACCTCTGGACCAGAAACACAAGAGTGGAGTAACACACACTGCAAAGCTGTGCTTC
CTTGTTTCAGCCTGTGAATCCTCACCTTGTTTCCCATCTAGCCTATATTTTTCAAACTAA
CTTGGCCATAGAATCATGTCGTATTTAGGGTGGAAGCTGCCCCAGGTCTAGCGCGTCA
TTTAACAGATGAGGAAATGGAAGCTTGGGCAGTGGAAGTATCTTGCCGAGGTCACACA
GCAAGTCAGCAGCACAGCGTGTGTGACTCCGAGCCTGCTCCGCTAGCCCACATTGCC
CTCTGGGGGTGAGTATGTCTTCACATCCTCCAATACCCCTAATGACAGACAAACAGAA
CATGGCAAAGCCTCAGCTCTGCATGGTGAAAGTAAGAACCAGCAATTGCCACAAACAG
AAATACAGTGTTGGTCCGGCAGCCTCCGGGGGTTCTGCACAAGTGGATTACCAGTGA
ATACAAGGCTATCTATCTTCCGAAAAACCAAAGTTGTATTTATGCTATCTATTTTCTATA
AAATTTTATATTAATTTACTTGTCCTATTTTTGAACTCTTTCAAAAGCACACTTTATATTT
CCCCTGCTTAAACAGTCCCCCGAGGGTGGGTGCCCAAAAGGCTCTACACTTGTTATCA
TTCCCTCTCCACCACAGGCATATTGAGTAAGTTTGTATTTGGGTTTTTTTAAAACCTCCA
CTCTACAGTTAAGAAAACTAAGGCACAGAGCTTCAATAATTTGGTCAGAGCCAAGTAG
CAGTAATGAAGCTGGAGGTTAAACCCAGCAGCATGACTGCAGTTCTTAATCAATGCCT
TTTGAATTGCACATATGGGATGAACTAGAACATTTTCTCGATGATTCGCTGTCCTTGTT
ATGATTATGTTACTGAGCTCTGTTGTAGCACAGACATATGTCCCTATATGGGGCGGGG
GTGGGGGTGTCTTGATCGCTGGGCTATTTCTATACTGTTCTGGCTTTTCCCAAGCAGT
CATTTCTTTCTATCCTCCAAGCACCAGCAATTAGCTTTACCTTTTCAGCTTCTAGTTTGC
TGAAACTAATCTGCTATAGACAGAGACTCCGGTGAACCAATTTTATTAGGATTTGATCA
AATAAACTCTCTCTGACAAAGGACTGCTGAAAGAGTAACTAAGAGTTTGATGTTTACTG
AGTGCATAGTATGTGCTAGATGCTGGCCGTGGATGCCTCATAGAATCCTCCCAACAAC
TCATGAAATGACTACTGTCATTCAGCCCAATACCCAGACGAGAAAGCTGAGGGTAAGA
CAGGTTTCAAGCTTGGCAGTCTGACTACAGAGGCCACTGGCTTAGCCCCTGGGTTAG
TCTGCCTCTGTAGGATTGGGGGCACGTAATTTTGCTGTTTGGGGTCTCATTTGCCTTC
TTAGAGATCACAAGCCAAAGCTTTTTATTCTAGAGCCAAGGTCACGGAAGCCCAGAGG
ACATCTTGTGGCTCGGGAGTAGCTCTCTGCTGTCTTCTCAGCTCTGCTGACAATACTT
GAGATTTTCAGATGTCACCAACCGCCAAGAGAGCTTGATATGACTGTATATAGTATAGT
CATAAAGAACCTGAACTTGACCATATACTTATGTCATGTGGAAAATTTCTCATAGCTTC
AGATAGATTATATCTGGAGTGAAGGATCCTGCCACCTACGTATCTGGCATAGTGTGAG
TCCTCATAAATGCTTACTGGTTTGAAGGGCAACAAAATAGTGAACAGAGTGAAAATCC
CCACTAAGATCCTGGGTCCAGAAAAAGATGGGAAACCTGTTTAGCTCACCCGTGAGC
CCATAGTTAAAACTCTTTAGACAACAGGTTGTTTCCGTTTACAGAGAACAATAATATTG
GGTGGTGAGCATCTGTGTGGGGGTTGGGGTGGGATAGGGGATACGGGGAGAGTGGA
GAAAAAGGGGACACAGGGTTAATGTGAAGTCCAGGATCCCCCTCTACATTTAAAGTTG
GTTTAAGTTGGCTTTAATTAATAGCAACTCTTAAGATAATCAGAATTTTCTTAACCTTTTA
GCCTTACTGTTGAAAAGCCCTGTGATCTTGTACAAATCATTTGCTTCTTGGATAGTAAT
TTCTTTTACTAAAATGTGGGCTTTTGACTAGATGAATGTAAATGTTCTTCTAGCTCTGAT
ATCCTTTATTCTTTATATTTTCTAACAGATTCTGTGTAGTGGGATGAGCAGAGAACAAAA
ACAAAATAATCCAGTGAGAAAAGCCCGTAAATAAACCTTCAGACCAGAGATCTATTCTC
CAGCTTATTTTAAGCTCAACTTAAAAAGAAGAACTGTTCTCTGATTCTTTTCGCCTTCAA
TACACTTAATGATTTAACTCCACCCTCCTTCAAAAGAAACAGCATTTCCTACTTTTATAC
TGTCTATATGATTGATTTGCACAGCTCATCTGGCCAGAAGAGCTGAGACATCCGTTCC
CCTACAAGAAACTCTCCCCGGTAAGTAACCTCTCAGCTGCTTGGCCTGTTAGTTAGCT
TCTGAGATGAGTAAAAGACTTTACAGGAAACCCATAGAAGACATTTGGCAAACACCAA
GTGCTCATACAATTATCTTAAAATATAATCTTTAAGATAAGGAAAGGGTCACAGTTTGG
AATGAGTTTCAGACGGTTATAACATCAAAGATACAAAACATGATTGTGAGTGAAAGACT
TTAAAGGGAGCAATAGTATTTTAATAACTAACAATCCTTACCTCTCAAAAGAAAGATTTG
CAGAGAGATGAGTCTTAGCTGAAATCTTGAAATCTTATCTTCTGCTAAGGAGAACTAAA
CCCTCTCCAGTGAGATGCCTTCTGAATATGTGCCCACAAGAAGTTGTGTCTAAGTCTG
GTTCTCTTTTTTCTTTTTCCTCCAGACAAGAGGGAAGCCTAAAAATGGTCAAAATTAATA
TTAAATTACAAACGCCAAATAAAATTTTCCTCTAATATATCAGTTTCATGGCACAGTTAG
TATATAATTCTTTATGGTTCAAAATTAAAAATGAGCTTTTCTAGGGGCTTCTCTCAGCTG
CCTAGTCTAAGGTGCAGGGAGTTTGAGACTCACAGGGTTTAATAAGAGAAAATTCTCA
GCTAGAGCAGCTGAACTTAAATAGACTAGGCAAGACAGCTGGTTATAAGACTAAACTA
CCCAGAATGCATGACATTCATCTGTGGTGGCAGACGAAACATTTTTTATTATATTATTTC
TTGGGTATGTATGACAACTCTTAATTGTGGCAACTCAGAAACTACAAACACAAACTTCA
CAGAAAATGTGAGGATTTTACAATTGGCTGTTGTCATCTATGACCTTCTCTGGGACTTG
GGCACCCGGCCATTTCACTCTGACTACATCATGTCACCAAACATCTGATGGTCTTGCC
TTTTAATTCTCTTTTCGAGGACTGAGAGGGAGGGTAGCATGGTAGTTAAGAGTGCAGG
CTTCCCGCATTCAAAATCGGTTGCTTACTAGCTGTGTGGCTTTGAGCAAGTTACTCACC
CTCTCTGTGCTTCAAGGTCCTTGTCTGCAAAATGTGAAAAATATTTCCTGCCTCATAAG
GTTGCCCTAAGGATTAAATGAATGAATGGGTATGATGCTTAGAACAGTGATTGGCATC
CAGTATGTGCCCTCGAGGCCTCTTAATTATTACTGGCTTGCTCATAGTGCATGTTCTTT
GTGGGCTAACTCTAGCGTCAATAAAAATGTTAAGACTGAGTTGCAGCCGGGCATGGTG
GCTCATGCCTGTAATCCCAGCATTCTAGGAGGCTGAGGCAGGAGGATCGCTTGAGCC
CAGGAGTTCGAGACCAGCCTGGGCAACATAGTGTGATCTTGTATCTATAAAAATAAAC
AAAATTAGCTTGGTGTGGTGGCGCCTGTAGTCCCCAGCCACTTGGAGGGGTGAGGTG
AGAGGATTGCTTGAGCCCGGGATGGTCCAGGCTGCAGTGAGCCATGATCGTGCCACT
GCACTCCAGCCTGGGCGACAGAGTGAGACCCTGTCTCACAACAACAACAACAACAAC
AAAAAGGCTGAGCTGCACCATGCTTGACCCAGTTTCTTAAAATTGTTGTCAAAGCTTCA
TTCACTCCATGGTGCTATAGAGCACAAGATTTTATTTGGTGAGATGGTGCTTTCATGAA
TTCCCCCAACAGAGCCAAGCTCTCCATCTAGTGGACAGGGAAGCTAGCAGCAAACCT
TCCCTTCACTACAAAACTTCATTGCTTGGCCAAAAAGAGAGTTAATTCAATGTAGACAT
CTATGTAGGCAATTAAAAACCTATTGATGTATAAAACAGTTTGCATTCATGGAGGGCAA
CTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAACAA
GATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCCCT
GCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCCGCTCTACTCACT
GGTGTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAACTGCA
AAAGGCTGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTT
TTTCCTTCTTACTGTCCCCTTCTGGGCTCACTATGCTGCCGCCCAGTGGGACTTTGGA
AATACAATGTGTCAACTCTTGACAGGGCTCTATTTTATAGGCTTCTTCTCTGGAATCTT
CTTCATCATCCTCCTGACAATCGATAGGTACCTGGCTGTCGTCCATGCTGTGTTTGCTT
TAAAAGCCAGGACGGTCACCTTTGGGGTGGTGACAAGTGTGATCACTTGGGTGGTGG
CTGTGTTTGCGTCTCTCCCAGGAATCATCTTTACCAGATCTCAAAAAGAAGGTCTTCAT
TACACCTGCAGCTCTCATTTTCCATACAGTCAGTATCAATTCTGGAAGAATTTCCAGAC
ATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGCTAC
TCGGGAATCCTAAAAACTCTGCTTCGGTGTCGAAATGAGAAGAAGAGGCACAGGGCT
GTGAGGCTTATCTTCACCATCATGATTGTTTATTTTCTCTTCTGGGCTCCCTACAACATT
GTCCTTCTCCTGAACACCTTCCAGGAATTCTTTGGCCTGAATAATTGCAGTAGCTCTAA
CAGGTTGGACCAAGCTATGCAGGTGACAGAGACTCTTGGGATGACGCACTGCTGCAT
CAACCCCATCATCTATGCCTTTGTCGGGGAGAAGTTCAGAAACTACCTCTTAGTCTTCT
TCCAAAAGCACATTGCCAAACGCTTCTGCAAATGCTGTTCTATTTTCCAGCAAGAGGCT
CCCGAGCGAGCAAGCTCAGTTTACACCCGATCCACTGGGGAGCAGGAAATATCTGTG
GGCTTGTGACACGGACTCAAGTGGGCTGGTGACCCAGTCAGAGTTGTGCACATGGCT
TAGTTTTCATACACAGCCTGGGCTGGGGGTGGGGTGGGAGAGGTCTTTTTTAAAAGG
AAGTTACTGTTATAGAGGGTCTAAGATTCATCCATTTATTTGGCATCTGTTTAAAGTAGA
TTAGATCTTTTAAGCCCATCAATTATAGAAAGCCAAATCAAAATATGTTGATGAAAAATA
GCAACCTTTTTATCTCCCCTTCACATGCATCAAGTTATTGACAAACTCTCCCTTCACTC
CGAAAGTTCCTTATGTATATTTAAAAGAAAGCCTCAGAGAATTGCTGATTCTTGAGTTT
AGTGATCTGAACAGAAATACCAAAATTATTTCAGAAATGTACAACTTTTTACCTAGTACA
AGGCAACATATAGGTTGTAAATGTGTTTAAAACAGGTCTTTGTCTTGCTATGGGGAGAA
AAGACATGAATATGATTAGTAAAGAAATGACACTTTTCATGTGTGATTTCCCCTCCAAG
GTATGGTTAATAAGTTTCACTGACTTAGAACCAGGCGAGAGACTTGTGGCCTGGGAGA
GCTGGGGAAGCTTCTTAAATGAGAAGGAATTTGAGTTGGATCATCTATTGCTGGCAAA
GACAGAAGCCTCACTGCAAGCACTGCATGGGCAAGCTTGGCTGTAGAAGGAGACAGA
GCTGGTTGGGAAGACATGGGGAGGAAGGACAAGGCTAGATCATGAAGAACCTTGACG
GCATTGCTCCGTCTAAGTCATGAGCTGAGCAGGGAGATCCTGGTTGGTGTTGCAGAA
GGTTTACTCTGTGGCCAAAGGAGGGTCAGGAAGGATGAGCATTTAGGGCAAGGAGAC
CACCAACAGCCCTCAGGTCAGGGTGAGGATGGCCTCTGCTAAGCTCAAGGCGTGAG
GATGGGAAGGAGGGAGGTATTCGTAAGGATGGGAAGGAGGGAGGTATTCGTGCAGC
ATATGAGGATGCAGAGTCAGCAGAACTGGGGTGGATTTGGTTTGGAAGTGAGGGTCA
GAGAGGAGTCAGAGAGAATCCCTAGTCTTCAAGCAGATTGGAGAAACCCTTGAAAAGA
CATCAAGCACAGAAGGAGGAGGAGGAGGTTTAGGTCAAGAAGAAGATGGATTGGTGT
AAAAGGATGGGTCTGGTTTGCAGAGCTTGAACACAGTCTCACCCAGACTCCAGGCTG
TCTTTCACTGAATGCTTCTGACTTCATAGATTTCCTTCCCATCCCAGCTGAAATACTGA
GGGGTCTCCAGGAGGAGACTAGATTTATGAATACACGAGGTATGAGGTCTAGGAACA
TACTTCAGCTCACACATGAGATCTAGGTGAGGATTGATTACCTAGTAGTCATTTCATGG
GTTGTTGGGAGGATTCTATGAGGCAACCACAGGCAGCATTTAGCACATACTACACATT
CAATAAGCATCAAACTCTTAGTTACTCATTCAGGGATAGCACTGAGCAAAGCATTGAG
CAAAGGGGTCCCATATAGGTGAGGGAAGCCTGAAAAACTAAGATGCTGCCTGCCCAG
TGCACACAAGTGTAGGTATCATTTTCTGCATTTAACCGTCAATAGGCAAAGGGGGGAA
GGGACATATTCATTTGGAAATAAGCTGCCTTGAGCCTTAAAACCCACAAAAGTACAATT
TACCAGCCTCCGTATTTCAGACTGAATGGGGGTGGGGGGGGCGCCTTAGGTACTTAT
TCCAGATGCCTTCTCCAGACAAACCAGAAGCAACAGAAAAAATCGTCTCTCCCTCC CT
TTGAAATGAATATACCCCTTAGTGTTTGGGTATATTCATTTCAAAGGGAGAGAGAGAGG
TTTTTTTCTGTTCTTTCTCATATGATTGTGCACATACTTGAGACTGTTTTGAATTTGGGG
GATGGCTAAAACCATCATAGTACAGGTAAGGTGAGGGAATAGTAAGTGGTGAGAACTA
CTCAGGGAATGAAGGTGTCAGAATAATAAGAGGTGCTACTGACTTTCTCAGCCTCTGA
ATATGAACGGTGAGCATTGTGGCTGTCAGCAGGAAGCAACGAAGGGAAATGTCTTTC
CTTTTGCTCTTAAGTTGTGGAGAGTGCAACAGTAGCATAGGACCCTACCCTCTGGGCC
AAGTCAAAGACATTCTGACATCTTAGTATTTGCATATTCTTATGTATGTGAAAGTTACAA
ATTGCTTGAAAGAAAATATGCATCTAATAAAAAACACCTTCTA
HIV-1 Gag
ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATTAGATCGATGGGAA
AAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGG
CAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAG
GCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACG
TAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAA
AAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAA
AGCACAGCAAGCAGCAGCTGACACAGGACACAGCAGCCAGGTCAGCCAAAATTACCC
TATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTA
AATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGT
TTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGT
GGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGC
AGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAG
AGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGG
ATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCC
TGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAA
GGACCAAAGGAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCG
AGCAAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGC
GAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGGAGCGACACTAGAAGAA
ATGATGACAGCATGTCAGGGAGTGGGGGGACCCGGCCATAAAGCAAGAGTTTTGGCT
GAAGCAATGAGCCAAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTA
GGAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGCCAA
AAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCA
AATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCAC
AAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAA
GAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCCGATA
GACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAGCGACCCCTCGT
CACAATAA
HIV-1 Pol
TTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCCAGGGAATTTTCTT
CAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAAGA
GACAACAACTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCT
TCCCTCAGATCACTCTTTGGCAGCGACCCCTCGTCACAATAAAGATAGGGGGGCAATT
AAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAATTTGC
CAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACA
GTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAG
GACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTA
AATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGG
CCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTT
GTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAAT
ACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTT
CAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATC
CTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTT
TTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAA
ACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGG
ATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAA
ATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAA
ATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGA
TTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGA
ACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGG
ACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGC
AGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAA
GTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAA
AAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACA
GAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTG
AAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAA
CAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAA
ATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAA
GCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGT
ACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGC
CAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAA
GTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAG
CTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGG
AATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGC
AGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGG
AGGAAATGAACAAGTAGATAAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAG
ATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGAGAGCAAT
GGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGAT
AAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATAT
GGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGC
CAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAG CATAC
TTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCA
GCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGG
AATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAA
TTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTAC
AAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGC
AGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAAC
AAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGG
AAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATA
GTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACA
GATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAA
HIV-1 Tat
ATGGAGCCAGTAGATCCTAGACTAGAGCCCTGGAAGCATCCAGGAAGTCAG
CCTAAAACTGCTTGTACCAATTGCTATTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGT
TTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGA
AGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCC
AATCCCGAGGGGACCCGACAGGCCCGAAGGAATAG
HIV-1 Vif
ATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGG
ATTAACACATGGAAAAGATTAGTAAAACACCATATGTATATTTCAAGGAAAGCTAAGGA
CTGGTTTTATAGACATCACTATGAAAGTACTAATCCAAAAATAAGTTCAGAAGTACACA
TCCCACTAGGGGATGCTAAATTAGTAATAACAACATATTGGGGTCTGCATACAGGAGA
AAGAGACTGGCATTTGGGTCAGGGAGTCTCCATAGAATGGAGGAAAAAGAGATATAG
CACACAAGTAGACCCTGACCTAGCAGACCAACTAATTCATCTGCACTATTTTGATTGTT
TTTCAGAATCTGCTATAAGAAATACCATATTAGGACGTATAGTTAGTCCTAGGTGTGAA
TATCAAGCAGGACATAACAAGGTAGGATCTCTACAGTACTTGGCACTAGCAGCATTAA
TAAAACCAAAACAGATAAAGCCACCTTTGCCTAGTGTTAGGAAACTGACAGAGGACAG
ATGGAACAAGCCCCAGAAGACCAAGGGCCACAGAGGGAGCCATACAATGAATGGACA
CTAG
HIV-1 Env
ATGAGAGTGAAGGAGAAATATCAGCACTTGTGGAGATGGGGGTGGAGATG
GGGCACCATGCTCCTTGGGATGTTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTC
ACAGTCTATTATGGGGTACCTGTGTGGAAGGAAGCAACCACCACTCTATTTTGTGCAT
CAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCCACACATGCCTGTGT
ACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTAAC
ATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATC
AAAGCCTAAAGCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGAT
TTGAAGAATGATACTAATACCAATAGTAGTAGCGGGAGAATGATAATGGAGAAAGGAG
AGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGAGGTAAGGTGCAGAAAGA
ATATGCATTTTTTTATAAACTTGATATAATACCAATAGATAATGATACTACCAGCTATAC
GTTGACAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAG
CCAATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAA
GACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGA
ATTAGGCCAGTAGTATCAACTCAACTGCTGTTAAATGGCAGTCTAGCAGAAGAAGAGG
TAGTAATTAGATCTGTCAATTTCACGGACAATGCTAAAACCATAATAGTACAGCTGAAC
ACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAAAAATCCGTAT
CCAGAGGGGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGACAA
GCACATTGTAACATTAGTAGAGCAAAATGGAATGCCACTTTAAAACAGATAGCTAGCAA
ATTAAGAGAACAATTTGGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGG
ACCCAGAAATTGTAACGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCA
ACACAACTGTTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAAGGGTCAAATAA
CACTGAAGGAAGTGACACAATCACACTCCCATGCAGAATAAAACAATTTATAAACATGT
GGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGCGGACAAATTAGATGTTC
ATCAAATATTACAGGGCTGCTATTAACAAGAGATGGTGGTAATAACAACAATGGGTCC
GAGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATA
AATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAG
AGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTT
GGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCA
GACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGC
GCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAAT
CCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTC
TGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTC
TGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTA
CACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAAC
AAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAAT
TGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAAT
AGTTTTTGCTGTACTTTCTGTAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGT
TTCAGACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAG
AAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCAC
TTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTT
ACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCT
CAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTGCTGTTA
GCTTGCTCAATGCCACAGCTATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAG
TAGTACAAGAAGCTTATAGAGCTATTCGCCACATACCTAGAAGAATAGGACAGGGCTT
GGAAAGGATTTTGCTATAA
HIV-1 genome
GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAG
GGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGC
CCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGG
AAAATCTCTAGCAGTGGCGCCCGAACAGGGACATGAAAGCGAAAGGGAAACCAGAGG
AGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGG
CGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGAT
GGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATTAGATCGATGGGAAAAAATTCG
GTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGG
GAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGAC
AAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACGTAGATCATT
ATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCA
AGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCA
AGCAGCAGCTGACACAGGACACAGCAGCCAGGTCAGCCAAAATTACCCTATAGTGCA
GAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGG
GTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCAT
TATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGAC
ATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGA
TAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAG
GGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACA
CATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAA
TAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAG
GAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCTT
CACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGA
TTGTAAGACTATTTTAAAAGCATTGGGACCAGGAGCGACACTAGAAGAAATGATGACA
GCATGTCAGGGAGTGGGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATG
AGCCAAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAGGAACCAAA
GAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGCCAAAAATTGCAG
GGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGA
TTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGG
CCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTC
AGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAA
CTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAGCGACCCCTCGTCACAATAAAG
ATAGGGGGGCAATTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAG
AAGAAATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTT
TATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAG
GTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAG
ATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAG
CCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAG
CATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCT
GAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAG
AAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAAT
TAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGT
GGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTA
CCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCA
CAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGC
CTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAG
GATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCT
GTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTT
TGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAG
AAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGC
AAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCA
AAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAA
CAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTA
ATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGC
CATTTAAAAATCTGAAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGA
TGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGG
GGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGA
CAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTT
AGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTAT
GTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACA
GAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACA
AGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCA
CAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCA
GTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGC
ACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTGGTCAGTGCTGGAATCAGG
AAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAG
TAATTGGAGAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAG
TAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACT
GTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGT
AGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGG
CAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTAC
ATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGC
GGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAA
TCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCT
TAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATT
GGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACT
AAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAG
CAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGT
AGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCA
GGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGG
ATTAACACATGGAAAAGATTAGTAAAACACCATATGTATATTTCAAGGAAAGCTAAGGA
CTGGTTTTATAGACATCACTATGAAAGTACTAATCCAAAAATAAGTTCAGAAGTACACA
TCCCACTAGGGGATGCTAAATTAGTAATAACAACATATTGGGGTCTGCATACAGGAGA
AAGAGACTGGCATTTGGGTCAGGGAGTCTCCATAGAATGGAGGAAAAAGAGATATAG
CACACAAGTAGACCCTGACCTAGCAGACCAACTAATTCATCTGCACTATTTTGATTGTT
TTTCAGAATCTGCTATAAGAAATACCATATTAGGACGTATAGTTAGTCCTAGGTGTGAA
TATCAAGCAGGACATAACAAGGTAGGATCTCTACAGTACTTGGCACTAGCAGCATTAA
TAAAACCAAAACAGATAAAGCCACCTTTGCCTAGTGTTAGGAAACTGACAGAGGACAG
ATGGAACAAGCCCCAGAAGACCAAGGGCCACAGAGGGAGCCATACAATGAATGGACA
CTAGAGCTTTTAGAGGAACTTAAGAGTGAAGCTGTTAGACATTTTCCTAGGATATGGCT
CCATAACTTAGGACAACATATCTATGAAACTTACGGGGATACTTGGGCAGGAGTGGAA
GCCATAATAAGAATTCTGCAACAACTGCTGTTTATCCATTTCAGAATTGGGTGTCGACA
TAGCAGAATAGGCGTTACTCGACAGAGGAGAGCAAGAAATGGAGCCAGTAGATCCTA
GACTAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCAATTGCTA
TTGTAAAAAGTGTTGCTTTCATTGCCAAGTTTGTTTCATAACAAAAGCCTTAGGCATCTC
CTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCA
TCAAGTTTCTCTATCAAAGCAGTAAGTAATACATGTAATGCAACCTATACAAATAGCAAT
AGTAGCATTAGTAGTAGCAATAATAATAGCAATAGTTGTGTGGTCCATAGTAATCATAG
AATATAGGAAAATATTAAGACAAAGAAAAATAGACAGGTTAATTGATAGACTAATAGAA
AGAGCAGAAGACAGTGGCAATGAGAGTGAAGGAGAAATATCAGCACTTGTGGAGATG
GGGGTGGAGATGGGGCACCATGCTCCTTGGGATGTTGATGATCTGTAGTGCTACAGA
AAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAGGAAGCAACCACCACT
CTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCCAC
ACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACA
GAAAATTTTAACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAG
TTTATGGGATCAAAGCCTAAAGCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAA
AGTGCACTGATTTGAAGAATGATACTAATACCAATAGTAGTAGCGGGAGAATGATAAT
GGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGAGGTAAG
GTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATACCAATAGATAATGATACT
ACCAGCTATACGTTGACAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGG
TATCCTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAA
TGTAATAATAAGACGTTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAAT
GTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTAAATGGCAGTCTAGC
AGAAGAAGAGGTAGTAATTAGATCTGTCAATTTCACGGACAATGCTAAAACCATAATAG
TACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAA
AAAATCCGTATCCAGAGGGGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAA
ATATGAGACAAGCACATTGTAACATTAGTAGAGCAAAATGGAATGCCACTTTAAAACAG
ATAGCTAGCAAATTAAGAGAACAATTTGGAAATAATAAAACAATAATCTTTAAGCAATCC
TCAGGAGGGGACCCAGAAATTGTAACGCACAGTTTTAATTGTGGAGGGGAATTTTTCT
ACTGTAATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAA
GGGTCAAATAACACTGAAGGAAGTGACACAATCACACTCCCATGCAGAATAAAACAAT
TTATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGCGGACA
AATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACAAGAGATGGTGGTAATAACA
ACAATGGGTCCGAGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAA
GTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAG
GCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTC
CTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACG
GTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGG
CTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCC
AGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTT
GGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAG
TAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAA
ATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGA
AAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTA
ACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTA
GGTTTAAGAATAGTTTTTGCTGTACTTTCTGTAGTGAATAGAGTTAGGCAGGGATATTC
ACCATTATCGTTTCAGACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGG
AATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG
ATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGC
TTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGT
GGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAA
TAGTGCTGTTAGCTTGCTCAATGCCACAGCTATAGCAGTAGCTGAGGGGACAGATAG
GGTTATAGAAGTAGTACAAGAAGCTTATAGAGCTATTCGCCACATACCTAGAAGAATA
GGACAGGGCTTGGAAAGGATTTTGCTATAAGATGGGTGGCAAGTGGTCAAAAAGTAG
TGTGGTTGGATGGCCTGCTGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCAGCAGA
TGGGGTGGGAGCAGCATCTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAA
CACAGCAGCTAACAATGCTGCTTGTGCCTGGCTAGAAGCACAAGAGGAGGAGAAGGT
GGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTA
GATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAC
GAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTG
GCAGAACTACACACCAGGACCAGGGATCAGATATCCACTGACCTTTGGATGGCGCTA
CAAGCTAGTACCAGTTGAGCCAGAGAAGTTAGAAGAAGCCAACAAAGGAGAGAACAC
CAGCTTGTTACACCCTGTGAGCCTGCATGGAATGGATGACCCGGAGAGAGAAGTGTT
AGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCC
GGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGA
CTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCT
GCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAG
CCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCC
TTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCC
CTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT

Exemplary nucleotide sequences encoding human CCR5 can also be found, e.g., in Samson et al. Biochemistry 35:3362-3367 (1996), Raport et at J. Biol. Chem. 271:17161-17166 (1996); Combadiere et al. J. Leukoc. Biol. 60:147-152 (1996); Zhang et al. AIDS Res. Hum. Retroviruses 13:1357-1366 (1997), the contents of which are herein specifically incorporated by reference in its entirety.

Exemplary nucleotide sequences encoding HIV (e.g., HIV-1) Gap Env, Tat, Pol2, Pol1, or Vif can also be found in HIV Sequence Compendium 2013 Foley B, Leitner T, Apetrei C, Hahn B, Mizrachi I, Mullins J, Rambaut A, Wolinsky S, and Korber B, Eds. Published by Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, NM, LA-UR 13-26007, the contents of which are herein specifically incorporated by reference in its entirety

Exemplary Modified Pri-miRNA-Like Molecules

The nucleotide sequences of the exemplary modified pri-miRNA-like molecules are shown in Table 4 with the mature siRNA sequences are shown in bold. The exemplary 5′ flanking region, guide strand, terminal loop region, passenger strand, and 3′ flanking region sequences are summarized in Table 5.

TABLE 4
CCR5-miR-    AGGUAUAUUGCUGUUGACAGUGAGCGACUGUAAACUG
30a-         AGCUUGCUCUACUGUGAAGCCACAGAUGGGUAGAGCA
             AGCACAGUUUACCGCUGCCUACUGCCUCGGACUUCAA
             GGGGCUUGCGGCCGC
Gag-miR 21   CAUCUCCAUGGCUGUACCACCUUGUCGGCCUGCUAUG
             UCACUUCCCCUACUGUUGAAUCUCAUGGAGGGGAAGU
             GCCAUAGCAGCUCUGACAUUUUGGUAUCUUUCAUCUG
             ACCA
Pol1-miR-185 GGGCCUGGCUCGAGCAGGGGGCGAGGGAUUGACUUUG
             GGGAUUGUAGGGGAUGGUCCCCUCCCCCCCUACAAUC
             GCCAAAGUCCGUCCUUCCCUCCCAAUGACCGCGUCUU
             CGUC
Vif-miR-150  CAGCGGCGGCUCCUCUCCCCAUGGCCCUGGGGAUGUG
             UACUUCUGAACUUGCUGGGCUCAGACCAGUUCAGAAG
             AACACAUCCGCAGGGACCUGGGGACCCCGGCACCGGC
             AGGCC
Env-miR-20a  CUCUAUCUGAUGUGACAGCUUCUGUAGCACUUCUUCU
             GCUAGACUGCCAUAGUGUUUAGUUAUCUAUGGCAGUC
             UCGCAGAAGACGUACUGCUAGCUGUAGAACUCCAGCU
             UCGGCCUU
Tat-miR-16-1 AACUUAUGAUAGCAAUGUCAGCAGUGCCUUCCGCUUC
             UUCCUGCCAUAGCGUUAAGAUUCUAAAAUUAUCUCUA
             UGGCAGGCAGAAGCGGCAAGUAAGGUUGACCAUACUC
             UACAGUUGUU
Pol2-miR-122 UUCGUGGCUACAGAGUUUCCUUAGCAGAGCUGUAUCA
             UCUGCUCCUGUAUCUUGUGUCUAAACUAUCAAGAUAC
             AGGACCAGAUGAUCUAGCUACUGCUAGGCAAUCCUUC
             CCUCGAUAAAUG

TABLE 5
Modified pri-
miRNA-like	5′ flanking		Terminal loop	Passenger	3′ flanking
Molecule	region	Guide strand	region	strand	region
CCR5-miR-30a	AGGUAUAUU	UGUAAACUGAG	GUGAAGCCACAG	UAGAGCAAGCACA	UCGGACUUCAAG
	GCUGUUGAC	CUUGCUCUACU	AUGGGGCUGCCU	GUUUACC	GGGCUUGCGGCC
	AGUGAGCGA		ACUGCC		GC
	C
Gag-miR-21	CAUCUCCAU	CCUGCUAUGUC	UGUUGAAUCUCA	AGGGGAAGUGCCA	UCUGACAUUUUG
	GGCUGUACC	ACUUCCCCUAC	UGG	UAGCAGC	GUAUCUUUCAUC
	ACCUUGUCG				UGACCA
	G
Pol1-miR-185	GGGCCUGGC	UGACUUUGGGG	UGGUCCCCUCCC	CCCUACAAUCGCC	CGUCCUUCCCUC
	UCGAGCAGG	AUUGUAGGGGA	C	AAAGUC	CCAAUGACCGCG
	GGGCGAGG				UCUUCGUC
	GAU
Vif-miR-150	CAGCGGCGG	GGGAUGUGUAC	CUGGGCUCAGAC	AGUUCAGAAGAAC	CAGGGACCUGGG
	CUCCUCUCC	UUCUGAACUUG	C	ACAUCCG	GACCCCGGCACC
	CCAUGGCCC				GGCAGGCC
	UG
Env-miR-20a	CUCUAUCUG	UUCUUCUGCUA	UGUUUAGUUAUC	AUGGCAGUCUCGC	GUACUGCUAGCU
	AUGUGACAG	GACUGCCAUAG	U	AGAAGAC	GUAGAACUCCAG
	CUUCUGUAG				CUUCGGCCUU
	CAC
Tat-miR-16-1	AACUUAUGAU	UCCGCUUCUUCC	UUAAGAUUCUAAAA	CUAUGGCAGGCAG	AAGUAAGGUUGA
	AGCAAUGUCA	UGCCAUAGCG	UUAUCU	AAGCGGC	CCAUACUCUACA
	GCAGUGCCU				GUUGUU
Pol2-miR-122	UUCGUGGCU	UAUCAUCUGCU	UGUCUAAACUAUC	AGAUACAGGACCA	UAGCUACUGCUA
	ACAGAGUUU	CCUGUAUCUUG	A	GAUGAUC	GGCAAUCCUUCC
	CCUUAGCAGA				CUCGAUAAAUG
	GCUG

Pharmaceutical Compositions and Kits

This disclosure provides compositions, e.g., pharmaceutical compositions, which can include a nucleic acid molecule (e.g., an shRNA cluster or artificial miRNA duster), an artificial RNA molecule (e.g., RNAi molecule), a cell, or a viral particle, as described herein, formulated together with a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable, e.g., for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). In certain embodiments, the shRNA clusters, nucleic acid molecules (e.g., artificial miRNA molecules), artificial RNA molecules (e.g., RNAi molecules), cells, or viral particles are at least about 90%, e.g., at least about 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% pure in the pharmaceutical composition.

The compositions, e.g., pharmaceutical compositions, described herein may include a “therapeutically effective amount”, “prophylactically effective amount”, or “diagnostically effectively amount” of shRNA clusters, nucleic acid molecules (e.g., artificial miRNA clusters), cells, or viral particles.

A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), artificial RNA molecule (e.g., RNAi molecule), cell, or viral particle may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), cell, or viral particle is outweighed by the therapeutically beneficial effects. A “therapeutically effective dosage” typically inhibits a measurable parameter by at least about 20%, e.g., by at least about 40%, by at least about 60%, or by at least about 80% relative to untreated subjects. The measurable parameter may be, e.g., viral load, fever, headache, muscle or joint pains, skin rash, bleeding, reduced platelet levels, and reduced blood pressure. The ability of a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), cell, or viral particle to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in reducing, inhibiting, or preventing an HIV infection. Alternatively, this property of a composition can be evaluated by examining the ability of a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), cell, or viral particle to inhibit or reduce the viability of HIV, e.g., by an in vitro assay described herein.

A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

A “diagnostically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired diagnostic result. Typically, a diagnostically effective amount is one in which a disorder (e.g., an HIV infection or AIDS) can be diagnosed in vitro, ex vivo, or in vivo.

Also within this disclosure is a kit that comprises a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), artificial RNA molecule (e.g., RNAi molecule), cell, or viral particle, described herein. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label (e.g., a radioactive label), a therapeutic agent, or an agent useful for chelating, or otherwise coupling, nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), artificial RNA molecule (e.g., RNAi molecule), cell, viral particle; devices or other materials for preparing the nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), artificial RNA molecule (e.g., RNAi molecule), cell, or viral particle for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

Methods of Treatment or Prevention

The disclosure provides methods for inhibiting HIV infection, e.g., using a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), artificial RNA molecules (e.g., RNAi molecules), cell, or viral particle, as described herein. The disclosure also provides methods for treating or preventing HIV infection and/or AIDS, e.g., using a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), artificial RNA molecule (e.g., RNAi molecule), cell, or viral particle, as described herein.

A nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), cell, or viral particle, as described herein, can be formulated for in vivo administration to a subject, such as a human or veterinary subject. A composition so formulated can comprise a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster), cell, or viral particle, as described herein, designed to express artificial RNA molecules (e.g., RNAi molecules) to decrease the expression of a target gene, e.g., a target gene described herein. A composition can also comprise a pharmaceutically acceptable excipient.

For example, artificial RNA molecules can be reliably expressed in vivo in a variety of cell types. In some embodiments, a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) expressing an artificial RNA molecule is introduced (e.g., transduced or transfected) into a cell, e.g., a cell isolated from a subject. In certain embodiments, the transduced or transfected cell is administered to the subject in order to treat a condition. There are a variety of mechanisms by which transduced or transfected cells can be useful for treating HIV and AIDS. For example, a condition can be caused, in part, by a population of cells expressing an undesirable gene. These cells can be ablated and replaced with administered cells expressing an artificial RNA molecule that decreases expression of the undesirable gene. The artificial RNA molecules described herein can be targeted to essentially any gene, the decreased expression of which can be helpful in treating or preventing HIV infection and/or AIDS.

Any suitable cell can be used. For example, cells to be transfected or transduced can be essentially any type of cell for implantation into in a subject. The cell having the target gene can be germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchymal or epithelial, immortalized or transformed, or the like. The cell can be a stem cell or a differentiated cell. After transduction or transfection, stem cells can be administered to a subject, or cultured to form differentiated stem cells (e.g., embryonic stem cells cultured to form neural, hematopoietic, or pancreatic stem cells) or cultured to form differentiated cells.

Stem cells can be stem cells recently obtained from a donor, and in certain embodiments, the stem cells are autologous stem cells. Stem cells can also be from an established stem cell line that is propagated in vitro. Suitable stem cells include embryonic stems and adult stem cells, whether totipotent, pluripotent, multipotent, or of lesser developmental capacity. Stem cells can be derived from mammals, such as rodents (e.g., mouse or rat), primates (e.g., monkeys, chimpanzees, or humans), pigs, or ruminants (e.g., cows, sheep, and goats). Examples of mouse embryonic stem cells include: the JM1 ES cell line described in M. Qiu et al., Genes Dev 9, 2523 (1995), and the Rid SA line described in G. Friedrich, P. Soriano, Genes Dev 5, 1513 (1991), and mouse ES cells described in U.S. Pat. No. 6,190,910. Many other mouse ES lines are available from Jackson Laboratories (Bar Harbor, Me.), In addition, examples of embryonic stem cells are described in the following patents and published patent applications: U.S. Pat. Nos. 8,647,872, 6,245,566; 6,200,806; 6,090,622; 6,331,406; 6,090,622; 5,843,780; US 2002/0045259; US 2002/0068045. Examples of human adult stem cells include those described in the following patents and patent applications: U.S. Pat. Nos. 8,999,706, 7,259,011, 6,387,367; 6,265,175; 6,242,252; 6,184,035; 6,129,911; 5,968,829; 5,958,767; 5,958,767; 5,958,767; 5,958,767; 5,958,767; 5,789,246; 5,766,948; 5,486,359; US 2014/0170122, US 2002/0016002, US 2002/0076400, US 2002/0098584; and; for example, in International Publication No. WO 01/11011. Examples of making and using hematopoietic stem cells are described, e.g., in U.S. Pat. Nos. 8,617,885, 8,383,404, 7,927,785, 7,807,464, 7,767,453, 5,763,197, U.S. Publication No. 2012/0071397, International Publication Nos. WO 1996/033281 and WO 1996/022693. In certain embodiments, a suitable stem cell can be derived from a cell fusion or dedifferentiation process, such as described in U.S. Publication No. US 2002/0090722, in International Publication Nos. WO 02/38741; WO 01/51611, WO 99/63061, and WO 96/07732.

Transduced or transfected cells can also be used in the manufacture of a medicament for the treatment of subjects. Examples of pharmaceutically acceptable excipients include matrices, scaffolds, or other substrates to which cells can attach (optionally formed as solid or hollow beads, tubes, or membranes), as well as reagents that are useful in facilitating administration (e.g., buffers and salts), preserving the cells (e.g., chelators such as sorbates, EDTA, EGTA, or quaternary amines or other antibiotics), or promoting engraftment. Cells can be encapsulated in a membrane or in a microcapsule. Cells can be placed in microcapsules composed of alginate or polyacrylates. (Sugamori et at (1989) Trans. Am. Soc. Artif. Intern, Organs 35:791; Levesque et al, (1992) Endocrinology 130:644; and Lim et al. (1992) Transplantation 53:1180).

Additional methods for encapsulating cells are known in the art. (Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al, U.S. Pat. No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res. 82:4146; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183, U.S. Pat. Nos. 4,391,909; 4,353,888; Sugamori et al. (1989) Trans. Am. Artif Intern. Organs 35:791-799; Sefton et al. (1987) Biotehnol. Bioeng. 29:1135-1143; and Aebischer et al. (1991) Biomaterials 12:50-55).

The site of implantation of cell compositions can be selected by one skilled in the art depending on the type of cell and the therapeutic objective. Exemplary implantation sites include intravenous or intra-arterial administration, administration to the liver (via portal vein injection), the peritoneal cavity, the kidney capsule, or the bone marrow.

In certain embodiments, the disclosure provides for modification of nucleic acids and delivery (e.g., ex vivo or in vivo delivery) of modified nucleic acids. These modified nucleic acids can include a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein. These modified nucleic acids can also include, e.g., shRNA-like molecules, modified pri-miRNA-like molecules, and artificial RNA molecules, as described herein. Modification and delivery of modified nucleic acids, including RNAs incorporating modified nucleotides, such as those with chemical modifications to the 2′-OH group in the ribose sugar backbone, such as 2′-O-methyl (2′OMe), 2′-fluoro (2′F) substitutions, and those containing 2′OMe, or 2′F, or 2′-deoxy, or “locked nucleic acid” (LNA) modifications can be accomplished as described in U.S. Pat. Nos. 6,627,616, 6,897,068, 6,379,966; in U.S. Patent Application Publication Nos. US. 2005/0107325, US 2007/0281900 and US 2007/0293449; and in Vorhies and Nemunaitis J J, Methods Mol Biol. 2009; 480:11-29, Lopez-Fraga M et al., Infect Disord Drug Targets, 2008 December; 8(4):262-73, Watts et al., Drug Discov Today. 2008 October; 13 (19-20):842-55, Lu and Woodle, Methods Mol Biol. 2008; 437:93-107, de Fougerolles et al., Hum Gene Ther, 2008 February; 19(2):125-32, Rossi J J, Hum Gene Ther. 2008 April; 19(4):313-7, Belting M and Wittrup A. Methods Mol Biol. 2009; 480:1-10, Pushparaj et al., J. Dent, Res. 2008; 87: 992-1003, Shrivastava and Srivastava, Biotechnol J. 2008 March; 3(3):339-53, and Raemdonck K. et al., Drug Discov Today, 2008 November; 13 (21-22):917-31, Castanotto D & Rossi J J, Nature 2009 January; 457:426-433, Davis M et al., Nature 464, 1067-1070 (15 Apr. 2010), each of which are incorporated by reference in their entireties.

Combination Therapies

The nucleic acid molecules (e.g., shRNA dusters or artificial miRNA dusters), artificial RNA molecules (e.g., RNAi molecules), cells, viral particles, and compositions (e.g., pharmaceutical compositions) described herein can be used in combination with other therapies. For example, the combination therapy can include a nucleic acid molecule (e.g., shRNA duster or artificial miRNA duster), artificial RNA molecule (e.g., RNAi molecule), cell, and composition described herein co-formulated with, and/or co-administered with, one or more additional therapeutic agents, e.g., anti-HIV agents, vaccines, or agents that enhance an immune response. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject before or during the course of the subject's affliction with a disease, e.g., an HIV infection or AIDS. In one embodiment, two or more treatments are delivered prophylactically, e.g., before the subject is infected or diagnosed with HIV. In another embodiment, the two or more treatments are delivered after the subject has been infected or diagnosed with HIV infection or AIDS. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to an infection or disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

Exemplary HIV or AIDS therapies that can be combined with an shRNA duster, nucleic acid molecule (e.g., artificial miRNA cluster), artificial RNA molecule (e.g., RNAi molecule), cell, viral particle, and composition (e.g., pharmaceutical composition) described herein include, but are not limited to, a non-nucleoside reverse transcriptase inhibitor (NNRTI) (e.g., nevirapine, delavirdine, efavirenz, etravirine, rilpivirine, IDX899, RDEA-428, or lersivirine), a nucleoside reverse transcriptase inhibitor (NRTI) (e.g., zidovudine, didanosine, zalcitabine, stavudine, lamivudine; abacavir; emtricitabine, or entecavir), a nucleotide reverse-transcriptase inhibitor (NtRTI) (e.g., tenofovir), a protease inhibitor (PI) (e.g., saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, fosamprenavir, atazanavir, tipranavir, or darunavir), a fusion inhibitor (e.g., enfuvirtide), a CCR5 antagonist (also called an entry inhibitor) (e.g., maraviroc), an integrase strand transfer inhibitor (INSTI) (e.g.; elvitegravir, dolutegravir, or raltegravir), or any combination thereof.

In certain embodiments; the additional therapeutic agent is a second shRNA cluster, nucleic acid molecule (e.g., artificial miRNA cluster), artificial RNA molecule (e.g., RNAi molecule), cell, viral particle, or composition (e.g., pharmaceutical composition), as described herein.

Screening Methods

Constructs containing a nucleic acid molecule (e.g., shRNA cluster or artificial miRNA cluster) described herein, or libraries of such constructs can be introduced into intact cells/organisms and can be used in screening, such as high throughput screening (HTS). For example, potential targets for pharmaceuticals can be identified or studied using such constructs or libraries. A panel of nucleic acid molecules (e.g., shRNA clusters or artificial miRNA dusters) that affect target gene expression by varying degrees may be used. In particular, it may be useful to measure any correlation between the degree of gene expression decrease and a particular phenotype.

Libraries of nucleic acid molecules (e.g., shRNA dusters or artificial miRNA dusters) can be produced using methods known in the art. For example, libraries of nucleic acid molecules (e.g., shRNA clusters or artificial miRNA clusters) can be based on existing libraries, such as existing shRNA libraries. Existing materials and methods for design and construction of expression cassettes, selection and modification of vectors and vector backbones, library construction, design of target sequences, and library validation, as applied to conventional shRNA libraries may be applied in the construction of libraries comprised of the shRNA clusters or nucleic acid molecules (e.g., artificial miRNA clusters) of the present disclosure. As non-limiting examples, such materials and methods are described in Chang et al, Nature Meth. 3; 707-714 (2006), International Publication No. WO/2009/055724, the contents of which are specifically incorporated herein by reference.

In certain aspects, the disclosure provides methods for screening/evaluating gene function in vivo. A cell containing a construct for expression of an shRNA-like molecule, modified pri-miRNA-like molecule, or artificial RNA molecule may be introduced into an animal and a phenotype may be assessed to determine the effect of the decreased gene expression. An entire animal may be generated from such cells (e.g., ES cells) containing the construct of the nucleic acid molecule (e.g., shRNA duster or artificial miRNA duster) described herein, A phenotype of the animal may be assessed. The animal may be essentially any experimentally tradable animal such as a non-human primate, a rodent, a canine, a feline, etc. Populations of animals expressing different members of a library of nucleic acid molecules (e.g., shRNA dusters or artificial miRNA dusters) may also be generated. The phenotypes of such animals may be assessed to determine, for example, the effect of a target gene on a disease phenotype, stem cell differentiation, drug sensitivity, susceptibility to a viral infection, or any other phenotype of interest.

EXAMPLES

Example 1: Multiplexing Seven miRNA-Based shRNAs to Suppress HIV Replication

This example describes a strategy to express a large number of shRNA-mills using optimal flanking sequences from multiple endogenous miRNAs. It was found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. Multiple shRNAs were inserted in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singlely expressed counterpart. This system was used to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc−/− mice (Hu-PBL model), efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.

Design of Multiplexed shRNA-miRs

The essential difference between conventional shRNA and shRNA-miR is that while the former resembles pre-miRNA that is processed by Dicer in the cytoplasm, the latter resembles pri-miRNA that is first processed by Drosha/DGCR8 in the nucleus (Han et al., Genes Dev 18: 3016-3027; Yi et al. Genes Dev 17; 3011-3016; Chendrimada Nature 436: 740-744). Although based on in vitro processing of select miRNAs, it has been suggested that Drosha cuts the pri-miRNA ˜11 nt from the lower stem-single stranded RNA (ssRNA) junction, this may not always hold true for all miRNAs, particularly in vivo (Ma et al., Proc Natl Acad Sci USA 110: 20687-20692). Moreover, whether including just these flanking sequences will enable pri-miRNA processing is not known. Thus, the minimal flanking sequences required for processing of different miRNAs were examined. Starting with miR-30a backbone with 150-nt flanking sequences, the flanking sequence length was shortened to 30, 20, and 15-nt and tested the impact on the functionality of the miRNA. For this, plasmids expressing miRNA were co-transfected with different flank lengths along with psiCHECK vector containing the relevant target site in the 3′ UTR of Rluc and measured activity by dual luciferase assay 24 h later. FIG. 1A shows that the functionality of pri-miR-30a with 150-nt flanking sequence is similar to that with 30 or 20-nt flanking sequence, while a 15-nt flanking sequence slightly diminishes functionality. The functionality of pri-miR-150 was also tested with 60- or 30-, 20-, or 15-nt flanking sequences and no difference between 60 and 30 nt flanks was detected (FIG. 1B), suggesting that a 30-nt flanking sequence is long enough to ensure proper Drosha processing to retain the full functionality of miRNA. Thus, 30-nt flanks were used for all miRNA backbones.

Multiplexing seven shRNA-miRs into a single construct without decreasing functionality of individual shRNAs. Multiple shRNAs cannot be expressed using tandem repeats of the same miRNA backbone because homologous recombination at the flanking sequences is likely to eliminate the insert. An artificial miRNA duster was designed to multiplex shRNA-miRs under different miRNA backbones. First, it was determined that tandem expression of 2 shRNA-miRs with or without a spacer sequence between them did not affect the shRNA functionality, indicating that 2 tandem miRNAs can be directly combined. Thus, different numbers (1, 2, 4, and 7) of shRNA-miRs were expressed, each with a different miRNA backbone (without any spacer sequence between the individual miRNAs) under the control of EF-1 a promoter, as illustrated in FIG. 1C. The shRNA sequences were picked as they had shown the efficacy of conventional shRNAs targeting the CCR5 gene and highly conserved regions in the HIV-1 genome.

First it was tested if the shRNA-miRs in the multiplex are correctly processed and whether their expression is affected by the position of each individual shRNA-miR with respect to the promoter. siRNAs produced from single, 2, 4, or 7 shRNA-miRs were determined using deep sequencing. For this, 293 T cells were transfected with either of the single, 2, 4, or 7 shRNA-miR expression constructs and small RNAs isolated 48 h later were cloned and deep sequenced. siRNAs were detected for each unit in all the constructs. Moreover, analysis of the dominant reads show accurate and predicted processing of 6/7 shRNAs (FIG. 7). Only in 1 (Env), there were 2 forms, one as predicted, but one with 1 bp shift. However, even with some of the endogenous miRNAs, alternative processing is known to occur. Thus, the siRNAs generated appears to be the right ones.

Analysis of the read number frequencies revealed that the expression of individual shRNA-miRs in the multiplex is not affected by the position of that shRNA-miR with respect to the promoter. Although the small RNA reads showed 3-8-fold lower from the 7 shRNA-miR construct as compared to single shRNA-miR (FIG. 8, Table 1), the reads of all siRNAs in the construct (CCR5, Gag, Env, Tat, Pol1, Pol2, and Vif) were significantly higher than one of the most abundant endogenous miRNA, miR-16. Thus, the level of each of the 7 shRNAs appears to be adequate to suppress all targeted genes.

TABLE 1
Dominant small RNA reads in transfected cells
mature	Vector	CCR5	Gag	Env	Tat	Pol2	Pol1	Vif	2	4	7
sequences	alone	shRNA	shRNA	shRNA	shRNA	shRNA	shRNA	shRNA	shRNA	shRNA	shRNA
CCR5	0	141914	0	0	0	0	0	0	51630	27053	9891
Gag	0	0	19925	0	0	0	0	0	0	3749	889
Env	0	0	0	38766	0	0	0	0	0	0	1979
Tat	0	0	0	0	100301	0	0	0	0	0	5351
Pol2	0	0	0	0	0	45911	0	0	0	0	1991
Pol1	0	0	0	0	0	0	33988	0	0	4088	738
Vif	0	0	0	0	0	0	0	24859	13596	5757	1289
Inner	1627	2046	1556	1917	2235	2306	2589	1558	1594	1134	449
reference
(miR-16)

Next, individual shRNA-miR functionality in the multiplex was tested. For this, plasmids expressing single or multiple shRNA-miRs were co-transfected along with psiCHECK vector containing relevant shRNA-miR target sites in the 3′ UTR of Rluc and measured activity by dual luciferase assay 24 h later. AH individual shRNA-miRs within all multiplexed vectors (expressing 2, 4, or 7 shRNA-miRs) showed similar functionality compared to plasmids expressing only the individual shRNA-miRs (FIG. 2A). This was seen at all different concentration of plasmids used for transfection. The toxicity of single, dual, and multiple shRNA-miR constructs was also evaluated. Transfection of Jurkat cells with multiple snRNA-miR expression construct was not toxic to the cells as determined by annexin V staining (FIG. 2B).

The different shRNA-miR expression cassettes (expressing 1, 2, 4, and 7 shRNAs) were cloned in a lentiviral vector that also expresses mCherry or Zs Green as markers. The vectors were pseudotyped with HIV-1 envelope and the resultant lentivirus was used for transduction of primary CD4 T cells. The lentiviral titer with multiplexed shRNA-miRs was around 10 fold lower than single shRNA-miRs, but still sufficient to perform experiments after concentration by ultracentrifugation.

To monitor toxicity, the cells we re Cu I t u red for and 12 days after lentivirus transduction and followed the percentage of mCherry+ cells by flow cytometry. Although mCherry expression levels varied between different constructs, the expression levels did not decline with time for any of the constructs (FIG. 9). Cell viability of the transduced CD4 T cells was also tested by the MTS assay on days 2 and 4 after transduction. No decrease in the viability of transduced cells was observed (FIG. 2C). Thus, these data indicate that single and multiple snRNA-miR constructs were stable and had no obvious adverse effects on cell viability in vitro.

Functionality of shRNA-miRs targeting CCR5 and multiple HIV-1 genes in cell lines.

The ability of single, dual, and multiple snRNA-miR constructs to knockdown CCR5 was tested in the TZM-bl cells, Cells were transduced with lentiviruses encoding 2, 4, and 7 shRNA-miRs and after 48 h, CCR5 expression was tested by flow cytometry. As shown in FIG. 3A, all 3 constructs could effectively silence CCR5 expression as compared to cells transduced with lentivirus expressing mCherry alone. To test the efficacy of antiviral shRNA-miRs, 293 T cells were co-transfected with X4 tropic HIV-1 molecular clone pNL4-3 plasmid along with 2, 4, or 7 shRNA-miR encoding vectors. Culture supernatants obtained 48 h later were tested for viral replication by infecting TZM-bl cells encoding Tat-dependent luciferase. While as expected, supernatants from CCR5 shRNA-miR transfected cells had no effect on viral levels compared to control, that from antiviral shRNA treated samples showed increasingly (7shRNA-miR<4shRNA-miR<2shRNA-miR) lower levels of infectivity, suggesting antiviral shRNA-miRs were also functional (FIG. 3B). To evaluate the antiviral effect of CCR5 and antiviral shRNA-miRs in the context of lentivirus transduction, TZM-bl cells were transduced with HIV-1-pseudotyped lentiviruses encoding 1, 2, 4, or 7 shRNA-miRs and 24 h later infected with either X4-tropic NL4-3 or R5 tropic HIV-1Bal at an MOI of 0.01. p24 levels in culture supernatants were tested 9 days after infection. Again, CCR5 shRNA-miR transduction had no effect on X4 tropic virus, but inhibited R5 tropic virus by 50% and antiviral shRNA-miRs showed increasing levels of inhibition, with 7shRNA-miR virtually abrogating infection with both X4 tropic and R5 tropic viruses (FIG. 3C). Thus, the artificial miRNA clusters appears to be capable of expressing multiple functional shRNA-miRs and therefore can serve as good candidates for anti-HIV-1 therapy.

Resting T Cells can be Effectively Transduced with HIV-1-Env Pseudotyped shRNA-miR Expressing Lentivirus

Having established that multiplexed shRNA-miRs are functional, the efficacy of CCR5 only versus CCR5+6 antiviral shRNA-miRs was next tested to resist HIV-1 infection in activated and resting primary CD4 T cells. The goal was to develop a method to confer HIV-1 resistance to all T cells, regardless of activation status. Thus, the transduction efficiency of VSV-G and HIV-1 env-pseudotyped lentiviruses expressing mCherry was first tested. Primary CD4 T cells were transduced either before or after activation with PHA for 2 days and examined for mCherry expression 2 days after transduction. Flow cytometric analysis revealed that while both VSV-G and HIV-1 envelope-pseudotyped viruses transduced activated T cells with equal efficiency, effective transduction of resting T cell could be achieved only with the latter (FIG. 10), Thus, the HIV-1 envelope pseudotyped virus was used for testing antiviral efficacy. To evaluate the shRNA-miRs, resting or activated CD4 T cells were transduced with HIV-1 pseudotyped lentiviruses expressing no shRNA-miR, CCR5 shRNA-miR alone, or CCR5+6 antiviral shRNA-miRs. In this experiment, GFP-expressing lentivirus was used to track the transduced cells. After 48 h, the cultures were infected with HIV-1 strains, NL4-3, or Bal (transduced resting T cells were activated with PHA before infection). Culture supernatants harvested on days 0, 3, 9, and 15 were tested for released virus levels by p24 ELISA. The transduction levels were comparable between the three LV constructs in both activated and resting CD4 T cells (FIGS. 4A-4B). Again, similar to results obtained for TZM-bl cells, while CCR5 shRNA-miR was effective only against R5 virus, the 7 shRNA-miR expressing LV was able to nearly abrogate infection of both X4 and R5 tropic viruses in both activated and resting T cells (FIGS. 4A-4B). Moreover, GFP expressing cells increased over time in the protected CCR5 shRNA-miR and 7 shRNA-miR transduced cultures. The high enrichment of viable GFP positive cells and the well preserved cell viability at the end of the experiment on day 15 points to a clear survival advantage of the shRNA-miR transduced cells following HIV-1 infection (FIGS. 4A-4B). It was concluded that 7 shRNA-miR expressing lentivirus, pseudotyped with HIV-1 envelope provides a means to confer HIV-1 resistance in primary CD4 T cells.

Inhibition of HIV-1 Replication in HIV-Seropositive Donor PBMCs Transduced with HIV-1 Env-Pseudotyped 7 shRNA-miR Encoding Lentivirus

In addition to the effectiveness of shRNA-miRs to prevent infection, it was next tested if the replication of HIV-1 can be inhibited from already infected cells. For this, PBMC obtained from HIV-1 seropositive individuals, either treatment naive or on ART were used. Since in this case, antiviral shRNA-miRs are mainly relied on, shRN-miR targeting Tat only or 7shRNA-miRs were used to test efficacy. First, it was ensured that the transduction efficacy of all the vectors to be similar by determining mCherry expression by flow cytometry. To test shRNA efficacy, PBMCs from HIV-1 seropositive donors were depleted of CD8 T cells, transduced with shRNA-miR encoding lentiviruses, and activated the next day (FIG. 5A). The cells were cultured for 31 days and p24 antigen levels determined on days 0, 3, 15, and 31. As shown in FIG. 5B, in the control lentivirus (expressing no shRNA-miR) transduced cells, HIV-1 replication showed a peak on day 15 and declined by day 31, presumably due to depletion of CD4 T cells by infection. In contrast, in the 7 shRNA-miR transduced cultures, HIV-1 replication by day 15 was dramatically reduced in all 4 donor PMBCs and by day 31, p24 was virtually undetectable, even though the cultures contained healthy looking cells. In the Tat snRNA-miR only transduced cells, at day 15 HIV-1 replication was reduced in all four donors PBMCs. However at later time points. Tat shRNA-miR transduced cell cultures showed increased HIV-1 replication (FIG. 5B), indicating either inadequacy of Tat shRNA-miR antiviral activity or emergence of escape mutants. The presence of HIV-1 escape mutants were tested by sequencing Tat snRNA-miR target regions in the virus present in the supernatants. Of the 4 donors, cultures from two showed emergence of viral escape mutants. Donor 2 had a mutation at nucleotide position 18 and donor 4 had mutation at position 4 of the Tat-targeted sequence (FIG. 5C). In the 7shRNA-miR treated cultures, virus could not be recovered to perform similar studies.

Multiplexed miRNA-Based 7 shRNA-miRs Prevent CD4 T Cell Loss and HIV-1 Replication in Hu-PBL Mice Reconstituted with PBMCs from HIV-1+ Patients

Transplantation of gene modified T cells is being tried as a potential treatment in HIV-1 infected individuals (Surabhi and Gaynor (2002). J Viral 76: 12963-12973, Thus, it was next tested if reinfusion of 7 snRNA-miR treated cells provides a therapeutic possibility by preclinical testing in the Hu-PBL mouse model. For this, 2 HIV-1 infected individuals, one on ART (donor #1, viral load <20 copies/ml; CD4/CD3 ratio=0.34) and the other, treatment naïve (donor #4, viral load: 67,420 copies/nil; CD4/CD3 ratio=0.13), were selected. PBMCs were transduced with HIV-1 env-pseudotyped lentivirus expressing either no snRNA-miR (mock), only Tat snRNA-miR or 7 shRNA-miRs and injected into NOD/SCID/IL2-Ryc−/− mice to evaluate their ability to engraft, expand and resist HIV-1 replication. CD4 T cell counts were monitored on days 7, 28, and 42 and serum p24 levels on day 42 (FIG. 6A). In mice reconstituted with lentivirus only (no shRNA-miR) transduced T cells, for both donors, few CD4 T cells were found at all-time points tested and the cell numbers progressively declined over time, consistent with HIV-1 mediated depletion of xenogenically activated CD4 T cells that became productively infected with endogenous virus. In the Tat shRNA-miR only transduced group, CD4 T cells expanded (particularly noticeable in donor #1 on ART) by day 28, but declined by day 42, reflecting the initial effectiveness of a single antiviral shRNA-miR that failed at later time points. In contrast in the 7shRNA-miR transduced group, for both donors, CD4 T cells continued to increase and by day 42, constituted 87% (donor #1, on ART) and 52% (donor #4, treatment naïve) of PBMCs. Correspondingly, viral load was highest in no shRNA-miR control, intermediate in Tat shRNA-miR only and lowest in the 7 shRNA-miR group (FIG. 6B,C). Taken together, these results show that multiplexed miRNA-based 7shRNA can control HIV-1 replication and reverse CD4 T cell loss, thus providing a potential clinically viable treatment strategy.

In summary, a general platform to easily express large numbers of shRNA-miRs using minimal flanking sequences from different endogenous miRNAs was developed to express individual shRNA-miRs. Using this system, seven shRNA-miRs targeting CCR5 and six regions in the HIV-1 genome were expressed and it was shown that this affords better protection in vitro as well as in vivo in Hu-PBL mice.

To confer HIV-1 resistance in a therapeutic setting in any meaningful manner, it is desired to silence host factors and HIV-1 genes simultaneously. However, because HIV-1 is known to escape by mutating the target sites, it is important to be able to target multiple, highly conserved viral regions. If seven shRNAs are simultaneously expressed, it could cover nearly all HIV-1-strains by ensuring that at least four shRNAs are active against any given viral strain.

Advances in understanding Drosha processing of pri-miRNA should allow for rational design of shRNA-miRs. Although Drosha cleavage was previously reported to occur ˜11 nt from the lower stem-snRNA junction (Han et al. Cell 125: 887-901), it was recently found that the microprocessor measures the distances from both the lower and upper stem-ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing (Ma et al. Proc Natl Acad Sci USA 110: 20687-20692). The results shown in this Example suggest that incorporation of ˜30 nt of flanking sequences can be enough to ensure processing of different shRNA-miRs. In this example, efficacy was seen for all seven shRNA-miRs in the backbone of other miRNA flanks of ˜30 nt. This finding allows easy multiplexing by expression of snRNA-miRs in different miRNA backbones in tandem. Here, each shRNA-miR will be an independent module that can be easily changed and manipulated. Importantly, the functionality of individual snRNA-miRs within the multiplexed constructs did not decrease compared with constructs containing only single, nonmultiplexed shRNAmiR. In addition, the multiplexed snRNA-miRs were stable and had no obvious adverse effects on cells. More importantly, the seven shRNA-miR transduced resting T cells from HIV-1 seropositive individuals, when reconstituted in Hu-PBL mice led to restoration of CD4 T cell decline, indicating the feasibility of using such therapy in humans, Thus, the lentiviral platform to express seven snRNA-miRs provides a significant advancement towards using RNAi for HIV-1 gene therapy.

Conventionally, VSV-G is used for pseudotyping lentiviruses because of its broad tropism for many different cell types, Nonetheless, cells in the GO stage of the cell cycle, such as resting CD4 T cells are highly recalcitrant to transduction with VSV-G pseudotyped lentivirus (Agosto, et al. (2009). J Virol 83: 8153-8162), This study shows that lentivirus packaged with X4-tropic envelope from LAI efficiently delivered shRNAs into resting CD4 T cells. This is particularly important since in HIV-1 infection, resting memory CD4 T cells are the well-known reservoirs of latent HIV-1 infection and delivery of shRNAs might prevent viral reactivation in these cells. In addition, less differentiated T cells can persist longer after transfer because they have longer telomeres and are not prone to activation-induced cell death. Furthermore, in the absence of prolonged culture, perturbation of the T cell repertoire is also likely to be minimized. As ex vivo transduction is the only external manipulation required, the approach described herein would allow immediate reinfusion of the gene modified cells into the patient, which simplifies the therapy for wider clinical application.

Thus, this example illustrates a compact and flexible design to express multiple shRNAs without inducing toxic effects or compromising their expression and efficacy. This system was used to express seven snRNA-miRs targeting the CCR5 gene and six regions in the viral genome and showed its effectiveness in suppressing HIV-1 infection in vitro and in vivo in the Hu-PBL model. This strategy provides a clinically viable approach for gene therapy in HIV-1 infection.

Materials and Methods

Plasmids and Constructs

PsiCHECK2 vectors were modified to express shRNA target sites in the Renilla luciferase 3′ UTR. For this, synthetic oligonucleotides for the forward and reverse strands of the target sequences were annealed and cloned into psiCHECK2 at the XhoI and NotI site. The oligonucleotide target sequences are listed in Table 2. To determine the minimal flanking sequence required for efficient processing, we cloned miR-150 and miR-30a with different length of flanking sequences into pLB vector (Addgene plasmid 11619) at the HpaI and XhoI in front of the U6 promoter. The sequences inserted into the vectors are shown in Table 3. The shRNAs were inserted into pLVX vector (Clontech plasmid 631987) at the EcoRI and BamHI site. Multiplexed shRNAs were synthesized as ultramers (IDT Technologies) and cloned into pLVX vectors (FIG. 11). The sequences inserted into the vectors are shown in Table 3.

TABLE 2
Oligonucleotide sequences used to insert target
sites in the 3′ UTR of R-luc in psiCheck2 vector
Oligonucleotide 5′-3′ Sequence
psiCHECK-miR-30 AGCTTCCAGTCGAGGATGTTTACAGCTACAGT
                ACTGAGCTTCCAGTCGAGGATGTTTACA
psiCHECK-miR-   TCGACACTGGTACAACGGTTGGGAGAAGTCAC
150             GTGTACCACTGGTACAACGGTTGGGAGA
psiCHECK-Vif    AGTTCAGAAGTACACATCCCGCTACAGTACTA
                GTTCAGAAGTACACATCCC
psiCHECK-CCR5   CGAGCAAGCTCAGTTTACACCGCTACAGTACT
                GGCGAGCAAGCTCAGTTTACACCGC
psiCHECK-Gag    AGGGGAAGTGACATAGCAGGATGCTCAGAGGG
                GAAGTGACATAGCAGGC
psiCHECK-Env    ATGGCAGTCTAGCAGAAGAAATGCTCAGATGG
                CAGTCTAGCAGAAGAAGC
psiCHECK-Tat    CTATGGCAGGAAGAAGCGGAATGCTCAGCTAT
                GGCAGGAAGAAGCGGAGC
psiCHECK-Pol1   CCCTACAATCCCCAAAGTCAATGCTCAGCCCT
                ACAATCCCCAAAGTCAGC
psiCHECK-Pol2   AGATACAGGAGCAGATGATAATGCTCAGAGAT
                ACAGGAGCAGATGATAGC

TABLE 3
Sequences of the oligonucleotides
used in the generation shRNAs
Oligonucleotide 5′-3′ Sequence
miR-30a 150 nt  GACATTTTCTAGAATATTGCTGTTTGAATGAGG
                CTTCAGTACTTTACAGAATCGTTGCCTGCACAT
                CTTGGAAACACTTGCTGGGATTACTTCTTCAGG
                TTAACCCAACAGAAGGCTAAAGAAGGTATATTG
                CTGTTGACAGTGAGCGACTGTAAACATCCTCGA
                CTGGAAGCTGTGAAGCCACAGATGGGCTTTCAG
                TCGGATGTTTGCAGCTGCCTACTGCCTCGGACT
                TCAAGGGGCTACTTTAGGAGCAATTATCTTGTT
                TACTAAAACTGAATACCTTGCTATCTCTTTGAT
                ACATTTTTACAAAGCTGAATTAAAATGGTATAA
                ATTAAATCACTTTAAAACCATGTCTGTACATTT
miR-30a 30 nt   GAAGGTATATTGCTGTTGACAGTGAGCGACTGT
                AAACATCCTCGACTGGAAGCTGTGAAGCCACAG
                ATGGGCTTTCAGTCGGATGTTTGCAGCTGCCTA
                CTGCCTCGGACTTCAAGGGGCTAC
miR-30a 20 nt   TGCTGTTGACAGTGAGCGACTGTAAACATCCTC
                GACTGGAAGCTGTGAAGCCACAGATGGGCTTTC
                AGTCGGATGTTTGCAGCTGCCTACTGCCTCGGA
                CTTC
miR-30a 15 nt   TTGACAGTGAGCGACTGTAAACATCCTCGACTG
                GAAGCTGTGAAGCCACAGATGGGCTTTCAGTCG
                GATGTTTGC AGCTGCCTACTGCCTCGG
miR-150 60 nt   CGGGGAGGCAGCGTCCCCGAGGCAGCAGCGGCA
                GCGGCGGCTCCTCTCCCCATGGCCCTGTCTCCC
                AACCCTTGTACCAGTGCTGGGCTCAGACCCTGG
                TACAGGCCTGGGGGACAGGGACCTGGGGACCCC
                GGCACCGGCAGGCCCCAAGGGGTGAGGTGAGCG
                GGCATTGGGAC
miR-150 30 nt   GCAGCGGCGGCTCCTCTCCCCATGGCCCTGTCT
                CCCAACCCTTGTACCAGTGCTGGGCTCAGACCC
                TGGTACAGGCCTGGGGGACAGGGACCTGGGGAC
                CCCGGCACCGGCAGGCC
miR-150 20 nt   CTCCTCTCCCCATGGCCCTGTCTCCCAACCCTT
                GTACCAGTGCTGGGCTCAGACCCTGGTACAGGC
                CTGGGGGACAGGGACCTGGGGACCCCGGCA
miR-150 15 nt   CTCCCCATGGCCCTGTCTCCCAACCCTTGTACC
                AGTGCTGGGCTCAGACCCTGGTACAGGCCTGGG
                GGACAGGGACCTGGGGACCC

Cell Culture

293 FT/T and TZM-bl cells were cultured as described elsewhere (Perez et al., J Virol 83: 7397-7410), PBMCs were obtained from healthy and HIV infected adult volunteers under an IRB approved protocol. CD4 T cells were isolated from PBMCs using CD4 T cell enrichment kits (Stem cell Technologies, Vancouver, BC, Canada). CD8 Dynabeads (Invitrogen) were used for depleting CD8 T cells from PBMCs. The CD8 depleted PBMCs and isolated CD4 T cells were stimulated with PHA after transduction with lentivirus and were cultured at 37° C. in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml of penicillin-streptomycin with recombinant IL-2 (20 U/ml).

DNA Transfection and Dual-Luciferase Reporter Assay

293 FT cells were co-transfected using 10, 50, and 100 ng shRNA vector and 100 ng of psiCHECK2 plasmid harboring the target regions using Lipofectamine 2000. Dual luciferase assay was performed 24 hours later as reported earlier.

Generation of Lentiviral Vector and Transduction

The lentiviral vector, pLVX-IRES-mCherry, was purchased from Clontech. Oligonucleotides targeting viral Gag, Env, Tat, Pol, Vif, and cellular co-receptor CCR5 were cloned into the EF-1 α promoter-expressing lentiviral vector pLVX-IRES-mCherry. 293 T cells were plated to 70-80% confluence in 150 mm dishes one day before transfection. The lentiviral vector and the helper pHR8.9VPR and env plasmids pCMV-VSV-G or HIV LAI envelope (kindly donated by Dr. Una O'Doherty at University of Pennsylvania) were co-transfected in 293 T cells using calcium phosphate precipitation (Promega). Medium was replaced after 4 h and supernatants were harvested as described (Lee et al. (2005). Blood 106: 818-826; Agosto, et al. (2009), J Virol 83: 8153-8162), TZM-bl cells, resting/activated CD4 T cells, and CD8 depleted PBMC from healthy and HIV infected patients were transduced at a multiplicity of infection (MOI) of 5-50 as described earlier (Lee et al. (2005). Blood 106: 818-826). After transduction, the cells were washed twice with PBS and cultured in media for 48 h. Transduction efficiency was determined by examining for mCherry expression by flow cytometry.

Assay for HIV Replication in TZM-Bl Reporter Cells

2 μg of shRNA expression vectors and 100 ng of HIV-1 NL4-3 plasmid (NIH AIDS Research and Reference Reagent Program) were co-transfected into 293 T cells using Lipofectamine 2000 reagent. The supernatants harvested two days after transfection was used for infecting equivalent number of TZM-bl cells in presence of 10 μg/ml DEAE-D. Tat-induced luciferase activities were determined in cell lysates 48 h post-infections using the Luciferase assay system (Promega) as previously described.

Assay for Toxicity of Micro-RNA Based shRNAs

To determine the toxicity of shRNA constructs, Jurkat cells were transfected with single, dual, and multiple shRNA constructs by Neon transfection system (Life Technology). Transfected cells were harvested after 48 h, stained with anti-Annexin V FITC antibody analyzed by flow cytometry. To determine vector cytotoxicity in CD4 T cells, mCherry expression was followed in transduced cells over time by flow cytometry. Lentivirally transduced CD8+-depleted PBMCs were stimulated with PHA and cultured in presence of IL-2 and were monitored on day 0, 4, 7, and 12 for mCherry expression by flow cytometry. Lentivirus transduced cells harvested two and four days post-transduction were also subjected to MTS Assay (Promega) according to manufactures instructions.

HIV-1 Challenge Assays

Untransduced and lentiviral vector transduced TZM-bl cells were infected with R5-tropic BaL and X4-tropic NL4-3 strains of HIV-1 at an MOIs 0.01 for 4 hours at 37° C. 2×10⁵ resting CD4 T cells and CD8 depleted PBMCs from normal and HIV seropositive donors were activated with PHA (2 μg/ml) after transduction with corresponding lentiviruses and cultured in the presence of IL2. Cells were infected 48 h post activation with HIV BaL and NIA-3, at MOIs of 0.01 and 0.001, respectively. Supernatants from TZM-bl cells and T cells/PBMCs were collected and analyzed for HIV replication by p24 ELISA assay (Perkin Elmer) as described previously.

Sequence Analysis of the Tat shRNA Target Region of HIV-1

Viral RNAs from four different HIV-seropositive donors were analyzed for shRNA-induced mutations in the Tat-shRNA target region on day 15 (mock) or 31 (Tat/7shRNA) postinfection as previously described (Schopman et al. (2010). Retrovirology 7: 52; Sugiyama et al. (2011). Nucleic Acids Res 39: 589-598), Viral RNA was extracted using the QIAamp RNA Kit (Qiagen) and first strand cDNA was synthesized using Superscript III First Strand Synthesis System for RT-PCR (Invitrogen, USA) as per the manufacturer's instructions. DNA sequences were PCR amplified using Tat targeted primers sense 5′-TGT TGC TTT CAT TGC CAA GT-3′ and antisense primer 5-TGA TGA GTC TGA CTG CCT TGA-3′. PCR was performed using the following thermal program: 95° C. for 2 min and then 35 cycles at 95° C. for 30 s, 57.8° C. for 30 s, and 72° C. for 30 s, followed by 72° C. for 5 min. The PCR products were gel purified and cloned into the pCR2.1 TOPO vector and subsequently sequenced with the M13R primers.

NOD/SCID-Hu PBL Mouse Model

NOD/SCID IL2rγcnull mice were purchased from Jackson Laboratory (Bar Harbor, Me.) and maintained in specific pathogen free conditions at the Paul L. Foster School of Medicine, TTUHSC animal facility. Hu-PBL mice were generated as described (Kumar et al. (2008). Cell 134: 577-586). In brief, mice were conditioned with sublethal (2 Gy) whole-body irradiation. Lentivirus transduced HIV-seropositive donor PBMCs (2×10⁶) were intravenously injected via the tail vein (in 0.2 ml PBS) into 6 to 7 week-old mice. Cell engraftment was tested 7, 28, and 42 days after transplantation by staining mouse PBMCs with human CD45, CD3, CD4, and CD8 antibodies. All mouse experiments had been approved by the TTUHSC IACUC and animal infection experiments were performed in bio-safety level 2 animal facility at TTUHSC.

Flow Cytometry

Flow cytometry was performed to determine cell surface antigen expression by 30-min incubation on ice with pertinent antibodies. The following monoclonal antibodies were used: human-specific monoclonal antibodies used were anti-CCR5 conjugated with FITC or APC (2D7/CCR5; BD Pharmingen), anti-human CD45 (PE), CD3 (FITC), CD4 (PB), CD8 (APC), and corresponding isotype control mAbs (BD Pharmingen). Data were acquired by BD FACS Canto H and analyzed on BD FACS Diva software v3.0. Overlays were made using FlowJo software v3.0 where ever applicable.

Small RNA Deep Sequencing

Small RNA libraries were constructed and sequenced in a similar manner as described previously (Ma et al. (2013). Proc Natl Acad Sci USA 110: 20687-20692; Ma et al. (2014). Mol Ther Nucleic Acids 3: e161). Briefly, 48 hours after the constructs were transfected into 293FT cells, the small RNAs were purified with the miRNeasy kit (Qiagen, Valencia, Calif.) as per the manufacturer's instructions. Small RNA (50 ng) was ligated with 3′ and 5′ linkers (with barcode) using an improved ligation method that was optimized comprehensively to minimize the ligation bias between different small RNAs. The ligated small RNAs were reverse transcribed and amplified with the KAPA library amplification kit (KAPA Biosystems, Woburn, Mass.) for 10 cycles, and the library sequenced using the Iliumina MiSeq, Salt Lake City, Utah. All reads that were sequenced only once were discarded to lower the noise level.

This example is also illustrated in Choi et al. Mol Thar, 2015; 23(2):310-320, titled “Multiplexing seven miRNA-Based shRNAs to suppress HIV replication,” which is incorporated by reference in its entirety.

It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

All publications, patents, and accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

EQUIVALENTS

While specific embodiments of the compositions and methods herein have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. An shRNA cluster encoding a plurality of shRNA-like molecules, wherein each of the plurality of shRNA-like molecules comprises:

a stem region comprising an artificial RNA molecule comprising a guide strand and a passenger strand, wherein the guide strand is substantially complementary to a target mRNA, wherein the artificial RNA molecule, when expressed in a cell, substantially inhibits expression of the target mRNA; and

a backbone region comprising a 5′ flanking region, a terminal loop region, and a 3′ flanking region,

provided that the backbone region of at least one shRNA-like molecule is not repeated among the plurality of shRNA-like molecules and the backbone regions of the plurality of shRNA-like molecules are not identical to the backbone regions of pri-miRNAs encoded by a naturally-occurring miRNA cluster.

2. The shRNA cluster of claim 1, wherein the plurality of shRNA-like molecules comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more shRNA-like molecules.

3. The shRNA cluster of claim 1, wherein the plurality of shRNA-like molecules comprise 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more shRNA-like molecules.

4. The shRNA cluster of claim 1, wherein the shRNA cluster encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more miRNA-based shRNA molecules (shRNA-miR molecules) and/or the shRNA cluster encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more shRNA molecules that are not derived from naturally-occurring pri-miRNAs.

5. The shRNA cluster of claim 1, wherein the target mRNA is encoded by a human gene chosen from CCR5 or CXCR4 or a viral gene chosen from Gag, Env, Tat, Pol2, Pol1, or Vif of HIV-1.

6. An artificial miRNA cluster encoding a plurality of modified pri-miRNA-like molecules, the artificial miRNA cluster comprising:

a first nucleotide sequence encoding a first modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a first miRNA; and

a second nucleotide sequence encoding a second modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a second miRNA,

provided that the first and second miRNAs are not in the same naturally-occurring miRNA cluster.

7. The artificial miRNA cluster of claim 6, wherein each of the plurality of modified pri-miRNA-like molecules comprises:

a stem region comprising an artificial RNA molecule comprising a guide strand and a passenger strand, wherein the guide strand is substantially complementary to a target mRNA;

a terminal loop region that is at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, a terminal loop region of a naturally-occurring pri-miRNA;

a 5′ flanking region that is at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, a 5′ flanking region of a naturally-occurring pri-miRNA; and

a 3′ flanking region that is at least 50%, 60%, 70%, 80%, 90%, or 100% identical to, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, a 3′ flanking region of a naturally-occurring pri-miRNA.

8. The artificial miRNA cluster of claim 6, wherein the artificial miRNA cluster further comprises a third nucleotide sequence encoding a third modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a third miRNA, wherein the third miRNA is not in the same naturally-occurring miRNA cluster as the first, second, or both miRNAs.

9. The artificial miRNA cluster of claim 8, wherein the artificial miRNA cluster further comprises a fourth nucleotide sequence encoding a fourth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a fourth miRNA, wherein the fourth miRNA is not in the same naturally-occurring miRNA cluster as the first, second, or third miRNA.

10. The artificial miRNA cluster of claim 9, wherein the artificial miRNA cluster further comprises a fifth nucleotide sequence encoding a fifth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a fifth miRNA, wherein the fifth miRNA is not in the same naturally-occurring miRNA cluster as the first, second, third, or fourth miRNA.

11. The artificial miRNA cluster of claim 10, wherein the artificial miRNA cluster further comprises a sixth nucleotide sequence encoding a sixth modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a sixth miRNA, wherein the sixth miRNA is not in the same naturally-occurring miRNA cluster as the first, second, third, fourth, or fifth miRNA.

12. The artificial miRNA cluster of claim 11, wherein the artificial miRNA cluster further comprises a seventh nucleotide sequence encoding a seventh modified pri-miRNA-like molecule derived from a naturally-occurring pri-miRNA for a seventh miRNA, wherein the seventh miRNA is not in the same naturally-occurring miRNA cluster as the first, second, third, fourth, fifth, or sixth miRNA.

13. The artificial miRNA cluster of claim 7, wherein the plurality of artificial RNA molecules, when expressed in a cell, substantially inhibit the expression of 2, 3, 4, 5, 6, 7, or all of the genes chosen from human CCR5, human CXCR4, HIV-1 Gag, HIV-1 Env, HIV-1 Tat, HIV-1 Pol2, HIV-1 Pol1, or HIV-1 Vif.

14. A cell comprising the artificial miRNA cluster of claim 6.

15. A method of treating or preventing a disorder, the method comprising administering the cell of claim 14 to a subject, thereby treating or preventing the disorder.

16. The method of claim 15, wherein the disorder is an HIV-1 infection or AIDS.

17. A pharmaceutical composition comprising the cell of claim 14.

18. A method of producing a cell that expresses a plurality of artificial RNA molecules, the method comprising contacting a cell with the artificial miRNA cluster of claim 6, and culturing the cell under conditions that allow expression of the artificial RNA molecules.

19. A viral particle comprising the artificial miRNA cluster of claim 6.

20. A method of designing a nucleic acid molecule, the method comprising altering a backbone region of a naturally-occurring pri-miRNA, wherein the backbone region comprises a 5′ flanking region, a terminal loop region, and a 3′ flanking region, wherein the method comprises one, two, or all of the following:

1) adding one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region;

2) deleting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region; or

3) substituting one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides in one, two, or all of the 5′ flanking region, the terminal loop region, or the 3′ flanking region.