WO2010057152A2 - Cross-species chimeric rna molecules - Google Patents

Abstract

Methods to reduce the expression of at least one gene of an intracellular pathogen in a host cell are provided, wherein the method comprises introducing into a host cell a heterologous polynucleotide comprising or encoding a miRNA that fuses with at least one mRNA of the pathogen and reduces the translation of the mRNA. Pharmaceutical compositions comprising such polynucleotides, wherein the reduction in pathogenic gene expression leads to an inhibition of growth or differentiation of the pathogen or a reduction in the infection of a host cell with the pathogen are also provided, along with methods of treating pathogen-derived diseases in subjects by administering the pharmaceutical compositions.

Description

CROSS-SPECIES CHIMERIC RNA MOLECULES

FIELD OF THE INVENTION

The invention generally relates to the field of molecular biology and specifically relates to the involvement of microRNAs in host-parasite interactions.

BACKGROUND OF THE INVENTION

Intracellular pathogens contribute to various pathogen-derived diseases that affect large numbers of people all over the globe. For example, malaria parasites annually infect 300-500 million people, cause 100-120 million clinical cases, and kill approximately 2 million people living between the Tropic of Cancer and the Tropic of Capricorn. Including recent estimates of malaria infections in sub-Saharan Africa, these numbers could be as high as 500-900 million infections annually. Early attempts to control the disease led to insecticide resistance in the mosquitoes and drug resistance in the parasites, leading to a worldwide resurgence of malaria. With the recognition in the late 1960s that eradication was not feasible, attention has turned to control and newer approaches to combat this major cause of human suffering. Using malaria as a model of pathogen-derived disease, major scientific advances have been made in understanding the biology, molecular biology and immunology of host-parasite interactions.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the observation that host cell microRNAs are capable of translocating into intracellular pathogens and affecting the expression of pathogenic genes through the fusion of the host microRNA with pathogenic RNA transcripts to form cross-species chimeric RNA molecules. Consequently, the translation of these cross-species chimeric RNA molecules is inhibited. In some instances, the reduction of the expression of pathogenic genes by the host miRNA leads to an inhibition of growth or promotion of differentiation of the pathogen. Thus, provided herein are methods for reducing the expression of at least one gene of an intracellular pathogen by introducing into a host cell a heterologous polynucleotide comprising or encoding a microRNA capable of fusing with a pathogenic mRNA and reducing the translation thereof. Also provided herein are pharmaceutical compositions comprising a pharmaceutical carrier and polynucleotides comprising or encoding microRNAs capable of differentiating or inhibiting the growth of an intracellular pathogen infecting a host cell or reducing the infection rate of a host cell with a pathogen and methods for treating a pathogen-derived disease by administering to a subject in need thereof polynucleotides comprising or encoding a microRNA capable of differentiating or inhibiting the growth of the pathogen. The expression level of host microRNA molecules capable of differentiating or inhibiting the growth of an intracellular pathogen can be used to determine the susceptibility of a subject to a pathogen-derived disease or for determining the severity of the disease in a subject. In some embodiments, the intracellular pathogen is a Plasmodium, including but not limited to malaria parasites such as P. falciparum, P. vivax, P. ovales, and P. malariae, In some of these embodiments, the pathogen-derived disease that is treated using the methods and pharmaceutical compositions disclosed herein is malaria.

Methods for identifying pathogenic target RNAs that can fuse with a particular miRNA sequence are also provided herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:

Figure 1 demonstrates that erythrocyte miRNAs are found in P, falciparum. Figure IA shows the top 11 miRNA species found in the IDC of P. falciparum and their representation in the uninfected erythrocyte or P, falciparum at 8 and 32 hours post-infection. Figure IB shows the levels of the four indicated miRNAs as a percentage of the total miRNA composition of the uninfected erythrocytes or parasites at 8 and 32 hours after infection. Figure 1C shows the relative levels of erythrocytic miR-451 in both untreated normal (HbAA) and sickle-cell (HbSS) erythrocytes with the indicated treatments. Figure ID shows the relative intraparasitic miR-451 levels from parasites grown in HbAA erythrocytes, HbAA erythrocytes transfected with miR- 451, and HbSS erythrocytes.

Figure 2 demonstrates there is a minimal contribution of erythrocyte ghosts to the miR-451 levels detected in parasites. Figure 2 A shows the miR-451 levels in normal (HbAA) and sickle cell (HbSS) erythrocytes, with (ghosts) or without (erythrocytes) saponin lysis. Figure 2B shows miR-451 levels in uninfected normal and sickle cell erythrocytes after saponin lysis in RNAse A-treated (*) or untreated cells. Figure 2C shows miR-451 levels in enriched parasites from normal and sickle cell erythrocytes in RNAse A-treated (*) or untreated parasites. Figure 2D shows the levels of 18 S rRNA in purified parasites (RNAse A-treated * and untreated) grown in normal and sickle cell erythrocytes.

Figure 3 shows that miR-451 and miR-223 impair the growth of P. falciparum. Figure 3 A shows normalized parasitemia at different time points post-infection in erythrocytes transfected with control DNA or the indicated miRNA. Figure 3B shows parasite proliferation measured by [³H]-hypoxanthine incorporation in erythrocytes transfected with the indicated miRNA. Figure 3 C shows the intraparasitic miR-451 levels when the parasites are grown in the untreated HbSS or HbAA erythrocytes, or HbSS erythrocytes treated with 2'-O-methyl oligonucleotides. Figure 3D shows the daily increase in parasitemia when parasites were propagated in HbAA or HbSS erythrocytes transfected with the indicated 2'-O-methyl oligonucleotides to inhibit specific miRNAs. The /?- values for selective comparison are shown.

Figure 4 A shows a Northern blot analysis of total RNA from (left to right) human mammary epithelial cells (HMEC), whole blood, synthetic miR-451, and purified parasite (3D7) using locked nucleic acid (LNA) probes detecting miR-451 (left) or miR-181 (right). The size markers and the images of RNA samples stained with ethidium bromide are also shown. Figures 4B and 4C show the normalized parasitemia (Figure 4B) and level of miR-451 modification for PKA-R (Figure 4C) at the indicated time points when grown in HbAA erythrocytes transfected with miR- 181, miR-451 , or modified miR-451 biotinylated at the 5' or 3' end. Figure 4D shows the relative levels of one unmodified transcript (18S rRNA) and three miR-451 -modified transcripts (28S rRNA, PKA-R, and PEAMT) in the P. falciparum RNA which have been pulled down by either unmodified miR-451, desthiobiotin (Db-) miR-181, or Db- miR-451 transfected into erythrocytes. All error bars are standard error of the mean with p- values generated using a two-tailed Mest.

Figure 5 A shows the levels of miR-451 and miR-181 recovered from uninfected erythrocytes transfected with either Db-miR-181 or Db-miR-451. Figure 5B shows the relative levels of 28S rRNA enriched by desthiobiotin (Db) pulldowns from parasites grown in erythrocytes transfected with miR-451 (Mock), Db-miR-181 (miR-181), or Db-miR-451 (miR-451) in the presence or absence of excess miR-451 (+miR-451 samples) which was added to the parasite lysate prior to biotin pulldown. Figure 5 C shows the same treatments as in Figure 5B, except the relative enrichment of PKA-R transcripts has been measured. Figure 6 shows the effect of miR-451 modification on target RNA decay. The transcript level and decay half life (ti_/2) of total and miR-451 -modified PKA-R (Figure 6A) and 28S rRNA transcripts (Figure 6B) in P, falciparum are shown at different times following the inhibition of transcription by actinomycin D.

Figure 7 demonstrates that the P. falciparum RNAs modified by miR-451 exhibit decreased ribosomal loading. Figure 7 A shows the ribosomal profile of synchronized 3D7 P. falciparum 32 hours post-infection. The relative migration of the small and large ribosomal subunits and the 80S monosome are indicated by the *, t, and % symbols, respectively. Figure 7B shows the 18S and 28S P. falciparum rRNA distribution in the gradient fractions. 18S rRNA, representing the small subunit, was maximal in fraction 7; 28S rRNA, representing the large subunit, was maximal in fraction 9; the 80S monosome (18S and 28S rRNA) was maximal in fraction 13. Figure 7C shows the normalized levels of total and miR-451 -modified PKA-R. Figure 7D shows the amount of miR-451 -modified transcript expressed as a percentage of the total PKA-R transcript in each fraction. Figure 8 demonstrates decreased ribosomal loading of PEAMT mRNA modified by miR-451. Figure 8A shows the ribosomal profile of synchronized 3D7 P. falciparum 32 hours post-infection. Figure 8B shows the level of 18S and 28S rRNA transcripts in the indicated fractions. Figure 8C shows the relative levels of total and miR-451 -modified PEAMT mRNA in the indicated fractions determined over two separate gradients by SYBR green real-time PCR and normalized to 18S and the monosome fraction (fraction 13) using the ΔΔCt method. Figure 8D shows the amount of miR-451 -modified PEAMT transcript expressed as a percentage of the total PEAMT transcript at each fraction.

Figure 9 demonstrates an increase in the number of sexual stage parasites in miR-451-transfected erythrocytes. The normalized percentage (against mock- transfected) of sexual stage parasites grown in mock, miR-181, and miR-451- transfected erythrocytes at 10 days post-transfection, as determined by Giemsa staining, is presented. Figure 1OA shows a Northern blot analysis of parasite RNA (stars) revealing the presence of large sized parasite RNAs reacting with the indicated miRNAs. The migration position of ribosomal RNAs and miRNA is marked. Figure 1OB presents the anti-plasmodial activities for selected miRNAs at the indicated times. Figure 1 1 provides the sequence alignment (Figure 1 IA), the ability to modify

PKA-R (Figure 1 IB), and the anti-malarial activities (Figure 11C) of five indicated natural and chimeric miRNAs,

Figure 12 shows a graph depicting the ability of the liver-specific miRNAs miR-126 and miR-122 to inhibit parasitemia when transfected into erythrocytes. Figure 13 graphs the relative levels within P. falciparum of one unmodified transcript (18S rRNA) and two miR-451 modified transcripts (28 S rRNA and PKA-R) pulled down by either unmodified miR-451, desthiobiotin (Db-) miR-181 and Db-miR- 451 transfected into erythrocytes.

Figure 14 demonstrates that mosquito miRNAs from the Anopheles gambiae mosquito can reduce parasitemia in human erythrocytes. Figure 14A shows a reduction in parasitemia with the aga-Let-7, aga-miR-1174, and aga-miR-12 miRNAs. Figure 14B shows a reduction in parasitemia with the aga-miR-989 microRNA.

Figure 15 provides an alignment of sequences from real-time polymerase chain reaction (RT-PCR), rapid amplification of 5' complementary cDNA ends (5'-RACE), and unbiased sequencing (Solexa) of the P. falciparum transcriptome that comprise both human miR-451 sequence and P. falciparum PKA-R sequence. The miR-451 and PKA-R sequences are separated by either a 6-bp (PKA-R #1) or a 14-bp (PKA-R #2) junction sequence, suggesting that additional RNA editing occurs during or following miR-451 fusion to the PKA-R mRNA. The underlined residues within the sequences of PKA-R Wl represent those residues that are different than the known P. falciparum PKA-R sequence.

DETAILED DESCRIPTION OF THE INVENTION

The sickle cell allele is found at a high frequency in human populations where malaria is endemic (Aidoo et al. (2002) Lancet 359:1311-1312). The altered properties of sickle cell erythrocytes are thought to be partially responsible for malaria resistance (Friedman (1978) Proc NatlAcadSci USA 75;1994-1997; Pasvol et al. (1978) Nature 274:701-703), but the molecular basis of this resistance is largely unknown. Results presented elsewhere herein show that the dysregulated microRNA composition of sickle cell erythrocytes (Chen et al. (2008) PLoS ONE 3:e2360) contributes to resistance against the malarial parasite Plasmodium falciparum. During the intraerythrocytic growth of P. falciparum, a subset of erythrocyte microRNAs translocates into the parasite. Two of the sickle cell-enriched microRNAs, miR-451 and miR-223, reduced parasite growth when overexpressed in normal erythrocytes.

Conversely, blocking the erythrocyte-to-parasite translocation of miR-451 and miR-223 diminished malaria resistance in sickle cell erythrocytes. It was discovered that human microRNAs are covalently integrated into P. falciparum RNAs to form a novel class of cross-species fusion RNAs. Importantly, miR-451 integration impaired ribosomal loading of the modified target RNA, consistent with a model of translational inhibition. These data indicate that erythrocyte microRNAs directly participate in the parasite's posttranscriptional gene regulation. These results provide evidence of a novel and direct role for human microRNAs in the erythrocyte-parasite interaction and may lead to the development of new anti-malarial therapies. In addition to erythrocytic microRNAs, data provided elsewhere herein demonstrates that hepatocyte-specifϊc microRNAs are also capable of inhibiting the growth of P. falciparum. Additional data suggests that microRNAs from the Anopheles mosquito (the vector for the malarial parasite) can inhibit Plasmodial parasitemia. Without being bound by any theory or mechanism of action, preliminary results suggest the mosquito miRNAs serve as barriers to Plasmodial infection.

Provided herein are methods for modulating the expression of at least one gene of an intracellular pathogen in a host cell, wherein the method comprises introducing into the host cell at least one heterologous polynucleotide comprising or encoding a miRNA that fuses with at least one mRNA of the pathogen (e.g., Plasmodium) and reduces the translation of the mRNA. As used herein, the terms "microRNA,"

"miRNA," "mature microRNA," and "mature miRNA" refer to a single-stranded RNA molecule that is about 19 to about 25 nucleotides in length (including about 19, about 20, about 21, about 22, about 23, about 24, and about 25 nucleotides) that effectively reduces the expression level of target polynucleotides and polypeptides encoded thereby. A microRNA may be generated from various precursors, including but not limited to an endogenous primary microRNA transcript (pri-miRNA), an exogenously introduced hairpin RNA (including shRNA molecules), a transcript comprising a local hairpin structure comprising a microRNA that has been encoded by an exogenously introduced plasmid DNA, an exogenously introduced double-stranded siRNA that comprises a microRNA sequence, a transcript comprising a mature microRNA sequence that has been encoded by an exogenously introduced plasmid DNA, or a single-stranded, exogenously introduced oligonucleotide comprising a mature microRNA sequence, Endogenous miRNAs are generated through a series of steps beginning with the transcription of a primary miRNA transcript (pri-miRNA). The pri-miRNA transcript is a single-stranded RNA molecule that comprises at least one stem-loop structure. The pri-miRNA transcript is typically thousands of nucleotides long and is often capped, spliced, and poly-adenylated and can be polycistronic, comprising multiple microRNAs of the same or different sequence.

A "stem-loop structure" refers to a polynucleotide having a secondary structure that includes a region of nucleotides which are known or predicted to form a double stranded portion (the stem portion or stem region) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion or loop region). The term "hairpin" structures are also used herein to refer to stem-loop structures. The stem-loop structures of pri-miRNA and precursor miRNAs (pre-miRNAs) comprise the microRNA sequence, which is complementary (fully or partially) to the target polynucleotide, which base pairs with the microRNA* sequence (also referred to as the "star strand"). The microRNA and microRNA* sequences make up the stem of the stem-loop structure and can be fully or partially complementary to one another.

Primary microRNA transcripts can also be encoded by exogenously introduced plasmid DNA comprising the coding sequence for the primary microRNA transcript operably linked to regulatory sequences that regulate the expression of the transcript. Alternatively, primary microRNA transcripts can also refer to exogenously introduced hairpin-comprising single- stranded RNAs comprising a microRNA sequence that can be recognized and cleaved by the enzyme Drosha to generate a pre- raiRNA.

The primary microRNA transcript is cleaved by the RNase III enzyme Drosha to release the stem-loop structure comprising the microRNA, which is now referred to as the precursor miRNA or pre-miRNA. Thus, the terms "precursor-microRNA," "pre- miRNA," and "precursor-miRNA" refer to a nucleic acid having a stem-loop structure with a microRNA sequence incorporated therein. Pre-miRNAs are exported from the nuclear compartment into the cytoplasm by exportin-5, where the pre-miRNAs are further processed into miRNA duplexes by the cytoplasmic RNase III Dicer. The resultant miRNA duplexes (double-stranded RNA) comprise the mature microRNA, which is the strand that will bind to the complementary (fully or partially) target polynucleotide, and the microRNA* strand or "star strand," which is complementary to the microRNA itself (fully or partially). Traditionally, microRNAs have been shown to function through the incorporation of the mature miRNA into the RNA- induced silencing complexes (RISC), which is guided to complementary RNA molecules, wherein the RISC either nucleolytically degrades the target messenger RNA (mRNA) or blocks the translation of the target mRNA, thereby inhibiting the expression. Thus, the traditional target RNA of a miRNA is one that comprises a complementary sequence to the miRNA.

Data provided herein, however, demonstrates that miRNA can also effect inhibition of translation of target mRNAs through the fusion of the miRNA molecule with the target mRNA. Thus, this method of translational inhibition does not require any sequence complementarity between the miRNA and the target RNA. As used herein, the terms "target polynucleotide," "target RNA," "target transcript,"or "target mRNA" of a particular miRNA is intended the polynucleotide, RNA, or mRNA whose expression is reduced by the miRNA molecule. The target polynucleotide either comprises a complementary sequence to the miRNA (generally, in the 3' untranslated region) or is capable of fusing with the miRNA molecule. The term "polynucleotide" is intended to encompass a singular nucleic acid, as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, e.g., messenger RNA (mRNA), plasmid DNA (pDNA), or short interfering RNA (siRNA). A polynucleotide can be single- stranded or double-stranded, linear or circular. A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond {e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. Polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. Polynucleotides can also include expression vectors, expression constructs, or populations thereof. "Polynucleotide" also can refer to amplified products of itself, as in a polymerase chain reaction. The "polynucleotide" can contain modified nucleic acids, such as phosphorothioate, phosphate, ring atom modified derivatives, and the like. The "polynucleotide" can be a naturally occurring polynucleotide (i.e., one existing in nature without human intervention), or a recombinant polynucleotide (i.e., one existing only with human intervention). While the terms "polynucleotide" and "oligonucleotide" both refer to a polymer of nucleotides, as used herein, an oligonucleotide is typically less than 100 nucleotides in length.

The presently disclosed methods involve the introduction of a heterologous polynucleotide. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention and/or is removed from its native environment or synthesized by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

The term "expression" has its meaning as understood in the art and refers to the process of converting genetic information encoded in a gene or a coding sequence into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of a polynucleotide (e.g., via the enzymatic action of an RNA polymerase ), and for polypeptide-encoding polynucleotides, into a polypeptide through "translation" of mRNA. Thus, an "expression product" is, in general, an RNA transcribed from the gene or coding sequence (e.g., either pre- or post-processing) or polynucleotide or a polypeptide encoded by an RNA transcribed from the gene or coding sequence (e.g., either pre- or post-modification).

The heterologous polynucleotides useful in reducing the expression of at least one gene of an intracellular pathogen or inhibiting its growth can comprise or encode a miRNA (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17). As used herein, the terms "encoding" or "encoded" when used in the context of a specified nucleic acid mean that the nucleic acid comprises the requisite information to direct transcription of the nucleotide sequence into a specified RNA molecule and in some instances, translation of the nucleotide sequence into a specified polypeptide. It is noted that a polynucleotide that encodes a particular sequence can also encode additional sequences that are 5 ' or 3' to the particular sequence or can comprise additional non-coding or coding sequences.

The expression of a gene of an intracellular pathogen is modulated through the introduction or increase in the level of a miRNA molecule in the host cell infected by the pathogen. The term "modulate" refers to either an increase or a decrease in expression when compared to a control. In these embodiments, the control may be the same or similar pathogen (e.g., same species, strain, stage in life cycle) that is not infecting a host cell or a same or similar pathogen that is infecting the same or similar host cell (e.g., same species, differentiation state) in the absence of the exogenously introduced miRNA sequence. In general, the expression of a pathogenic gene is reduced by the introduction of the miRNA molecule and generation of the cross-species chimeric RNA molecule.

By "reduces" or "reducing" the expression level of a polynucleotide or a polypeptide encoded thereby is intended to mean, the level of the polynucleotide or the encoded polypeptide is statistically lower than the target polynucleotide level or encoded polypeptide level in an appropriate control which is not exposed to the exogenously introduced microRNA. In particular embodiments, reducing the target polynucleotide level and/or the encoded polypeptide level according to the presently disclosed subject matter results in less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less of the target polynucleotide level, or the level of the polypeptide encoded thereby in an appropriate control.

Methods to assay for the level of the RNA transcript include Northern blots, nuclease protection assays, reverse transcription (RT)-PCR, real-time RT-PCR, microarray analysis, and the like. Alternatively, the ability of the miRNA to reduce the level or inhibit the translation of the target polynucleotide can be measured using a variety of affinity-based approaches (e.g., using a ligand or antibody that specifically binds to the target polypeptide) including, but not limited to, Western blots, immunoassays, ELISA, flow cytometry, protein microarrays, and the like. In still other methods, the ability of the miRNA to reduce the level or inhibit the translation of the target polynucleotide can be assessed by measuring a functional activity of the polypeptide encoded by the transcript or by measuring a signal produced by the polypeptide encoded by the transcript, the level of the encoded polypeptide, or the activity of the polynucleotide or polypeptide,

In some embodiments wherein the expression of at least one gene of an intracellular pathogen in a host cell is reduced, a heterologous polynucleotide comprising or encoding a microRNA sequence is introduced into the host cell, wherein the microRNA is capable of fusing with at least one mRNA of the pathogen and reducing the translation of the mRNA. In some of these embodiments, the heterologous polynucleotide that is introduced into the host cell comprises or encodes a nucleotide sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 22, wherein the polynucleotide or an expression product thereof when introduced into or expressed in the host cell fuses with at least one mRNA of the pathogen and reduces the translation of the at least one mRNA. An active fragment of the recited sequences that is capable of fusing with the target polynucleotide and inhibiting its translation can also be used, including fragments comprising at least 5 nucleotides of the recited sequences (SEQ ID NO: 1, 2, 3, 4, 6, 8, 9, 1O₅ 11, 12, 13, 14, 15, 16, 17, 22), including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, and up to the full-length of the recited sequence. In some embodiments, the active fragment of the recited sequences comprises the first 7 nucleotides of the recited sequences. In certain embodiments, the heterologous polynucleotide comprises the consensus sequence CXGUU (SEQ ID NO: 18; wherein X can be any nucleotide). In some of these embodiments, the polynucleotide comprises CCGUU (SEQ ID NO: 19). In other embodiments, the polynucleotide comprises CAGUU (SEQ ID NO: 20). The sequence set forth in SEQ ID NO: 18, 19, or 20 can be comprised within a polynucleotide of about 19 to about 25 nucleotides in length, and in some embodiments, can impart the ability of the polynucleotide to inhibit the translation of a target polynucleotide. In some of the embodiments, translation is inhibited through the fusion of the polynucleotide comprising the sequence set forth in SEQ ID NO: 18, 19, or 20 with a pathogenic RNA molecule. As used herein, "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a fragment or the entirety of a specific sequence; for example, as a segment of a full-length cDNA sequence, or the complete cDNA sequence.

As used herein, the term "comparison window" refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4: 11- 17; the local alignment algorithm of Smith et al. (1981)ΛΛ. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J MoI. Biol. 48:443- 453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Set 85 :2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Set USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2,0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. MoI. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAMl 20 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. MoI. Biol 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection. Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J MoI. Biol. 48:443- 453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. ScL USA 89:10915).

Data presented elsewhere herein demonstrate that the host miRNAs fuse to the 5' end of target polynucleotides through the 3' end of the miRNA molecule. As used herein, the term "fuse" refers to the linkage of one polynucleotide (miRNA) to a second polynucleotide (e.g., mRNA, rRNA) through a phosphodiester bond between the 3' end of the first polynucleotide and the 5' end of the second polynucleotide. In some embodiments, the polynucleotide that is introduced into a host cell to reduce the expression of a gene of an intracellular pathogen comprises or encodes a polynucleotide having a free 3' hydroxyl group. By "free" is intended the 3' hydroxyl group of the nucleic acid molecule is capable of forming a phosphodiester bond with the 5' end of a second nucleic acid molecule. In some embodiments, a junction sequence can be found between the miRNA sequence and the target RNA. The junction sequence may be derived from the target RNA and may be the result of RNA editing or the ligation process. In some embodiments, the junction sequence has a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides. In certain embodiments, the junction sequence is about 6 nucleotides in length and can have the sequence set forth in SEQ ID NO: 44. In other embodiments, the junction sequence is about 14 nucleotides in length and can have the sequence set forth in SEQ ID NO: 45.

As used herein, the term "intracellular pathogen" refers to an organism that is capable of survival within a cell of a host organism. Non-limiting examples of intracellular pathogens include parasites such as those that belong to the genus

Plasmodium. Non-limiting examples of Plasmodium include Plasmodium falciparum, Plasmodium vivax, Plasmodium ovales, and Plasmodium malariae. These species of Plasmodium (P. falciparum, P. vivax, P, ovales, P. malariae) can infect humans and cause the disease malaria. These species of Plasmodium and other that can invade human red blood cells, leading to the clinical, molecular, and cellular hallmarks of malaria are referred to herein as malaria parasites.

The terms "host" and "host cell" refer to the organism and cell, respectively, that is infected by an intracellular pathogen. In some embodiments, the host is a vector. A "vector" is an organism that can carry and transmit an intracellular pathogen to another organism. In some embodiments, the vector is a mosquito. In those embodiments, wherein the intracellular pathogen comprises a Plasmodium (e.g., P. falciparum, P. vivax, P. ovales, P. malariae), the host can be an Anopheles mosquito. In some of the embodiments wherein the host is an Anopheles mosquito, the mosquito is an Anopheles gambiae. In some embodiments including those wherein the host is a human and the intracellular pathogen is a Plasmodium (e.g., P. falciparum, P, vivax, P. ovales, P. malariae), the host cell can be an erythrocyte or a hepatocyte.

In certain embodiments, the pathogenic gene whose expression is reduced by the introduced nucleic acid molecule or expression product thereof encodes the cAMP- dependent regulatory subunit (PKA-R) or phosphoethanolamine N-methyltransferase (PEAMT).

In some embodiments, the microRNA sequence that is introduced into the host cell will inhibit the growth or promote the differentiation of an intracellular pathogen infecting the cell. The growth or differentiation state of the intracellular pathogen can be measured using any method known in the art. For example, in those embodiments wherein the intracellular pathogen comprises a Plasmodium, methods including those described elsewhere herein such as Giemsa staining to evaluate the progression to ring, trophozoite, schizont, and gametocyte stage and the percentage of infected cells, FACS analysis of YoYo-I staining to assay the level of parasitemia, [³H]hypoxanthine incorporation for DNA replication, and TUNEL assay to monitor apoptosis can be used.

The presently disclosed subject matter, therefore, provides methods for differentiating or inhibiting the growth of an intracellular pathogen in a host cell by introducing into the host cell a heterologous polynucleotide comprising or encoding a microRNA that is capable of reducing the expression of at least one gene of the pathogen, wherein the reduction in the expression of the gene leads to the differentiation or inhibition of growth of the pathogen. In some embodiments, the microRNA sequence is capable of fusing to a pathogenic polynucleotide to generate a cross-species chimeric RNA molecule and, therefore, inhibit the translation of the target polynucleotide. In certain embodiments, the heterologous polynucleotide that is introduced into the host cell comprises or encodes a nucleotide sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, or 17, wherein the polynucleotide or an expression product thereof when introduced into or expressed in the host cell reduces the expression of at least one gene of the pathogen. An active fragment of the recited sequences that is capable of reducing the expression of at least one gene of the pathogen can also be used, including fragments comprising at least 5 nucleotides of the recited sequences (SEQ ID NO: 1 , 2, 4, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17), including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, and up to the full-length of the recited sequence. In some embodiments, the active fragment of the recited sequences comprises the first 7 nucleotides of the recited sequences. In certain embodiments, the heterologous polynucleotide comprises the consensus sequence CXGUU (SEQ ID NO: 18; wherein X can be any nucleotide). In some of these embodiments, the polynucleotide comprises CCGUU (SEQ ID NO: 19). In other embodiments, the polynucleotide comprises CAGUU (SEQ ID NO: 20). The sequence set forth in SEQ ID NO: 18, 19, or 20 can be comprised within a polynucleotide of about 19 to about 25 nucleotides in length, and in some embodiments, can impart the ability of the polynucleotide to inhibit the translation of a target polynucleotide. In some of the embodiments, translation is inhibited through the fusion of the polynucleotide comprising the sequence set forth in SEQ ID NO: 18, 19, or 20 with a pathogenic RNA molecule. In some embodiments, the polynucleotide or an expression product thereof comprises a free 3' hydroxyl group. In certain embodiments, the pathogen comprises a Plasmodium (e.g., P. falciparum, P, vivax, P, ovales, P. malariae). The host cell can comprise, for example, an erythrocyte or a hepatocyte and the host can be, for example, a human or a mosquito.

Methods are also provided for reducing the infection of a host cell by an intracellular pathogen by introducing into the host cell a heterologous polynucleotide comprising or encoding a miRNA that is capable of reducing the expression of at least one gene of the pathogen, wherein the reduction in the expression of the gene leads to the reduction in infection of a host cell by the pathogen. In some embodiments, the microRNA sequence is capable of fusing to a pathogenic polynucleotide to generate a cross-species chimeric RNA molecule, and therefore, inhibit the translation of the target polynucleotide. In certain embodiments, the heterologous polynucleotide that is introduced into the host cell comprises or encodes a nucleotide sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14, 15, 16, or 17, wherein the polynucleotide or an expression product thereof when introduced into or expressed in the host cell reduces the expression of at least one gene of the pathogen. An active fragment of the recited sequences that is capable of reducing the expression of at least one gene of the pathogen can also be used, including fragments comprising at least 5 nucleotides of the recited sequences (SEQ ID NO: 14, 15, 16, 17), including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, and up to the full-length of the recited sequence. In some embodiments, the active fragment of the recited sequences comprises the first 7 nucleotides of the recited sequences. In certain embodiments, the heterologous polynucleotide comprises the consensus sequence CXGUU (SEQ ID NO: 18; wherein X can be any nucleotide). In some of these embodiments, the polynucleotide comprises CCGUU (SEQ ID NO: 19). In other embodiments, the polynucleotide comprises

CAGUU (SEQ ID NO: 20). The sequence set forth in SEQ ID NO: 18, 19, or 20 can be comprised within a polynucleotide of about 19 to about 25 nucleotides in length, and in some embodiments, can impart the ability of the polynucleotide to inhibit the translation of a target polynucleotide. In some of the embodiments, translation is inhibited through the fusion of the polynucleotide comprising the sequence set forth in SEQ ID NO: 18, 19, or 20 with a pathogenic RNA molecule. In some embodiments, the polynucleotide or an expression product thereof comprises a free 3' hydroxy 1 group. In certain embodiments, the pathogen comprises a Plasmodium (e.g., P. falciparum, P. vivax, P. ovale s, P. malariae). The host cell can comprise, for example, an erythrocyte or a hepatocyte and the host can be, for example, a human or a mosquito.

As used herein, the "infection" of a host cell by an intracellular pathogen refers to the entry of the pathogen into the host cell and the initiation of proliferation or differentiation of the pathogen. While not being bound by any theory or mechanism of action, it is believed the mosquito miRNAs set forth in SEQ ID NO: 14, 15, 16, and 17 act as a barrier to infection and therefore, reduce the infection rate of the host cell by the intracellular pathogen (e.g., Plasmodium) by inhibiting the entry of additional pathogens into the host cell and/or preventing or reducing the initiation of proliferation or differentiation of a pathogen immediately following entry into the host cell. The methods presented herein require introducing a polynucleotide into a host cell (e.g., erythrocyte). "Introducing" is intended to mean presenting to a cell the polynucleotide in such a manner that the sequence gains access to the interior of the cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the polynucleotide sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion and so on. In some embodiments, the term "introducing" excludes naturally- occurring cell-to-cell transport of small polynucleotides, such as miRNAs. When the polynucleotide is introduced in vivo, the polynucleotide can be introduced through infection using defective or attenuated retrovirals or other viral vectors, or the polynucleotide can be coated with lipids or cell-surface receptors or transfecting agents, encapsulated in liposomes, microparticles, or microcapsules to facilitate uptake by the cell. Alternatively, the polynucleotides can be introduced into a cell by linking the polynucleotide to a peptide which is known to enter cells (or the nucleus) or to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu (1987) J Biol. Chem. 262:4429-4432, which is herein incorporated by reference in its entirety) Numerous techniques are known in the art for the introduction of foreign sequences into cells and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique can provide for the stable transfer of the polynucleotide to the cell, so that the polynucleotide is expressible by the cell and preferably heritable and expressible by its cell progeny. In some embodiments, the polynucleotide is introduced into the cell through transfection. Methods for transfecting host cells, such as erythrocytes are known in the art. See, Bacchetti and Graham (1977) Proc Natl Acad Sci USA

74(4):1590-4; Straus and Raskas (1980) J Gen Virol 48:241-5; and Deitsch et al. (2001) Nucleic Acids Res 29(3):850-3; all of which are herein incorporated by reference. Transfection typically is carried out by mixing a cationic lipid with the polynucleotide(s) to produce liposomes which fuse with the cell plasma membrane and deposit the genetic material inside the cell.

As discussed above, the miRNA employed in the methods of the invention can comprise a DNA molecule which when transcribed produces a miRNA or a precursor thereof (e.g., primary transcript, precursor miRNA, miRNA duplex). In such embodiments, the DNA molecule encoding the miRNA or precursor thereof is found in an expression cassette.

The expression cassette comprises one or more regulatory sequences, selected on the basis of the cells to be used for expression, operably linked to a polynucleotide encoding the miRNA or precursor thereof. "Operably linked" is intended to mean that the nucleotide sequence of interest (i.e., a DNA encoding a microRNA or precursor thereof) is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a cell when the expression cassette or vector is introduced into a cell). "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in En∑ymology 185 (Academic Press, San Diego, California). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression cassette can depend on such factors as the choice of the host cell to be transformed, the level of expression of the miRNA or precursor thereof, and the like. Such expression cassettes typically include one or more appropriately positioned sites for restriction enzymes, to facilitate introduction of the nucleic acid into a vector. It will further be appreciated that appropriate promoter and/or regulatory elements can readily be selected to allow expression of the relevant miRNA or precursor thereof in the cell of interest. In certain embodiments, the promoter utilized to direct intracellular expression of a miRNA or precursor thereof is a promoter for RNA polymerase III (Pol III). References discussing various Pol III promoters, include, for example, Yu et al (2002) Proc, Natl Acad. Set 99(9), 6047-6052; Sui et al. (2002) Proc. Natl Acad. Sci. 99(8), 5515-5520 (2002); Paddison et al. (2002) Genes andDev. 16, 948-958; Brummelkamp et al. (2002) Science 296, 550-553; Miyagashi (2002) Biotech. 20, 497-500; Paul et al (2002) Nat. Biotech. 20, 505-508; Tuschl etal (2002) Nat. Biotech. 20, 446-448, each of which is herein incorporated by reference in its entirety. According to other embodiments, a promoter for RNA polymerase I, e.g., a tRNA promoter, can be used. See McCown et al (2003) Virology 313(2):514-24; Kawasaki (2003) Nucleic Acids Res. 31 (2):700-7, each of which is herein incorporated by reference in its entirety. The regulatory sequences can also be provided by viral regulatory elements.

For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, California), In vitro transcription can be performed using a variety of available systems including the T7, SP6, and T3 promoter/polymerase systems (e.g., those available commercially from Promega, Clontech, New England Biolabs, and the like). Vectors including the T7, SP6, or T3 promoter are well known in the art and can readily be modified to direct transcription of miRNAs or precursors thereof. When double-stranded miRNA precursors are synthesized in vitro, the strands can be allowed to hybridize before introducing into a cell or before administration to a subject. As noted above, miRNAs or precursors thereof can be delivered or introduced into a cell as a single RNA molecule including self-complementary portions (e.g., an shRNA that can be processed intracellularly to yield a miRNA), or as two strands hybridized to one another. In other embodiments, the miRNAs or precursors thereof are transcribed in vivo. As discussed elsewhere herein, regardless of if the miRNA or precursor thereof is transcribed in vivo or in vitro, in either scenario, a primary transcript can be produced which can then be processed (e.g., by one or more cellular enzymes) to generate the miRNA that accomplishes gene inhibition.

The expression level of a host miRNA capable of modulating the differentiation state or growth rate of an intracellular pathogen within a host can be used to determine the susceptibility of the host to the pathogen-induced disease or to determine the severity of the disease in a subject.

As used herein, the term "pathogen-derived disease" is a pathological state characterized by symptoms that are directly or indirectly due to the infection of at least one cell in a subject by an intracellular pathogen. As used herein, the term "susceptibility" refers to the likelihood that the subject has the disease in question (clinical or subclinical) or will contract or develop the disease at any point during the subject's lifetime. In some cases wherein a subject's susceptibility to disease refers to the likelihood that the subject has the disease in question, the patient may not be presenting with clinical symptoms typically associated with the disease at the time the subject's susceptibility to the disease is being assessed and thus has a subclinical form of the disease.

There may exist a positive correlation or negative correlation between the expression level of the host miRNA and a subject's susceptibility to or severity of a pathogen-induced disease. A positive correlation defines a relationship between two variables, wherein a change in one variable in one direction (e.g., increase or decrease) results in a change in the second variable in the same direction. For example, the level of a particular miRNA is positively correlated with the severity of a certain pathogen-induced disease if an increase in the level of the miRNA is associated with an increase in the severity of the disease and conversely, a decrease in the level of the miRNA is associated with a decrease in the severity of the disease.

On the other hand, a negative correlation defines a relationship between two variables, wherein a change in one variable in one direction results in a change in the second variable in the opposite direction. Thus, the level of a particular miRNA is negatively correlated with the severity of a certain pathogen-induced disease if an increase in the level of the miRNA is associated with a decrease in the severity of the disease and conversely, a decrease in the level of the miRNA is associated with an increase in the severity of the disease. To determine a subject's susceptibility to an erythrocyte disease, the level of the subject's erythrocyte miRNA can be compared to a control. In some instances, the control can be one or more subjects not having or not suspected of having the pathogen-induced disease or the control can be a previously assayed value for the same subject. In other instances, for example, wherein one is classifying a subtype of a particular pathogen-induced disease, wherein a subtype is associated with a more severe disease state than other subtypes and the subtypes can be classified based on the level of a particular miRNA in these patients, the control would be the average value of the level of the miRNA across a population of patients with the pathogen-induced disease that one is attempting to classify. Thus, in some of those embodiments wherein one is determining the severity of a disease relative to a control, a control may comprise one or more patients with the disease that exhibit more or less severe symptoms that would indicate a more or less severe disease.

In some embodiments, the expression level of host miRNAs capable of differentiating or inhibiting the growth of a malaria parasite can be used to determine the susceptibility of a subject to malaria or the severity of the disease in a subject. These methods comprise obtaining a sample of erythrocytes from the subject, determining the level of at least one miRNA in the erythrocytes, wherein the miRNA is capable of differentiating or inhibiting the growth of a malaria parasite, and comparing the level to that of a control subject, wherein a decrease in the level of the miRNA compared to the control indicates an enhanced susceptibility of the subject to malaria or a more severe disease relative to the control subject. In some of these embodiments, the miRNA is capable of fusing to a target polynucleotide of the malaria parasite and reducing the expression of the target polynucleotide, leading to the differentiation of the parasite or inhibition of its growth. In some embodiments, the miRNA comprises at least one of hsa-miR-451 (SEQ ID NO: 1), hsa-miR-223 (SEQ ID NO: 2), hsa-miR-92 (SEQ ID NO: 5), hsa-miR-25 (SEQ ID NO: 6), hsa-miR-15a (SEQ ID NO: 7), hsa- miR~15b (SEQ ID NO: 8), hsa-let-7i (SEQ ID NO: 9), hsa-miR-126 (SEQ ID NO: 12), and hsa-miR-122 (SEQ ID NO: 13). In some of these embodiments, the miRNA capable of inhibiting the growth of the malaria parasite is capable of translocating into the malaria parasite. By "translocate" when referring to a pathogen is intended the transfer of a host material (e.g., miRNA) from the host into a pathogen infecting the host. The miRNA can translocate into the parasite during any stage of infection, particularly later stages of infection. The ability of a miRNA to translocate into the parasite can be determined using assays provided in U.S. Patent Application Publication No. US2009/0124566, which is herein incorporated in its entirety. The translocated miRNA can inhibit the growth or survival of the parasite and, therefore, the levels of these miRNAs in the erythrocyte are negatively correlated with the susceptibility to malaria.

As demonstrated elsewhere herein, erythrocytes overexpressing hsa-miR-451, hsa-miR-223, hsa-miR-92, hsa-miR-25, hsa-miR-15a, hsa-miR-15b, and hsa-let-7i exhibit a reduced parasitemia when infected with P. falciparum. Thus, a subject that comprises an increased level of at least one of these miRNAs in the erythrocytes compared to a control has a decreased susceptibility to malaria relative to the control. Conversely, a subject that has a reduced level of at least one of these miRNAs in the erythrocytes compared to a control has an enhanced susceptibility to malaria relative to the control.

It was also shown that the liver miRNAs hsa-miR-126 and hsa-miR-122 can impair the growth and/or differentiation of P. falciparum in erythrocytes. It is expected that these miRNAs can also inhibit the growth and/or differentiation of P. falciparum in hepatocytes. Thus, the levels of hsa-miR-126 and hsa-miR-122 are negatively correlated with susceptibility to malaria. Accordingly, a subject that comprises an increased level of at least one of these miRNAs in the erthrocytes and/or hepatocytes compared to a control has a decreased susceptibility to malaria relative to the control. Conversely, a subject that has a reduced level of at least one of these miRNAs in the erythrocytes and/or hepatocytes compared to a control has an enhanced susceptibility to malaria relative to the control.

In some embodiments, the increase in the level of at least one of hsa-miR-451, hsa-miR-223, hsa-miR-92, hsa-miR-25, hsa-miR-15a, hsa-miR-15b, hsa-let-7i, hsa- miR-126, and hsa-miR-122 over that of a control that indicates a reduced susceptibility to malaria, or the reduction in the level of at least one of hsa-miR-451, hsa-miR-223, hsa-miR-92, hsa-miR-25, hsa-miR-15a, hsa-miR-15b, hsa-let-7i, hsa-miR-126, and hsa- miR-122 over that of a control that indicates an enhanced susceptibility to malaria relative to the control can be a fold change greater than one, including but not limited to at least about 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, or greater. The severity of a subject's disease generally refers to the level and frequency of disease-associated symptoms and the progression of the disease overall. The severity of a particular subject's disease can be assessed using any type of diagnostic and prognostic procedures known in the art. Those patients that are determined to be particularly susceptible to malaria can further be treated with any preventative treatment for malaria known in the art or disclosed herein, including the administration of the presently disclosed polynucleotides that are capable of inhibiting the growth or differentiating the Plasmodium or reducing infection by Plasmodium. According to the methods of the invention, a subject's susceptibility to malaria or severity of the disease is determined by measuring the expression of a given miRNA in erythrocytes.

The term "erythrocyte" refers to a mature red blood cell that is CD71^". The term "red blood cell" can refer to either a mature red blood cell (i.e., erythrocyte) or an immature red blood cell (i.e., reticulocyte). Under normal physiological conditions, reticulocytes generally represent a minor fraction of red blood cells throughout the body. Reticulocytes differentiate into mature erythrocytes, which make up the majority of the cells in the blood, typically having a life span of about 120 days.

Erythrocytes can be obtained from a subject using any suitable purification method known in the art to isolate the erythrocytes from whole blood, including but not limited to density gradient purification, FACS, filtration, and antibody depletion. In certain embodiments, the erythrocyte purification scheme described in U.S. Patent Application Publication No. US2009/0124566 can be used to isolate erythrocytes. In some embodiments, erythrocytes are substantially pure and are substantially free from platelets, leukocytes, or reticulocytes. Thus, in some embodiments, the percentage of reticulocytes found within the purified erythrocyte population comprises less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, or lower of the total cell population.

Total RNA can be isolated from the purified population of erythrocytes. General methods for RNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999. In particular, RNA isolation can be performed using the mirVana microRNA isolation kit, which is commercially available from Ambion (Austin, TX), according to the manufacturer's instructions. This kit allows the capture of RNAs as small as 10 nucleotides. In some embodiments, the isolated total RNA can be size-fractionated using methods known in the art to enrich the population of RNAs for RNAs of a small size. In certain embodiments, the isolated (and in some embodiments, size- fractionated) RNA population is enriched for RNAs that have a length of less than 400 bp, less than 300 bp, less than 200 bp, less than 100 bp, less than 50 bp, less than 40 bp, less than 30 bp, less than 25 bp, or less than 20 bp.

Isolated RNA can be used in hybridization or amplification assays that include, but are not limited to, PCR analyses and probe arrays. One method for the detection of RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to the miRNA being detected. The nucleic acid probe can be of sufficient length and specificity to specifically hybridize under stringent conditions to a miRNA of the present invention, or any derivative DNA or RNA. Hybridization of a miRNA with the probe indicates that the miRNA in question is present. In one embodiment, the miRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probes are immobilized on a solid surface and the RNA is contacted with the probes, for example, in an array. A skilled artisan can readily adapt known miRNA detection methods for use in detecting the level of miRNAs useful for the present invention.

An alternative method for determining the level of a miRNA in a sample involves the process of nucleic acid amplification, for example, by RT-PCR (U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. ScL USA 88: 189-93), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. ScL USA 87:1874-78), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. ScL USA 86:1173-77), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1 197), rolling circle replication (U.S. Pat. No. 5,854,033), each of which is herein incorporated by reference in its entirety, or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, miRNA levels are assessed by quantitative RT-PCR. In some embodiments, a multiplexing quantitative PCR assay, such as a stem- loop RT-PCR assay, such as that described previously (Lao et al. (2006) Biochem. Biophys. Res. Commun. 343:85-89, which is herein incorporated by reference in its entirety) and elsewhere herein (see Experimental Example 1), is used to assess the levels of miRNAs. For PCR analysis, well known methods are available in the art for the determination of primer sequences for use in the analysis.

Thermal cyclers are often employed for the specific amplification of polynucleotides. The cycles of denaturation, annealing and polymerization for PCR may be performed using an automated device, typically known as a thermal cycler. Thermal cyclers that may be employed are described elsewhere herein as well as in U.S. Pat. Nos. 5,612,473; 5,602,756; 5,538,871 ; and 5,475,610, the disclosures of which are herein incorporated by reference.

MicroRNA microarrays provide one method for the simultaneous measurement of the expression levels of multiple miRNAs. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, for example, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316, each of which is herein incorporated by reference in its entirety. High-density oligonucleotide arrays are particularly useful for determining the miRNA expression profile for a large number of miRNAs in a sample.

Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, for example, U.S. Patent No. 5,384,261, which is herein incorporated by reference in its entirety. Although a planar array surface is generally used, the array can be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays can be nucleic acids on beads, gels, polymeric surfaces, fibers (such as fiber optics), glass, or any other appropriate substrate. See, for example, U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of which is herein incorporated by reference in its entirety. Arrays can be packaged in such a manner as to allow for diagnostics or other manipulation of an all- inclusive device. See, for example, U.S. Pat. Nos. 5,856,174 and 5,922,591 , each of which is herein incorporated by reference in its entirety.

In a specific embodiment of the microarray technique, oligonucleoties with sequences that are complementary to miRNAs are applied to a substrate in a dense array. The microarrayed oligonucleotides, immobilized on the microchip, are suitable for hybridization under stringent conditions, Fluorescently labeled miRNAs can be generated through incorporation of fluorescent nucleotides using any method known to one of skill in the art. In particular embodiments, the miRNAs are labeled using the mirVana miRNA labeling kit that is commercially available from Ambion and amine-reactive dyes according to the manufacturer's instructions. Alternatively, in other embodiments, the miRNAs are labeled with the mercury LNA Array Labeling Kit from Exiqon (Vedbaek, Denmark) according to the manufacturer's instructions. Labeled miRNAs applied to the chip hybridize with specificity to each spot comprising a complementary oligonucleotide on the array. After stringent washing to remove non-specifϊcally bound RNA, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.

With dual color fluorescence, separately labeled miRNAs generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the miRNAs from the two sources corresponding to each specified miRNA is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of miRNAs. Such methods have been shown to have the sensitivity required to detect low levels of miRNA, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al (1996) Proc. Natl. Acad. Sci. USA 93 : 106-49). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Agilent ink-jet microarray technology. The development of microarray methods for large-scale analysis of miRNA levels makes it possible to identify the miRNA composition of erythrocyte samples from various subjects.

To aid in the interpretation of the results from a miRNA microarray, the data can be normalized. "Normalization" may be used to remove sample-to-sample variation. For microarray data, the process of normalization aims to remove systematic errors by balancing the fluorescence intensities of the two labeling dyes. The dye bias can come from various sources including differences in dye labeling efficiencies, heat and light sensitivities, as well as scanner settings for scanning two channels. Some commonly used methods for calculating normalization factor include: (i) global normalization that uses all miRNAs on the array; (ii) normalization RNA normalization, wherein the normalization RNA is constantly expressed; and (iii) internal controls normalization that uses known amount of miRNAs added during hybridization (Quackenbush (2002) Nat. Genet. 32 (Suppl.), 496-501). In one embodiment, the miRNAs disclosed herein can be normalized to at least one normalization RNA, which is an RNA whose level is constant and abundantly expressed across multiple tissues, such as U6 snoRNA. In certain embodiments, the normalization RNA is a miRNA whose level does not change throughout erythroid differentiation, including but not limited to hsa-miR-152. It will be understood by one of skill in the art that the methods disclosed herein are not bound by normalization to any particular normalization RNA, and that any suitable normalization RNA known in the art can be used. In some embodiments, the data is normalized to the geometric mean of a set of multiple normalization RNAs.

Similarly, the level of a particular miRNA can be measured using a real-time RT-PCR assay, such as the stem-loop RT-PCR assay described in U.S. Patent Application Publication No. US2009/0124566 The real-time PCR data can also be normalized to at least one normalization gene.

The present invention also provides kits useful for determining the susceptibility of a subject to a pathogen-derived disease (e.g., malaria), for determining the severity of a pathogen-derived disease, or for monitoring the progression of a pathogen-derived disease. These kits comprise reagents (e.g., primers) sufficient for the detection of at least one of hsa-miR-451, hsa-raiR-223, hsa-miR-92, hsa-miR-25, hsa-miR-15a, hsa- miR-15b, hsa-let-7i, hsa-miR-126, and hsa-miR-122.

In another embodiment, the kit comprises a set of oligonucleotide primers sufficient for the detection and/or quantitation of at least one of the miRNAs listed above. The oligonucleotide primers may be provided in a lyophilized or reconstituted form, or may be provided as a set of nucleotide sequences. In one embodiment, the primers are provided in a microplate format, where each primer set occupies a well (or multiple wells, as in the case of replicates) in the microplate. The microplate may further comprise primers sufficient for the detection of one or more normalization RNAs as discussed infra. The kit may further comprise reagents and instructions sufficient for the amplification of miRNAs.

The present invention also contemplates methods for treating a pathogen- derived disease in a subject in need thereof by administering a polynucleotide comprising or encoding a microRNA capable of inhibiting the growth of the intracellular pathogen. In some embodiments, the subject is administered a polynucleotide comprising or encoding a microRNA that fuses to a polynucleotide of the intracellular pathogen and inhibits its translation, leading to an inhibition of growth or differentiation of the pathogen.

By subject or patient is intended an animal, including a mammal, such as a human, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use. In particular embodiments, the subject is a human.

As used herein, the terms "treatment" or "prevention" refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a particular infection or disease or sign or symptom thereof and/or may be therapeutic in terms of a partial or complete cure of an infection or disease and/or adverse effect attributable to the infection or the disease. Accordingly, the method "prevents" (i.e., delays or inhibits) and/or "reduces" (i.e., decreases, slows, or ameliorates) the detrimental effects of a disease or disorder in the subject receiving the compositions of the invention. Therefore, the subject may have the pathogenic disease or may be particularly susceptible, which could be determined using the methods disclosed herein.

In some embodiments, the pathogen-derived disease comprises malaria. In some of these embodiments, the subject is administered a polynucleotide that comprises or encodes a nucleotide sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, or 17, wherein the polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth of a malaria parasite infecting the host ceil or reducing the infection of the host cell by a malaria parasite. An active fragment of the recited sequences that is capable of differentiating or inhibiting the growth or infection rate of a malaria parasite can also be used, including fragments comprising at least 5 nucleotides of the recited sequences (SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17), including about 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, and up to the full-length of the recited sequence. In some embodiments, the active fragment of the recited sequences comprises the first 7 nucleotides of the recited sequences. In certain embodiments, the heterologous polynucleotide comprises the consensus sequence CXGUU (SEQ ID NO; 18; wherein X can be any nucleotide). In some of these embodiments, the polynucleotide comprises CCGUU (SEQ ID NO: 19). In other embodiments, the polynucleotide comprises CAGUU (SEQ ID NO: 20). The sequence set forth in SEQ ID NO: 18, 19, or 20 can be comprised within a polynucleotide of about 19 to about 25 nucleotides in length, and in some embodiments, can impart the ability of the polynucleotide to differentiate or inhibit the growth of a malaria parasite. In some embodiments, the promotion of parasitic differentiation or growth inhibition or reduction in the infection rate is due to the ability of the polynucleotide or expression product thereof to reduce the expression of at least one parasitic gene, wherein the reduction leads to the differentiation or growth inhibition or reduction in infection rate. In some of the embodiments, the reduction in expression is through the fusion of the polynucleotide with a parasitic mRNA and subsequent translational inhibition,

In specific embodiments, a subject is administered a polynucleotide comprising or encoding a nucleotide sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 (hsa-miR-15a), wherein the polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth of a malaria parasite infecting the host cell. An active fragment of the recited sequences that is capable of differentiating or inhibiting the growth of a malaria parasite infecting the host cell, including fragments comprising at least 5 nucleotides of the recited sequence (SEQ ID NO: 7), including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, and up to the full-length of the recited sequence. In some embodiments, the active fragment of the recited sequence comprises the first 7 nucleotides of the recited sequences. Without being bound to any particular theory or mechanism of action, preliminary observations suggest hsa-miR-15a does not function to reduce the expression of a pathogenic gene by fusing to target RNA molecules, but rather functions through the traditional miRNA-mediated translational inhibition pathway involving the binding of the miRNA to a complementary sequence within the pathogen RNA transcript and subsequent translational inhibition mediated by the RISC complex. In certain embodiments, the polynucleotide or an expression product thereof comprises a free 3' hydroxyl group.

In alternative embodiments, a subject can be administered a polynucleotide comprising or encoding a mosquito miRNA wherein the polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth or reducing the infection rate of a malaria parasite (e.g., P. falciparum, P. vivax, P. ovales, P. malariae) or is capable of reducing the infection of the host cell by a Plasmodium. In some of these embodiments, the polynucleotide that is administered to the subject comprises or encodes a nucleotide sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of aga-Let-7 (SEQ ID NO: 14), aga-miR-1174 (SEQ ID NO: 15), aga-miR-12 (SEQ ID NO: 16), or aga-miR-989 (SEQ ID NO: 17) microRNAs, wherein the polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth of a malaria parasite infecting the host cell or is capable of reducing the infection of the host cell by a malaria parasite. An active fragment of the recited sequences that is capable of differentiating or inhibiting the growth of a malaria parasite or is capable of reducing the infection of a host cell by a malaria parasite can also be used, including fragments comprising at least 5 nucleotides of the recited sequences (e.g., SEQ ID NO: 14, 15, 16, or 17), including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, and up to the full-length of the recited sequence. In some embodiments, the active fragment of the recited sequences comprises the first 7 nucleotides of the recited sequences. Delivery of a therapeutically effective amount of a polynucleotide can be obtained via administration of a pharmaceutical composition comprising a therapeutically effective dose of the polynucleotide. By "therapeutically effective amount" or "dose" is meant the concentration of a polynucleotide that is sufficient to elicit the desired therapeutic effect. As used herein, "effective amount" is an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times.

The effective amount of the polynucleotide will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount can include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the polynucleotide, and, if desired, the adjuvant therapeutic agent being administered along with the polynucleotide. Methods to determine efficacy and dosage are known to those skilled in the art. See, for example, Isselbacher et al. (1996) Harrison 's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference.

Toxicity and therapeutic efficacy of polynucleotides can be determined by standard pharmaceutical methods in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population).

The dose ratio between toxic (e.g., immunotoxic) and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Polynucleotides which exhibit high therapeutic indices are preferred. While polynucleotides that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such polynucleotides to the site of the affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the polynucleotide lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the presently disclosed methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the formulation which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

The pharmaceutical formulation can be administered at various intervals and over different periods of time as required, e.g., multiple times per day, daily, every other day, once a week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, and the like. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, disorder, or unwanted condition, previous treatments, the general health and/or age of the subject, and other diseases or unwanted conditions present. Generally, treatment of a subject can include a single treatment or, in many cases, can include a series of treatments.

One of ordinary skill in the art upon review of the presently disclosed subject matter would appreciate that the presently disclosed pharmaceutical compositions can be administered directly to a cell, a cell culture, a cell culture medium, a tissue, a tissue culture, a tissue culture medium, and the like. When referring to the polynucleotides of the invention, the term "administering," and derivations thereof, comprises any method that allows for the polynucleotide to contact and gain entry into a cell. The presently disclosed polynucleotides can be administered to (or contacted with) a cell or a tissue in vitro or ex vivo. The presently disclosed polynucleotides also can be administered to (or contacted with) a cell or a tissue in vivo by administration to an individual subject, e.g., a patient, for example, by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial administration) or topical application, as described elsewhere herein.

The present invention provides pharmaceutical compositions that can be used for the treatment of pathogen-derived diseases. The polynucleotides described herein are useful in mammalian tissue culture systems, in animal studies, and for therapeutic purposes. The presently disclosed compositions can be formulated for delivery, i.e., administering to the subject, by any available route including, but not limited, to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, opthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes. In some embodiments, the route of delivery is intravenous, parenteral, transmucosal, nasal, bronchial, vaginal, and oral. The presently disclosed pharmaceutical compositions include a polynucleotide with a pharmaceutically acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds also can be incorporated into the compositions.

Pharmaceutical compositions for the treatment of a pathogen-derived disease include those comprising a pharmaceutical carrier and a polynucleotide comprising or encoding a miRNA that is capable of differentiating or inhibiting the growth of an intracellular pathogen infecting a host cell or reducing the infection rate of the host cell by the pathogen. In some embodiments, the pharmaceutical composition comprises a polynucleotide comprising or encoding a nucleotide sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, or 17, wherein the polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth of a pathogen infecting the host cell or reducing the infection rate of the host cell by a pathogen. An active fragment of the recited sequences that is capable of differentiating or inhibiting the growth or infection rate of a pathogen can also be used, including fragments comprising at least 5 nucleotides of the recited sequences (SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17), including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, and up to the full-length of the recited sequence. In some embodiments, the active fragment of the recited sequences comprises the first 7 nucleotides of the recited sequences. In certain embodiments, the microRNA comprises the consensus sequence CXGUU (SEQ ID NO: 18; wherein X can be any nucleotide). In some of these embodiments, the sequence comprises CCGUU (SEQ ID NO: 19). In other embodiments, the sequence comprises CAGUU (SEQ ID NO: 20). The sequence set forth in SEQ ID NO: 18, 19 or 20 can be comprised within a polynucleotide of about 19 to about 25 nucleotides in length, and in some embodiments, can impart the ability of the polynucleotide to differentiate or inhibit the growth of a pathogen.

In some embodiments, the promotion of pathogenic differentiation or growth inhibition or reduction in infection rate is due to the ability of the polynucleotide or expression product thereof to reduce the expression of at least one pathogenic gene, wherein the reduction leads to the differentiation or growth inhibition or reduction in infection rate. In some of the embodiments, the inhibition is through the fusion of the polynucleotide with a pathogenic mRNA and subsequent translational inhibition.

In certain embodiments, the pathogen comprises a Plasmodium (e.g., P. falciparum, P. vivax, P, ovales, P. malariae). The host cell can comprise, for example, an erythrocyte or a hepatocyte and the host can be, for example, a human or a mosquito.

Additional pharmaceutical compositions provided by the presently disclosed subject matter include those comprising a pharmaceutical carrier and a polynucleotide comprising or encoding a mosquito rm^'RNA wherein the polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth of a malaria parasite (e.g., P, falciparum, P. vivax, P. ovales, P. malariae) or is capable of reducing the infection of the host cell by a Plasmodium are provided. In some of these embodiments, the polynucleotide comprises or encodes a nucleotide sequence having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of aga-Let-7 (SEQ ID NO: 14), aga-miR-1174 (SEQ ID NO: 15), aga-miR-12 (SEQ ID NO: 16), or aga-miR-989 (SEQ ID NO: 17) microRNAs, wherein the polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth of a malaria parasite infecting the host cell or is capable of reducing the infection of the host cell by a malaria parasite. An active fragment of the recited sequences that is capable of differentiating or inhibiting the growth of a malaria parasite or is capable of reducing the infection of a host cell by a malaria parasite can also be used, including fragments comprising at least 5 nucleotides of the recited sequences (e.g., SEQ ID NO: 14, 15, 16, or 17), including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, and up to the full-length of the recited sequence. In some embodiments, the active fragment of the recited sequences comprises the first 7 nucleotides of the recited sequences. In some embodiments, the polynucleotide or an expression product thereof comprises a free 3' hydroxyl group.

As one of ordinary skill in the art would appreciate, a presently disclosed pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). The composition should be sterile and should be fluid to the extent that easy syringability exists. In some embodiments, the pharmaceutical compositions are stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars, polyalcohols, such as manitol or sorbitol, or sodium chloride are included in the formulation. Prolonged absorption of the injectable formulation can be brought about by including in the formulation an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., polynucleotide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. In certain embodiments, solutions for injection are free of endotoxin.

Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In those embodiments in which sterile powders are used for the preparation of sterile injectable solutions, the solutions can be prepared by vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions also can be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically or cosmetically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient, such as starch or lactose, a disintegrating agent, such as alginic acid, Primogel, or corn starch; a lubricant, such as magnesium stearate or Sterotes; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring. Compositions for oral delivery can advantageously incorporate agents to improve stability within the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the presently disclosed compositions can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Liquid aerosols, dry powders, and the like, also can be used.

Systemic administration of the presently disclosed compositions also can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds also can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical or cosmetic carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Guidance regarding dosing is provided elsewhere herein.

Depending on the route of administration, the agent may be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the agent. For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

To administer an agent by other than parenteral administration, it may be necessary to coat the agent with, or co-administer the agent with, a material to prevent its inactivation. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in- water emulsions as well as conventional liposomes (Strejan et al. (1984) J Neuroimmunol 7:27).

A polynucleotide can be injected directly as naked DNA or RNA, by infection using defective or attenuated retrovirals or other viral vectors, or can be coated with lipids or cell-surface receptors or transfecting agents, encapsulated in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu (1987) J Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors) and so on. In another embodiment, polynucleotide-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the polynucleotide to avoid lysosomal degradation. In yet another embodiment, the polynucleotide can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor. Alternatively, the polynucleotide can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies (1989) Proc. Natl. Acad. Sci. USA

86:8932-8935; Zijistra et al. (1989) Nature 342:435-438).

The presently disclosed subject matter also provides for methods for identifying at least one miRNA of a host organism that is capable of translocating into an intracellular pathogen infecting a cell within said host organism and fusing with at least one mRN A of said intracellular pathogen to produce a cross-species chimeric mRNA.

The steps of the method comprise comparing the sequence of pathogenic mRNAs to pathogenic genomic sequences (for example, the PlasmoDB comprising the P. falciparum genome) to identify pathogenic mRNAs having an unalignable 5' untranslated region (UTR; for which the genomic sequence encoding the mRNA does not comprise the complementary sequence to the 5' UTR of the pathogenic mRNA).

The sequence of the pathogenic unalignable 5' UTR is then used to screen a database of mature microRNA sequences of the host organism (for example, the miRBase database;

Griffiths- Jones et al. (2008) Nucleic Acids Res 36:D154-158) to identify mature microRNA sequences of the host organism with an identical or similar (e.g., 1 nucleotide difference, 2 nucleotide difference, 3 nucleotide difference, 4 nucleotide difference) sequence, signifying that the host microRNA is capable of fusing with the pathogenic mRNA having the unalignable 5'UTR.

Alternatively, the presently disclosed subject matter also provides for methods of identifying RNA transcripts of an intracellular pathogen that are fused to a host miRNA to generate a cross-species chimeric RNA molecule. This assay is referred to herein as the "in vivo capture assay." The method comprises the steps of: a) providing an oligonucleotide having the nucleotide sequence of a host miRNA and a tag on the 5' end of the oligonucleotide; b) introducing the oligonucleotide into a host cell; c) infecting the host cell of step b) with an intracellular pathogen; d) extracting RNA from the pathogen in said host cell; e) capturing the oligonucleotide via the tag from the extract of RNA from the pathogen; f) eluting the captured oligonucleotide; and g) determining the sequence of the at least one pathogenic target RNA fused to the captured oligonucleotide to identify the pathogenic target RNA.

In some embodiments, the intracellular pathogen comprises a Plasmodium (e.g., P. falciparum, P. vivax, P. ovales, P. malariae), In certain embodiments, the host cell comprises an erythrocyte or a hepatocyte. In particular embodiments, the host is a human or a mosquito.

The oligonucleotide comprises a tag to facilitate the capture of the target RNAs. As used herein, a "tag" comprises a molecule that can be used to capture a molecule attached to the tag (e.g., through immobilization to a solid support). In some embodiments, the tag is a molecule that can reversibly bind to another molecule (that, in some embodiments, is bound to a solid support). In particular embodiments, the tag comprises biotin, desthiobiotin, or an analog thereof capable of binding to molecules such as avidin, streptavidin, neutravidin, or an analog thereof. In some of these embodiments, the oligonucleotide is captured by contacting a solid support comprising streptavidin or an analog thereof with the extract of RNA from the pathogen to immobilize the oligonucleotide on the solid support. In particular embodiments, the immobilized oligonucleotide can then be eluted with biotin or an analog thereof.

The sequence of the pathogenic RNA can be determined using any method known in the art, including but not limited to, polymerase chain reaction or microarray analysis. All of the pathogenic RNAs eluted from the captured oligonucleotides can be added to a microarray comprising oligonucleotides having a nucleotide sequence that is complementary to RNA expressed by the pathogen.

It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a polynucleotide" is understood to represent one or more polynucleotides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

Throughout this specification and the embodiments, the words "comprise," "comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

As used herein, the term "about," when referring to a value is meant to encompass variations of, in some embodiments ± 50%, in some embodiments ± 40%, in some embodiments ± 30%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ± 1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1 : Erythrocytic microRNAs as genetic determinants of malaria resistance in sickle cell diseases. An abundant and diverse population of erythrocytic microRNAs were previously identified, whose expression was dramatically altered in HbSS erythrocytes (Chen et al. (2008) PLoS ONE 3:e2360). Since significant material exchange (Kirk (2001) Physiol Rev 81 :495-537; Deitsch et al. (2001) Nucleic Acids Res 29:850-853) occurs between the host and P. falciparum during the intraerythrocytic developmental cycle (IDC), the altered miRNA profile in HbSS erythrocytes may contribute to malaria resistance. To examine this possibility, it was first determined whether erythrocytic miRNAs are present in the parasite during the IDC. RNA >10 nt was obtained from both uninfected erythrocytes and synchronized parasites at 8 and 32 hours post- invasion (Chen et al. (2008) PLoS ONE 3:e2360). The RNA from uninfected erythrocytes and isolated erythrocyte -free parasites was assayed by a multiplexed miRNA real-time assay capable of analyzing up to 336 individual miRNAs (Taqman® Low Density Array (TLDA), detailed in Methods). The specificity and dynamic range (Chen et al. (2005) Nucleic Acids Res 33:el79) of the real-time assay are ideal for detecting human miRNAs amidst P. falciparum RNA. The presence of -90 human miRNAs was detected in parasites at various points of the IDC (Table 1). Table 1. Relative expression level of human microRNAs in uninfected red blood cells (RBCs)₅ and in P. falciparum 8 hour and 32 hour post infection.

Since no standard miRNA controls are presently validated for P. falciparum, the abundance of individual human miRNAs were normalized as a percentage of the total human miRNA pool found in the parasite (Fig. IA). Of the 11 most abundant human miRNAs in uninfected normal erythrocytes, three miRNAs (miR-451, miR-223, miR-19b) were significantly enriched in parasite versus uninfected erythrocytes. The presence of miR-451 and several other human miRNAs in P. falciparum has been reported previously (Rathjen et al. (2006) FEBS Lett 580:5185-5188; Xne et aL (2008) Malar J 7:47). Interestingly, compared to normal erythrocytes (HbAA), miR-451 and miR-223 levels were elevated in 100% and 42 % of HbSS erythrocytes, respectively (Chen et al. (2008) PLoS ONE 3:e2360). While intraparasitic miR-223 and miR-19b levels increased at both 8 and 32 hours post-infection, the dramatic increase in miR-451 was only seen at the later time point, in both TLDA and additional single miRNA real- time assays (Fig. IB). There was also a higher level of miR-451 when the cell number was normalized between HbSS and HbAA erythrocytes (data not shown). Additionally, transfection of miR-451 into HbAA erythrocytes via electroporation resulted in a higher level of miR-451 in approximately 50% of erythrocytes and led to the intraparasitic accumulation of miR-451 to levels comparable to those observed in parasites grown in HbSS erythrocytes (Fig. 1C and ID). When similar lysis and RNA purification methods were applied to uninfected HbAA and HbSS erythrocytes, <20% of miR-451 in erythrocytes was recovered (Fig. 2A). To further confirm that miRNAs found within the parasite did not result from erythrocyte contamination, saponin lysis- purified parasites and lysed erythrocyte membrane ghosts were treated with RNAse A to remove extracellular RNA, RNAse A treatment decreased the miR-451 level by

95% in uninfected erythrocytes (Fig. 2B) and only by 16-25% in purified parasites (Fig. 2C). RNAse A treatment had minimal effect on levels of parasite 18S ribosomal RNA (Fig. 2D). Taken together, these data strongly suggest that miR-451 and other erythrocytic microRNAs are robustly transported into the parasite. It was then examined whether increased miRNA levels could directly affect parasite growth in synchronized parasite culture. Over the course of 10 and 20 days, transfection of erythrocytes with miR-451 reduced parasitemia by 18% (p=0.0006, n=24) and 25% (p<0.0001 , n=24), while miR-223 reduced parasitemia by 23% (p=0.0002, n=24) and 30% (pO.OOOl, n=24), respectively (Fig. 3A). Moreover, an 8-day hypoxanthine incorporation assay revealed that elevated levels of either miR-451 or miR-223 decreased parasite proliferation by -25% (p=0.006, n=4) (Fig. 3B). This effect was specific to miR-451 and miR-223, as transfection of miR-181 or a ssDNA control showed no significant effect on either parasitemia or proliferation (Fig. 3A and 3B). This effect is unlikely to be due to altered parasite invasion, since the infection rate after the first invasion cycle was nearly identical in all erythrocyte treatments.

Given the anti-plasmodial activities of miR-451 and miR-223, their high expression in HbSS erythrocytes (Chen et al (2008) PLoS ONE 3:e2360)may directly contribute to malaria resistance. Transfection of miR-451 -specific 2'-O-methyl antisense oligonucleotides (Hutvagner et al. (2004) PLoS Biol 2:E98) into HbSS erythrocytes strongly reduced intraparasitic miR-451 levels (Fig. 3C). Using this tool, the effect of inhibiting specific miRNAs on malaria resistance of HbSS erythrocytes was deterimined. Inhibition of miR-451 , but not miR-181 , in HbSS erythrocytes led to a 2.1 fold increase (p=0.0005, n=18) in parasite growth rate by day 6 (Fig. 3D). Although inhibition of miR-223 alone did not significantly affect parasite growth, simultaneous inhibition of both miR-451 and miR-223 increased parasite growth ~3- fold (pO.OOOl, n=5), reaching 50% of the rate observed in HbAA erythrocytes (Fig. 3D). These results indicate that higher levels of miR-451/-223 in HbSS erythrocytes are an important determinant of malaria resistance. Mammalian miRNAs typically regulate their target mRNAs through non- canonical base-pairing with the 3'UTR, as enabled by a riboprotein complex composed of Dicer and Ago proteins (Bartel (2004) Cell 1 16:281-297). Since P. falciparum lacks orthologues for Dicer/Ago (Hall et al. (2005) Science 307:82-86), it is unclear how human miRNAs might affect parasites. While no sequences in the P. falciparum genome were found to have significant homology to miR-451 , we identified 12 P. falciparum ESTs whose 5' ends are identical/nearly identical to miR-451 via BLAST (Table 2).

Table 2. Cross-species chimeric RNA molecules comprised of human miRNA and P. falciparum RNA

CO

Oi

The miR-451 sequence is contiguous via its 3' end with several P. falciparum sequences, including 28S rRNA and several protein coding genes. Two P. vivax ESTs also have miR-451 -like sequences at their 5' end. A further systematic search was performed by aligning all 77783 P. falciparum ESTs from PlasmoDB (v.5.4) (Bahl et al. (2003) Nucleic Acids Res 31:212-215) against the P. falciparum genome to identify unalignable 5' EST regions. Unaligned 5' ends were then searched for matches to any of the 838 unique, mature human miRNAs in miRBase (release 11.0) (Griffiths-Jones et al. (2008) Nucleic Acids Res 36:D154-158). In total, we identified 7 human miRNAs (miR-451, miR-92, let-7a, miR-15b, miR-26b miR-15a, and let-7i) in 19 P. falciparum ESTs, of which miR-451 was found most frequently (Table 2). These data indicate that human miRNAs likely join to the 5' end of P. falciparum transcripts to form chimeric (human/parasite) fusion RNAs.

To determine whether these putative chimeric ESTs represent authentic P. falciparum transcripts, parasite RNAs were reverse transcribed and amplified with primers encoding the sequences of miR-451 and three P. falciparum ESTs

(XPF2nO413_s XPF2n3060, and AU086436). Indeed, all three chimeric transcripts (XPF2nO413 - 28S rRNA, XPF2n3060 -c AMP -dependent kinase regulatory subunit (PKA-R), and AU086438 - phosphoethanolamine N-methyltransferase (PEAMT)) amplified and were confirmed by sequencing. The percentage of miR-451 -modified transcripts was determined via real-time

PCR comparing the amplification products of primers targeting miR-451 (chimeric) to primers targeting only the P. falciparum sequence (total) (Methods). In normal HbAA erythrocytes, 4.7% (±0.53) of PKA-R mRNAs, 0.14% (±0.04) of 28S rRNAs, and 0.36% (±0.05) of PEAMT mRNAs were modified by miR-451 (Fig. 4B). Parasites grown in HbSS erythrocytes displayed higher percentages of miR-451 -modified transcripts: up to 37.1% (±0.55) for PKA-R, 1.26% (±0.12) for 28S rRNA, and 4.47% (±0.28) for PEAMT. We also investigated whether the degree of miR-451 modification could be altered by manipulating the amount of erythrocytic miR-451. Indeed, parasites grown in miR-451-transfected erythrocytes showed an increase in 5' miR- 451 -modified transcripts (14.9% ±0.80 for PKA-R, 0.37% ± 0.11 for 28S rRNA and 0.62%±0.08 for PEAMT) (Fig. 4B). Moreover, transcripts from parasites grown in HbSS erythrocytes where miR-451 is inhibited showed decreased 5 'modification versus untreated HbSS erythrocytes (24.2% ±2.71 for PKA-R, 0.59% ± 0.15 for 28S rRNA, and 2.39% (±0.31) for PEAMT) (Fig. 4B). Additionally, parasite RNA was examined by Northern blot analysis using locked nucleic acid (LNA) detection probes for miR-451 and miR-181 sequences. A number of parasite transcripts >1 kb were found to react with miR-451 (Fig. 4A). One prominent band roughly co-migrated with parasite 28S rRNA. Taken together, these results demonstrate the presence of multiple miR-451 modified transcripts in parasites and that the extent of this modification is sensitive to the miR-451 level in the host erythrocyte. Since the 3' end of miR-451 was linked to P. falciparum RNAs in all identified chimeric RNAs, biotin was covalently linked to either the 5' or 3' end of miR- 451 to determine the effect of terminal biotinylation on anti-plasmodial activity and the extent of target mRNA modification. While 5' biotin-miR-451 and unmodified miR- 451 strongly reduced parasite growth while increasing 5' PKA-R modification, 3'- biotin-miR-451 was ineffective in both respects (Fig. 4B and 4C). This indicates that miR-451 requires a free 3' hydroxyl for integration with the target parasite RNAs to exert anti-plasmodial activity. To formally establish erythrocytes as the source of miR- 451 in the chimeric transcripts, we set up an in vivo capture assay using 5 '-tagged miRNAs. Since 5 '-biotinylation of miR-451 did not affect its anti-plasmodial activity, we synthesized miR-451 and miR-181 5'-tagged with desthiobiotin (Db-miR-451 and Db-miR-181), a biotin analogue that binds with less affinity to streptavidin and is easily displaced by free biotin (Hirsch et a (2002) Anal Biochem 308:343-357). Erythrocytes transfected with these oligonucleotides were infected with P. falciparum. After 4 days, parasite RNA was extracted and Db-miR-451 and Db-miR-181 -tagged transcripts were captured with streptavidin and eluted using biotin. It was found that while an untargeted transcript (18S rRNA) was present at similar levels in all samples, the target RNAs (28S rRNA, PKA-R, and PEAMT) were all highly enriched only in the RNA captured by Db-miR-451 (Fig. 4D). Similar levels of Db-miR-451 and Db-miR- 181 were recovered from electroporated erythrocytes, suggesting this enrichment is specific to miR-451 and not due to differences in available miRNA (Fig. 5A). Moreover, the addition of excess Db-miR-451 after parasite lysis did not change the enrichment levels for both tested target RNAs, further indicating that the miRNA-RNA ligation occurs in vivo, within the parasite (Fig. 5B and 5C).

In additional studies, P. falciparum were grown in human erthrocytes and then the P. falciparum mRNA was sequenced in an unbiased manner using technology from Illumina (San Diego, CA). Figure 15 presents those chimeric sequences that were identified and comprise sequence from both human miR-451 and the P. falciparum PKA-R gene. Interestingly, the sequences contained either a 6- or a 14 -bp junction sequence (which are set forth in SEQ ID NO: 42 and 43, respectively), which suggests that further RNA editing occurs during or following miR-451 fusion.

To determine whether miR-451 modification affects target RNA stability, de novo transcription was inhibited in synchronized parasites with actinomycin D (Reich et al. (1962) Proc Natl Acad Sci USA 48: 1238-1245; Shock et al. (2007) Genome Biol 8:R134) and the decay kinetics of miR-451 -modified and total transcripts were compared. The modified transcripts exhibited no significant differences in decay kinetics relative to total transcript (Fig. 6). The observed half-lives were comparable to those previously reported (Bahl et al. (2003) Nucleic Acids Res 31 :212-215). Global translation rates were unaffected in parasites grown in miR-451 enriched erythrocytes, which was not surprising since only 0.14 % of the 28S rRNA is miR-451 -modified. To determine whether miR-451 modification influenced the translation status of its target mRNAs, the ribosomal loading profiles in synchronized parastite lysates were examined. Velocity sedimentation was used to resolve the lysate ribosomal subunits (4OS and 60S), 80S monosome, and polyribosomes (Fig. 7A and 7B), but no ribosomes were detected in uninfected erythrocytes. The ribosomal occupancy of miR-451- modified versus total PKA-R transcripts was compared by determining the relative transcript levels through the gradient via real-time RT-PCR (Fig. 7C). While the majority of PKA-R transcripts were present in the polyribosome fractions, the miR- 451 -modified transcripts were largely found in the monosome and 4OS subunit fractions (Fig. 7C and 7D), suggesting that translation of miR-451 -modified PKA-R transcripts is suppressed. Analysis of PEAMT demonstrated the same effect on ribosomal loading as the PKA-R transcript (Fig. 8). Decreased PKA-R protein levels should result in increased PKA activity, which has been implied in the induction of gametocyto genesis in P. falciparum cultures (Trager and Gill (1989) J Protozool 36:451-454). Parasites grown in miR-451 -enriched erythrocytes show a 2-fold increase in sexual stage parasites by day 10, consistent with elevated PKA activity due to decreased PKA-R levels (Fig. 9). Importantly, regulation of PKA-R was recently shown to also be essential for parasite survival (Merckx et al. (2008) PLoS Pathog 4:el9). Thus, in contrast to the canonical pathway by which miRNAs have been reported to suppress translation in mammalian systems (Bartel (2004) Cell 116:281- 297), direct miR-451 modification of parasite transcripts may reduce the ribosomal loading of target RNAs through a novel mechanism. Although these findings are reminiscent of the trans-splicing processes in trypanosomatid protozoans and nematodes (Nilsen (1993) Annu Rev Microbiol 47:413- 440), it is believed the the formation of such cross-species RNA modifications as a means of gene regulation has not been previously described. Consistent with the observation that microRNAs can function as inhibitors of malaria, knockout of

Dicer 1 /Ago 1 in the mosquito Anopheles gambiae leads to an increased sensitivity to Plasmodium infection (Winter et al. (2007) Nucleic Acids Res 35:6953-6962). Therefore, the suppression of parasite growth by human miRNAs may also represent a novel mechanism of innate immunity.

Materials and Methods for Example 1 Plasmodium falciparum Culture

Plasmodium falciparum (3D7 strain) were maintained in normal human erythrocytes (type B+) according to previously published methods (Cranmer et al. (1997) Trans R Soc Trop Med Hyg 91 :363-365). RNA extraction from malaria parasites

Parasite-infected red blood cells were washed with IX PBS and lysed in 0.15% Saponin for 30 minutes on ice. The parasite pellet was washed 3 times with IX PBS and the erythrocyte membrane removed from the parasite pellet. Total parasite RNA, including small RNA >10 nt, was extracted from purified parasites using Ambion's miRVANA RNA isolation kit according to the manufacturer's protocols.

Multiplex microRNA real-time assays

Total RNA was extracted from uninfected erythrocytes and parasites 8 and 32 hours post infection. The level of the human microRNAs in these samples was determined with microRNA real-time assays on Taqman Low Density Array (TLDA) cards (Applied Biosystems) which can detect 334 human microRNAs configured in a 96-well format and spotted on a microfluidic card. Eight pools of multiplex reverse transcription were performed on RNA collected from each time point. The RT samples were loaded into the TLDA cards and run on an ABI PRISM 7900HT according to the ABI user bulletins (PN4371129 and PN4351684). Due to the lack of validated endogenous controls between infected and uninfected blood, TLDA samples were analyzed by comparing the Ct value of an individual microRNA to the total weighted Ct value for the total microRNA. Individual real-time micro RNA assays

Confirmatory assays within the parasite were performed using TaqMan microRNA assays, following the manufacturer's protocols. Input for uninfected erythrocyte assays was normalized either by loading equal amounts of RNA (100 ng per sample), or by using RNA from an equal number of cells (as counted by complete blood count) as indicated in Figures 1 and Sl . For parasite RNA samples, both Rab GTPase (PF08_0110: Rab GTPase which maintains steady expression across the lifecycle, DeRisi Lab Malarial Transcriptome Database) and 18S rRNA were used as endogenous controls. The data was recorded using the SDS 2.1 software package (ABI) and the results were quantified using the ΔCt method (Abruzzo et at. (2005) Biotechniques 38:785-792). ΔCt represents the threshold cycle (Ct) minus the endogenous control. Overexpression and inhibition of microRNA in parasites

Human erythrocytes were washed twice with RPMI and resuspended in complete cytomix at 50% hematocrit. Four hundred μl of packed RBCs (approximately 1.5xlO⁹ cells) were electroporated with 10 μg of the indicated nucleic acid oligonucleotide using a Gene Pulser II electroporator (Bio-Rad) at a setting of 310V/950 μF. The transfected erythrocytes were resuspended in complete malaria media and plated in 24 well plates. The transfection efficiency was determined using DNA oligonucleotides conjugated to FITC (FITC-TAAAGTGCTTATAGTGCAGGTAG; SEQ ID NO: 23) and flow cytometry. Transfected erythrocytes were infected with late trophozoites (15- 20% parasitemia) to an approximate final parasitemia of 1-2%, Freshly transfected erythrocytes were added every 5-6 days to the infected cultures and percent parasitemia was determined by flow cytometry using YoYo-I staining in the FL-I channel of a FACScan (Barkan et al. (2000) Int J Parasitol 30:649-653), The effects of a particular treatment were determined by examining the level of parasitemia at days 2, 6, 10, and 20 by flow cytometry (Barkan et al (2000) Int J Parasitol 30:649-653). The microRNA inhibition assays in HbSS erythrocytes

The HbSS erythrocytes were obtained at the Duke Comprehensive Sickle Cell Center (CSCC) following the approval protocol from the Duke Institution Review Board. The HbSS phenotypes are established in the CSCC with Hb electrophoresis. HbSS erythrocytes were electroporated with 2'-0-Me antisense oligonucleotides targeting particular microRNAs. The effects of the microRNA inhibition were determined by measuring the amount of parasitemia 2 and 6 days after infection. Here, infection rate was calculated as the percentage change in parasitemia per day ((D6 Parasitemia - D2 Parasitemia)/4 days), and normalized as a percentage of the HbSS growth rate.

Hypoxanthine incorporation assay Parasite proliferation in the erythrocytes transfected with the indicated ssDNA/microRNAs was assessed by ³H-hypoxanthine incorporation assay (Chulay et al. (1983) Exp Parasitol 55: 138-146). microRNA-transfected erythrocytes (2% hematocrit) were infected with synchronized trophozoite stage parasites. After 48 hours, the volume of erythrocytes (microRNA transfected, in the case of treated samples) required to attain 0.05% parasitemia in mock transfected cultures (control) was added to all cultures and then plated onto 96- well flat-bottomed microculture plates. At the same time, media containing ³H- hypoxanthine (0.5μCi/well) was added to allow incorporation. The total culture volume was maintained at 200 μl during the course of the hypoxanthine incorporation assay. Fifty percent of the media was replaced after 3 days, and after 6 days the parasites were harvested by saponin lysis. The total radioactivity within the parasite pellet was measured with a scintillation counter. Database search to identify P. falciparum ESTs containing human microRNAs

To identify P. falciparum sequences with close similarity to miR-451 , we used BLASTN to search the various databases in PlasmoDB. While a search for the P. falciparum genome and transcript database did not identify any sequences bearing similarity to miR-451, a search in the EST database led to the identification of 12 sequences. These 12 sequences were from two separate EST libraries (Watanabe et al. (2004) Nucleic Acids Res 32:D334-338). Interestingly, all miR-451 sequences are present at the extreme 5' end, forming chimeric RNAs with miR-451 fused at the 5'end to P. falciparum RNA. These ESTs mapped to the P. falciparum genomic regions, including ribosomal RNAs, as well as coding genes encoding putative cAMP- dependent protein kinase regulatory subunit (PKA-R), phosphoethanolamine N- methyltransferase (PEAMT), lipid/sterol :H+ symporter, and hypothetical proteins. In the same genome regions, there are also other ESTs, which do not bear miR-451 at their 5' end. Additional searches in other Plasmodium species further identified two ESTs from P. vivax which have miR-451 at their 5' end.

We downloaded the sequences for known human microRNAs in the miRBase database, Release 11.0 (Griffiths- Jones et al. (2008) Nucleic Acids Res 36:D154-158) from the Sanger Institute; this set included 838 unique mature microRNA sequences. We then downloaded all 77783 P. falciparum ESTs from PlasmoDB (version 5.4) and aligned them using BLAST to the P. falciparum genome to identify unalignable 5' portions of the ESTs. Unalignable 5' ends, hereafter called "overhangs", were then searched for matches to the microRNA sequences. Matches were ignored unless they occurred at the extreme 3' end of the overhang, with at most a 2 nt difference between the end of the microRNA and the end of the overhang; matches were allowed to overlap the beginning of the alignable portion of the EST by at most 2 nt. In addition to perfect matches detected in this way, we also considered "near matches" in which 1 or 2 residues differed between the microRNA and the portion of the overhang to which it was aligned. Both matches and near-matches were required to cover the full length of the mature microRNA sequence (i.e., -20 nt). We identified five microRNAs (mR451, miR-92, let-7a, miR-15b and miR-26b), which are present at the 5' end of chimeric RNAs with P. falciparum, but no other Plasmodium, RNAs (Table S2). In total, mir-451 (SEQ ID NO: 1) had the largest number of matches/near-matches, while mir-26b (SEQ ID NO: 22) had the second largest number. RT-PCR and sequence confirmation of the chimeric RNAs

The chimeric RNAs were amplified using a miR-451 forward primer and reverse primers for ESTs XPF0413, XPF2n3060, and AU086438 and the sequence was confirmed. The abundance of chimeric RNAs (XPF0413 - 28S rRNA, XPF2n3060 - PKA-R, and AU086438 - PEAMT) in the parasite samples was quantified by SYBR Green amplification using either internal or miR-451 specific primers. miR-451 tagged transcripts were amplified using a miR-451 forward primer (AAACCGTTACCATTACTGAGTT; SEQ ID NO: 24) and reverse primers for ESTs XPF0413 (TGAAC C AAC ACCTTTT ATGG; SEQ ID NO: 25), XPF2n3060 (CATAAGAACTTGTTTACTCATTTC; SEQ ID NO: 26), and AU086438 (CATCCGTATATTGATTATTTTCCA; SEQ ID NO: 27). Total transcript levels were determined using the same reverse primers along with gene specific forward primers for XPF0413 (GGCCATTTTTGGTAAGCAGAAC; SEQ ID NO: 28), XPF2n3060 (CCAAAACGGATAGTGAAATATTAG; SEQ ID NO: 29), and AU086438

(GACTTTGATTGAAAACTTAAACTCC; SEQ ID NO: 30). The RT-PCR reactions were run on an ABI 7900XT, and analyzed using SDS 2.2.1 and relative transcript levels quantified by the ΔΔCt method, RNAse A assay to assess RBC contamination Uninfected or parasite-infected erythrocytes were treated with 0.15% Saponin, incubated on ice for 30 minutes with intermittent vortexing, then washed 3 times with IX PBS. The pellet was treated with 0.5 μg RNAse in 200 μl of PBS at room temperature for 20 minutes. The RNAse was neutralized by washing the parasite pellet 3 times with IX PBS supplemented with ribonucleoside vanadyl complex (New England Biolabs) added to a final concentration of 10 raM. RNA was extracted and RT-PCR performed as previously described (for either miR-451 or 18 S rRNA). microRNA Northern blot analysis

RNA was extracted from purified parasites using Ambion's miRVANA RNA isolation kit. Five μg of total RNA from human or parasite samples was separated on a 1.2% formaldehyde-reducing agarose gel along with RNA size markers. RNA was transferred onto a charged nylon membrane overnight and blocked in ExpressHyb™ hybridization solution for 30 minutes at 42⁰C. Membranes were subsequently incubated with 10 pmol of ³²P end labeled locked nucleic acid (Exiqon) recognizing either miR- 451 or miR- 181 sequence overnight at 42⁰C. The membranes were washed at 37⁰C twice with 2X SSC/0.1% SDS, followed by two washes with 0.2X SCC/0.1% SDS. The membrane was wrapped in Saran Wrap and exposed to film for 3 hours. In vivo capture assay

The RNA oligonucleotides for miR-451 and miR-181 were synthesized with desthiobiotin covalently linked to the 5' end (Dharmacon). B+ erythrocytes were transfected with 5^r desthiobiotin miR-451, -181, or unmodified miR-451, and infected with P. falciparum (3D7). Eighty hours post-infection, parasite RNA was extracted using a miRVANA (Ambion) kit and incubated with streptavidin beads (GE Healthcare) at 4⁰C for 1 hour, After three washes under high stringency conditions (20 raM KCl), desthiobiotin 5'-miR-451 and miR- 181 and the fused P. falciparum RNA targets were eluted with 2 raM biotin overnight at 4⁰C. The enrichment of the indicated P. falciparum transcripts (18S, 28S, and PKA-R) in the eluted materials was quantified using real-time PCR as described herein.

To determine the levels of miR-181 and miR-451 available in this assay, the relevant microRNAs were transfected into uninfected erythrocytes. The electroporated erythrocytes were lysed, RNA extracted, and the capture assay performed. Subsequently, TaqMan microRNA real-time PCR was performed to determine the levels of available miR-181 and miR-451. For Figure 5B and 5C, 1.0 μg of miR-451 was added after saponin lysis, but before RNA extraction, to a matching set of samples (Db-miR-451, Db-miR-181, and miR-451-Mock), then the capture assay was performed as previously described, with levels of PKA-R and 28 S rRNA determined by real-time PCR. Parasite RNA decay kinetics

The rate of decay of miR451 -modified and total RNA was assessed at 0, 5, 10, 15, 30, 60, 90, 120, and 240 minutess after actinomycin D treatment (20 μg/ml) similar to a previous study (Shock et al. (2007) Genome Biol 8:R134). The level of miR-451- modified and total transcripts was measured by real-time PCR using primer pairs described above.

Isolation and velocity sedimentation ofribosomes

Either uninfected blood or blood with synchronized parasites grown at 2.5% hematocrit and at least 10% parasitemia was pooled and washed in 0.2 mM cycloheximide, then lysed (Stephens and Nicchitta (2007) Methods En∑ymol 431 :47- 60). Lysates were layered over a 0.5 M sucrose cushion and spun in an SW55 rotor (Beckman) for 146 min at 55,000 rpm. The supernatant and sucrose cushion was pooled and collected; the ribo some-containing pellet was re-suspended in lysis buffer. Linear 15-50% sucrose gradients were generated as previously described (Stephens and Nicchitta (2007) Methods Enzymol 431 :47-60). The ribosome suspension was layered over these sucrose gradients and spun in an SW41 rotor (Beckman) for 3 hrs at 35,000 rpm. Fractions of -330 ul were collected and absorbance at 254 ran (A₂₅₄) was continuously recorded using a Teledyne Isco according to the manufacturer's instructions. Plots of the A₂₅₄ readings of all runs fit quadratic functions of the form ax +bx+c with an R value of >0.99 (Microsoft Excel). Quadratic approximations were used to subtract out baseline values to generate normalized A₂₅₄ traces. 3⁵S-methionine translation assay

Two hundred μl of packed erythrocytes were transfected with 5 μg of miR-451 according to the previously mentioned protocol. Late stage schizonts were added to the transfected erythrocytes. Thirty-six hours post-infection, cultures were washed with methionine-free media and incubated in 5 ml of low-methionine RPMI (10%) supplemented with 0.5 mCi of ³⁵S-Methionine. Additionally, a positive control for translational inhibition with 10 μM cycloheximide was also performed. After 6 hours, parasites were isolated (saponin lysis) and lysed in RJPA buffer with 3 freeze-thaw cycles to solubilize protein. The lysates were spun at 14,000 xg and the supernatant was allowed to bind to 150 μl Stratabeads™ (Stratagene) for 2 minutes at room temperature. The beads were washed 3 times in IX PBS and protein was eluted with 250 μl Laemmli buffer. Two ml of scintillation fluid were added to 50 μl of eluted proteins, and counted in the scintillation counter. Sexual stage identification

During the overexpression assay previously described, blood smears were made on day 10 at the same point as the FACS measurement. After Giemsa staining, the number of gametocytes was counted in no less than 250 total parasites per sample (all samples measured in triplicate). The percentage of gametocytes was then calculated and normalized against the level in the mock transfected control.

Example 2: Additional miRNAs inhibit plasmodial growth

In addition to miR-451, seven other miRNAs were tested for their ability to affect parasite growth. Six of the microRNAs (miR-92, miR-25, miR-15b, miR-26b, let-7a/c, and let-7i) were found to be fused with P. falciparum RNAs and had many parasite targets using Northern blots (Fig 10A). The other miRNA (miR-15a) does not appear to tag mRNAs, but appears to have a miRNA target sequence to an niRNA. Each of the miRNAs were transfected into erythrocytes to test their ability to affect parasitic growth. As shown in Figure 1OB and Table 3, in preliminary experiments, several of these miRNAs exhibited strong anti -Plasmodial activities.

Table 3. MicroRNAs Found to Fuse to Plasmodial Transcripts and/or Inhibit Plasmodial Growth.

To further study the effect of these miRNAs on Plasmodial activity, additional assays are performed, including Giemsa staining to evaluate the progression to the ring, trophozoite, schizont and gametocyte stage as well as the percentage of infected erythrocytes, FACS analysis of YoYo-I staining to assay the level of parasitemia, [³H]hypoxanthine incorporation for DNA replication (Chulay et al. (1983) Exp Parasitol 55: 138-146), and the TUNEL assay to monitor apoptosis.

Since both miR-451 and miR-223 exhibit significant anti-Plasmodial effects, while miR-181 does not, the sequences have been compared to determine the relevant portions responsible for this activity. As an example, an anti-parasitic motif was discovered by comparing the sequences of miRNAs with (miR-451 and miR-223) or without (miR-181) anti-Plasmodial activity (Fig. 1 IA). This initial analysis, based on two chimeric miRNAs, showed that a chimeric miR-181 whose sequence at positions 4-8 has been replaced with the sequence of miR-451 at those positions can strongly suppress parasite growth while a reciprocal change of miR-451 sequence replaced by that of miR-181 fails to suppress parasite growth (Fig. 11C). This different anti- Plasmodial activity is also reflected in their ability to modify PKA-R (Fig. 1 IB).

Example 3: Liver-specific microRNAs inhibit intraervthrocytic P, falciparum growth Transfection of erthrocytes with oligonucleotides comprising the sequence of the human liver-specific miRNAs miR-122 and miR-126 led to a dramatic reduction in erythrocytic parasite growth upon infection with P. falciparum (Figure 12). While not being bound by any theory or mechanism of action, it is believed the presence of the liver-specific miRNAs provides false environmental cues and "fools" the parasites into undergoing inappropriate liver-stage specific development within the erythrocytes, ultimately leading to parasite death.

In order to determine the molecular cause of changes in parasite growth upon ectopic miRN A expression, microarrays are used. Expression levels of liver-stage specific antigens (LSA-I and LSA-3) are also measured to determine if these liver- specific miRNAs trigger the molecular features of the liver-stage specific form of P. falciparum (the sporozite).

Parasite RNA targets of these liver-specific miRNAs are identified on a genomic scale with the in vivo capturing assay described in Example 1. Briefly, the parasites are propagated in RBCs transfected with liver-specific miRNAs which have been des-biotinylated at the 5' end. P. falciparum RNAs fused to transfected des- biotinylated miRNA are purified by strepavidin pull-down and specifically eluted with biotin. The composition and abundance of these isolated P. falciparum RNAs are analyzed by P. falciparum microarrays. These newly identified human miRNA - P. falciparum RNA fusions are further confirmed, while enrichment for gene ontology categories, biological processes, 573'UTR lengths, and sequence motifs of the Plasmodium target mRNAs, are determined. Importantly, identified RNA targets are related to their anti -malaria activities to identify new P. falciparum RNA(s) and biological processes consistently altered as a result of miRNA anti-malarial activities. The RNA(s) targeted by the liver-specific miRNAs are over-expressed to determine if overexpression can rescue growth and proliferation of P. falciparum. Through this analysis, the relevant target P. falciparum pathways that are responsible for the anti- parasite activities of these two liver-specific miRNAs are identified.

Example 4: Identification of the parasite target RNAs of miR-451 and other miRNAs using microarrays

Northern blots and preliminary microarray analysis of the parasite RNAs pulled down by the miR-451 and -223 in vivo capture assays supports the existence of many parasite targets (Fig 4C and 13C), in addition to the 12 ESTs that were found to be modified by miR-451. The capture assays in conjunction with microarrays are used to determine, on a global level, the composition and abundance of all P. falciparum RNAs fused with tagged miRNAs. P. falciparum microarrays containing oligonucleotides corresponding to > 6000 P. falciparum genes are produced by the Duke Microarray Core and have been used successfully to identify parasite target RNAs for two miRNAs (Fig 13C). The asexual IDC stage is characterized by the progression through ring, trophozoite, and schizont stages with dramatic changes in gene expression (Bozdech et al. (2003) PLoS Biol 1 :E5). This changing population of potential targets presents a good opportunity to identify the sequence determinants of miR-451 and other miRNA target selection and any developmental stage-specificity inherent to the process. Des- biotinylated (Db)-miRNAs are then transfected into erythrocytes and the transfected cells are used to propagate parasites. Both the parasite total RNA as well as the RNA targets pulled down by the Db-raiRNA and mock transfected cells at 8, 16, 24, 32, and 40 hours after infection are collected. RNAs fused with Db-miRNAs are pulled down with the capture assay, amplified, labeled with Cy 5, and hybridized to microarrays together with time-course-equivalent total parasite RNA labeled with Cy3. Results are analyzed in at least two ways: firstly, the Cy5/Cy3 ratios are computed and used as measurements of target enrichment; and secondly, the relative abundance of the pulled down RNAs is ranked based on the hybridization signal of the pull down RNA alone (Cy5 only).

Once targets of miR-451, miR-223, and other miRNAs capable of 5' end modification are identified, the contribution of various features to target selection, such as UTR length, genomic location, UTR sequence composition, and function or pathway associations is determined. These features are then combined into a simple predictor such as a linear or quadratic support vector machine or logistic regression is used to determine how successfully Plasmodium targets can be predicted ab initio. This approach is analogous to previous successful sequence classification approaches, e.g., the miRNA gene finder miRscan (Ohler et al. (2004) Rna 10:1309-1322), which combined a set of 10 features in a Bayesian classifier. Overall, this work enables the determination of the intrinsic predisposition of Plasmodium genes to be miRNA targets; it also allows the comparison of features targeted by different miRNAs. With sufficiently discriminative predictors of target selection, promising novel candidates are selected for further experimental validation.

The P. falciparum RNA targets for four anti-Plasmodial miRNAs and four miR- 451 variants, two with and two without anti-Plasmodial activity (with closest sequence similarity as determined in aim 2), is determined globally by microarray analysis of the target RNAs captured with desthiobiotin-tagged synthetic RNA oligos. Each sample is performed in triplicate to control for sample-to- sample variations. Unsupervised analysis is first used to determine how these different miRNAs and miR-451 variants with or without anti-parasitic activity differ in their targets. If each mutation leads to a distinct set of targets, SAM (Tusher et al. (2001) Proc Natl Acad Sci USA 98:5116- 5121) and other software is implemented to perform supervised analysis to compare the pools of target RNAs that are consistently targeted by anti-Plasmodial miRNAs to those that are preferentially targeted by miR-451 variants with no anti-Plasmodial activity. These mRNA targets and the biological processes they represent are likely to be relevant targets through which miR-451 mediates its anti-Plasmodial activity. In addition, similar analyses are also performed on other miRNAs with or without anti- Plasmodial activity. These studies identify parasite RNAs likely to be essential for the anti-parasitic activities of miRNAs and elucidate whether different miRNAs mediate their anti-Plasmodial activity through a common or distinct subset of P. falciparum target RNAs or biological processes. The functional validation of these targets is determined by whether the over-expression of these genes can "rescue" the parasites from miRNA-mediated inhibition.

Example 5. MicroRNAs from Anopheles gambiense mosquito inhibit parasitemia of P. falciparum.

MicroRNAs from the Anopheles gambiae mosquito were tested for their ability to inhibit parasitemia of P. falciparum in human erythrocytes. Figure 14A and Figure 14B show that the aga-Let-7, aga-miR-1 174, aga-miR-12, and aga-miR-989 microRNAs are capable of reducing P. falciparum parasitemia in human erythrocytes. Preliminary results suggest that the mosquito miRNAs function by acting as a barrier to infection.

Claims

THAT WHICH IS CLAIMED:

1. A method for reducing the expression of at least one gene of a Plasmodium in a host cell, said method comprising introducing into said host cell at least one heterologous polynucleotide comprising or encoding a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 22, wherein said heterologous polynucleotide or an expression product thereof when introduced into or expressed in said host cell fuses with at least one mRNA of said Plasmodium and reduces the translation of said at least one mRNA, thereby reducing the expression of at least one gene of said Plasmodium.

2. The method of claim 1, wherein said host cell comprises an erythrocyte or a hepatocyte.

3. The method of claim 1 or claim 2, wherein said host is a human or a mosquito.

4. The method of any one of claims 1 -3, wherein said Plasmodium comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovales, or Plasmodium malariae.

5. The method of any one of claims 1-4, wherein said gene encodes cAMP- dependent kinase regulatory subunit (PKA-R) or phosphoethanolamine N- methyltransferase (PEAMT).

6. The method of any one of claims 1-5, wherein said reduction of the expression of said at least one mRNA of said Plasmodium differentiates or inhibits the growth of said Plasmodium.

7. The method of any one of claims 1-6, wherein said heterologous polynucleotide or an expression product thereof comprises a free 3' hydroxy 1 group.

8. A method for differentiating or inhibiting the growth of a Plasmodium in a host cell, said method comprising introducing into a host cell at least one heterologous polynucleotide comprising or encoding a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, or 17, wherein said heterologous polynucleotide or an expression product thereof when introduced into or expressed in said host cell reduces the expression of at least one gene of said Plasmodium, wherein said reduction of expression of said at least one gene differentiates or inhibits the growth of said Plasmodium.

9. The method of claim 8, wherein said host cell comprises an erythrocyte or a hepatocyte.

10. The method of claim 8 or claim 9, wherein said host is a human or a mosquito.

11. The method of any one of claims 8-10, wherein said Plasmodium comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovales, or

Plasmodium malar iae.

12. The method of any one of claims 8-11, wherein said gene encodes cAMP-dependent kinase regulatory subunit (PKA-R) or phosphoethanolamine N- methyltransferase (PEAMT).

13. The method of any one of claims 8-12, wherein said heterologous polynucleotide or an expression product thereof comprises a free 3' hydroxy 1 group.

14. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polynucleotide comprising or encoding a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, or 17, wherein said polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth of a Plasmodium infecting said host cell or reducing the infection of the host cell by a Plasmodium.

15. The pharmaceutical composition of claim 14, wherein said host cell comprises an erythrocyte or a hepatocyte.

16. The pharmaceutical composition of claim 14 or claim 15, wherein said Plasmodium comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovales, or Plasmodium malarias.

17. The pharmaceutical composition of any one of claims 14-16, wherein said polynucleotide or an expression product thereof comprises a free 3' hydroxyl group.

18. A method for treating malaria in a subject, said method comprising administering to said subject a pharmaceutical composition comprising a heterologous polynucleotide comprising or encoding a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, or 17, wherein said heterologous polynucleotide or an expression product thereof when introduced into or expressed in a host cell is capable of differentiating or inhibiting the growth of a malaria parasite infecting said host cell or reducing the infection of the host cell by a malaria parasite.

19. The method of claim 18, wherein said host cell comprises an erythrocyte or a hepatocyte.

20. The method of claim 18 or claim 19, wherein said malaria parasite comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovales, or

Plasmodium malariae.

21. The method of any one of claims 18-20, wherein said heterologous polynucleotide or an expression product thereof comprises a free 3' hydroxyl group.

22. A method for determining the susceptibility of a subject to malaria or for determining the severity of malaria in a subject, said method comprising obtaining a sample of erythrocytes or hepatocytes from said subject, determining the level of at least one miRNA in said erythrocytes or said hepatocytes, wherein said miRNA has the nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, 9, 12, or 13, and comparing said level to that of a control subject, wherein a decrease in the level of said at least one miRNA compared to said control subject indicates an enhanced susceptibility of said subject to malaria or a more severe malaria relative to said control subject.

23. A method for identifying at least one Plasmodium target RNA of a miRNA, wherein said miRNA fuses to said Plasmodium target RNA, said method comprising the steps of: a) providing an oligonucleotide having the nucleotide sequence of a host miRNA and a tag on the 5' end of said oligonucleotide; b) introducing said oligonucleotide into a host cell; c) infecting said host cell of step b) with a Plasmodium; d) extracting RNA from said Plasmodium in said host cell; e) capturing said oligonucleotide via the tag from the extract of RNA from said Plasmodium; f) eluting the captured oligonucleotide; and g) determining the sequence of said at least one Plasmodium target RNA fused to the captured oligonucleotide to identify said Plasmodium target RNA.

24. The method of claim 23, wherein said host cell comprises an erythrocyte or a hepatocyte.

25. The method of claim 23 or claim 24, wherein said host is a human or a mosquito.

26. The method of any one of claims 23-25, wherein said Plasmodium comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovales, or Plasmodium malariae.

27. The method of any one of claims 23-26, wherein said tag comprises biotin, desthiobiotin, or an analog thereof.

28. The method of claim 27, wherein said capturing said oligonucleotide comprises contacting a solid support comprising streptavidin or an analog thereof with said extract of RNA from said Plasmodium to immobilize said oligonucleotide on said solid support.

29. The method of claim 28, wherein said eluting the captured oligonucleotide comprises contacting said solid support with said immobilized oligonucleotide with biotin or an analog thereof.

30. The method of any one of claims 23-29, wherein determining the sequence comprises adding said eluted oligonucleotides to a microarray comprising oligonucleotides having a nucleotide sequence that is complementary to a RNA from a Plasmodium.