The invention provides a method for modulating gene expression by contacting a cellular system with a double-stranded ribonucleic acid molecule capable of associating with a regulatory machinery that controls transcription of one or more genes, wherein the association results in altered expression of the one or more genes. The invention is further directed to method for directing the differentiation of neuronal stem cells into neurons by contacting a cellular system with a double-stranded ribonucleic acid molecule capable of associating with a regulatory machinery that controls transcription of one or more genes involved in neuronal differentiation and directing the transcription of the one or more genes. In related embodiments, the invention provides particular compositions of double-stranded ribonucleic acid molecules as well as therapeutic and screening applications of the invention.

Description of the Invention

BACKGROUND OF THE INVENTION

The present invention relates generally to molecular and developmental biology and more specifically to methods of modulating gene expression and influencing cellular differentiation.

The expression of cell type-specific genes depends on both positive and negative gene expression controls, which are implemented throughout the developmental history of cells. Numerous genetic studies have provided evidence that cell type-specific activators and repressors of gene expression are essential components of the process. In addition to the conventional transcription machinery, gene expression control by small non-coding RNAs, at the post-transcriptional step, also appears to be essential to the regulation of gene expression. Non-coding RNA genes, which produce transcripts, can function directly as structural, catalytic or regulatory RNAs, rather than expressed mRNAs that encode proteins. Plants, flies, worms, mice and humans harbor significant numbers of small RNAs likely to play regulatory roles. Although most of the identified non-coding RNAs have unknown function, their sequences are typically conserved among different species, and many have intriguing expression patterns in different tissues or stages of development, pointing towards a general role for non-coding RNAs in modulating gene expression during development, such as tissue-specific patterning and cell fate specification.

The mammalian nervous system is composed of a remarkable number of different types of neurons and glia. While it has become clear over the past several years that specific genes control cellular differentiation, the molecular mechanisms by which neuron-specific gene expression is regulated in the central nervous system remain a focus of study. The regulatory mechanisms of gene expression that determine a stem cell's fate with regard to giving rise to a particular lineage remain largely unknown. Genes involved in regulating mammalian neural differentiation are just beginning to be discovered. Some important regulators identified to date include NeuroD, NeuroM, neurogenin, and the neuron restrictive silencing factor (NRSF).

Many of the genes important for neuronal differentiation and maintenance contain neuron-restrictive silencer element/Repressor Element 1 (NRSE/RE1) sequence, which is recognized by the protein neuron-restrictive silencer factor (NRSF), also known as RE-1 silencing transcription factor (REST). The maintainance of neuronal gene repression in non-neuronal cells depends on the ability of NRSF/REST to bind the NRSE sequence. To repress gene expression, NRSF/REST recruits negative transcriptional regulators such as HDACs and methyl-DNA binding proteins. Multipotent adult neural stem cells switch from actively repressing neuron-specific genes in the "stem cell state" to actively expressing neuron-specific genes in the "differentiated state" allowing the cell to proceed to become a neuron, a process that involves de-repression in order to activate transcription of genes having an NRSE. The mechanisms controlling the switch from active transcriptiponal repression to de-repression of neuron-specific genes are of great significance to the determination of cell fate.

Given the cellular diversity of the nervous system and the complexity of the underlying genetic mechanism, the understanding and treatment of nervous system disorders presents a unique challenge. The elucidation of the mechanisms that regulate the development of the nervous system and ability to modulate the regulation of genes that coordinate nervous system development is an important goal and will provide valuable information regarding the the causes and potential treatments of neurological and neurodegenerative disorders.

Thus, there exists a need to understand the processes that orchestrate gene expression within regulatory networks that coordinate cellular processes such as differentiation; to elucidate the molecular components involved in these processes; and to develop therapeutic tools for influencing cellular processes such as neuronal differentiation. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a method for modulating gene expression by contacting a cellular system with a double-stranded ribonucleic acid molecule capable of associating with the regulatory machinery that controls transcription of one or more genes, wherein the association results in altered expression of the one or more genes.

The invention is further directed to method for inducing the differentiation of neuronal stem cells into neurons by contacting a cellular system with a double-stranded ribonucleic acid molecule capable of associating with the regulatory machinery that controls transcription of one or more genes involved in neuronal differentiation and inducing neuronal differentiation.

In related embodiments, the invention provides particular compositions of ribonucleic acid molecules as well as therapeutic and screening applications of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to small non-coding double-stranded ribonucleic acid (dsRNA) molecules, termed small modulatory RNAs (smRNAs), capable of coordinating the regulation of large clusters of genes, thereby mediating cellular processes, including, for example, cell fate determination, cell differentiation, maintenance of the differentiated state, cell motility, cellular functions and apoptosis. As described herein, one example of the coordination of cellular processes by an smRNA is the stimulation of neuronal development and functions, for example, ion channels, neurotransmitter receptors and their synthesizing enzymes, receptor-associated factors, neurotrophins, synaptic vesicle proteins, growth-associated, and cytoskeletal and adhesion molecule-factors involved in axonal guidance, transport machinery, transcription factors, and cofactors.

The invention also provides particular ribonucleic acid molecules that are capable of modulating gene expression through association with the regulatory machinery, that controls transcription of one or more genes. The invention is based, in part, on the discovery of a new class of functional non-coding RNAs, termed small modulatory RNAs (smRNAs), that have primary roles in regulating gene expression at the transcriptional level. Although distinct in function from small interfering RNA molecules (siRNAs), smRNAs have approximately the same nucleotide length (21-25 bp), enabling them to diffuse in cells without limitation. A smRNA of the invention can therefore be sequestered in specific cellular compartments and can, through interaction with one or more cognate protein partner(s), mediate effects in a spatial-temporal and sequence-dependent manner of target mRNA, DNA and proteins. In particular, the presence of small double-stranded ribonucleic acid molecules provided by the invention affects the activation of genes involved in coordinating complex cellular processes.

A small non-coding smRNA of the invention can orchestrate the interactions of a large number of genes within a network. Cellular processes too complex to be coordinated by a single gene can be orchestrated by a dsRNA as described herein. Such cellular processes encompass cell fate choices at any stage of development, including initial fate determinations, for example, from oocyte to embryonic stem cell as well as early differentiation into cell lineages such as formation of the inner cell mass (ICM) and trophoblast from blastomeres. Thus, in addition to the cellular processes described herein, the skilled person will appreciate that transcriptional regulation by a smRNA of the invention can orchestrate complex cellular processes, for example, cell fate determination, differentiation, lineage determination and organ development.

Therefore, while exemplified herein with regard to the NRSE/RE1 dsRNA and the regulation of neuronal gene expression through association with the NRSF/REST transcriptional machinery, the present invention is based on the broader discovery of dsRNA-dependent gene activation, previously thought to be limited to regulation by small interfering RNA (siRNA) molecules at a post-transcriptional level, at a transcriptional level. Those skilled in the art will appreciate that the ability of small non-coding dsRNA molecules to associate directly with proteins to regulate transcription of specific genes represnets a discovery that is universal to transcriptional regulation rather than one that is unique the nervous system as exemplified herein. Therefore, the skilled person will appreciate that the transcriptional regulation by a small non-coding dsRNA described by the invention is applicable to modulation of entire gene clusters that are interconnected in a network for controlling cellular processes as well as modulation of indvidual genes. Global regulation of gene expression, which depends on the interplay within a network of genes and is orchestrated by regulatory elements and their corresponding trans-acting facors, can be modulated with a small non-coding dsRNA as described herein.

In a particular embodiment, the invention is based, in part, on the discovery that small double-stranded ribonucleic acid molecules participate in the global orchestration of gene expression, for example, neuronal gene expression, during early eukaryotic development. The presence of small double-stranded ribonucleic acid molecules provided by the invention affects the activation of genes involved in cell fate determination and, ultimately, modulates cell differentiation.

As described herein, the small non-coding double-stranded ribonucleic acid molecules provided herein have a role in coordinating the regulation of both large gene clusters and individual genes by modulating gene expression. Consequently, a small non-coding double-stranded ribonucleic acid molecule of the invention can have a global effect on a gene network as well as a focused effect on expression of one or more individual genes. As described herein, a small non-coding double-stranded ribonucleic acid molecule of the invention can facilitate neuronal differentiation by modulating the expression of genes involved in the cell fate committment of neural stem cells early in development. As demonstrated in the Examples below qualitatively by immunostaining with lineage-specific markers and, quantitatively, with promoter-driven luciferase constructs, an NRSE dsRNAs can act as an inducer of neuronal differentiation. The introduction of an NRSE dsRNA alone can be sufficient to activate NRSE/RE1-containing neuron-specific genes and induce neuronal differentiation. Furthermore, as exemplified herein by showing that introduction of a ribozyme targeted against the NRSE dsRNA has anti-neuronal differentiation effects, an NRSE dsRNA is necessary to induce neuronal differentiation. The observations described herein gave rise to the discovery that small non-coding double-stranded ribonucleic acid molecules can act as key mediators of neuronal differentiation.

Multipotent neuronal stem cells require a highly selective gene regulation system to achieve uniquely different fates. As schematically depicted in FIG. 8 (see Original Patent), during the uncommitted stem cell state, the genes required for neural differentiation are repressed, while committing to the neural lineage requires repression of stem cell, astrocyte, oligodendrocyte lineages and de-repression of the neural restricted genes. By association with the regulatory machinery consisting of the NRSE/RE1 and NRSF/REST, the non-coding double-stranded ribonucleic acid molecules of the invention which correspond to NRSE sequences modulate the expression of NRSE element-dependent genes by switching the function of NRSF/REST between repressor and facilitator of gene expression.

In one embodiment, the invention is directed to a small double-stranded ribonucleic acid molecule termed neuron-restrictive silencer element/Repressor Element 1 double-stranded RNA, referred to throughout this disclosure interchangeably as NRSE/RE1 dsRNA or NRSE dsRNA. The sequence defined by this small non-coding dsRNA is derived from NRSE/RE1, which is recognized by NRSF/REST, known primarily as a negative transcriptional regulator that restricts neuronal gene expression to neurons. As described herein, the NRSE dsRNA can trigger gene expression of neuron-specific genes through association with the NRSF/REST transcriptional regulatory machinery.

As used herein, the term "regulatory machinery" refers a transcriptional machinery consisting of at least one nucleic acid regulatory element and at least one trans-acting regulatory protein that interact and, as a result of the interaction, modulate gene expression by controlling transcription. The regulatory machinery can control transcription by further recruitment, interaction or other association with co-factor modulatory proteins. Whether transcription of one or more genes is repressed or activated can depend on the particular set of co-factor modulatory proteins that is recruited by the regulatory machinery. As described herein, a smRNA of the invention can modulate gene expression by associating with the regulatory machinery through direct or indirect interaction with the either or both of the nucleic acid and protein components of the regulatory machinery.

In one embodiment, the regulatory machinery encompasses the NRSE/RE1 nucleic acid regulatory element and the NRSF/REST trans-acting regulatory protein. In this embodiment, co-factor modulatory proteins include the histone deacetylases, in particular HDAC1 and HDAC2, and methyl-CpG binding protein (MeCP2) which form a repressive chromatin state. As described in further detail below, the NRSE dsRNA provided by the present invention associates with the regulatory machinery that encompasses the NRSE/RE1 nucleic acid regulatory element and the NRSF/REST as the trans-acting regulatory protein.

This association, which is mediated through a dsRNA/protein interaction, results in the transition from neural stem cells with neuron-specific genes silenced by NRSF/REST, into cells of neuronal identity which can express neuronal genes. The apparent gene activation effects of the NRSE dsRNA clearly distinguish it from the post-transcriptional and target gene sequence-specific gene silencing effects of cellular interfering RNAs (miRNA/siRNAs), and demonstrate a previously unknown function for non-coding RNAs at a transcriptional level.

In one embodiment, the invention is thus directed to a double-stranded ribonucleic acid molecule termed neuron-restrictive silencer element/Repressor Element 1 double-stranded RNA (NRSE/RE1 dsRNA). The present invention also provides methods of modulating the expression of known genes or known nucleic acid sequences in eukaryotic cells using nucleic acid molecules capable of forming a double-stranded RNA.

The nucleic acid molecules provided by the present invention are capable of forming an NRSE/RE1 dsRNA that can bind to a portion of a genome, for example, to a nucleic acid regulatory element located in the promoter region of a particular gene. The nucleic acid molecules of the invention also are capable of binding to both endogenous as well as exogenous nucleic acid sequences. The nucleic acid sequences corresponding to an NRSE/RE1 smRNA of the invention can be encoded by one or more nucleic acid molecules, the expression of which results in the ribonucleic acid sequences capable of forming the smRNA of the invention.

The present invention therefore relates to ribonucleic molecules capable of forming an NRSE/RE1 dsRNA, to DNA molecules encoding the ribonucleic acid molecules capable of forming a dsRNA, to vectors and cells encompassing these molecules, to compositions encompassing the molecules and vectors, and to prophylactic and therapeutic methods for administering the ribonucleic acid molecules, the deoxyribonucleic molecules and the dsRNA.

As described herein, the nucleic acid molecules and methods provided by the invention have numerous uses for mediating cellular processes, including, for example, cell fate determination, cell differentiation, maintenance of the differentiated state, cell motility, cellular functions and apoptosis. As described herein, one example of the coordination of cellular processes by a small non-coding dsRNA is neuronal development and functions, for example, ion channels, neurotransmitter receptors and their synthesizing enzymes, receptor-associated factors, neurotrophins, synaptic vesicle proteins, growth-associated, and cytoskeletal and adhesion molecule-factors involved in axonal guidance, transport machinery, transcription factors, and cofactors. It is understood that the NRSE dsRNA is one example of a small non-coding dsRNA and the NRSE is likewise one example of a nucleic acid regulatory element from which a smRNA of the invention can be derived. Those skilled in the art will appreciate that many small non-coding dsRNAs derived from different nucleic acid regulatory elements exist, which coordinate numerous cellular processes.

As a consequence of their capability to modulate gene expression, the small non-coding smRNA of the invention are useful for treating or inhibiting the onset of disorders characterized by suppression of expression of particular genes involved in cellular processes, for example, neuronal differentiation. In particular, the NRSE/RE1 smRNA of the invention has therapeutic utility for conditions characterized by aberrances in neuronal stem cell differentiation and morphogenesis.

In the central nervous system, the neuron-restrictive silencer factor (NRSF), also known as RE-1 silencing transcription factor (REST) plays a critical role as a key transcriptional repressor for neuron-specific genes in non-neuronal cells as described by Chen et al., Nature Genetics 20:136-42 (1998); Huang et al., Nature Neuroscience 2:867-72 (1999); Lunyak et al., Science 298:1747-52 (2002); Palm et al., Journal of Neuroscience 18:1280-96 (1998); Schoenherr and Anderson, Current Opinions in Neurobiology 5:566-71 (1995b); Schoenherr et al., Proc Natl Acad Sci USA 93:9881-6 (1996), each of which is incorporated herein by referewnce in its entirety. NRSF/REST is a kruppel family zinc finger protein and binds specifically to a 21- to 23-base pair (bp) conserved DNA response element (NRSE/RE1). NRSE/RE1 sequences are encoded within a broad range of genes involved in neuronal development and function, including ion channels, neurotransmitter receptors and their synthesizing enzymes, receptor-associated factors, neurotrophins, synaptic vesicle proteins, growth-associated, and cytoskeletal and adhesion molecule-factors involved in axonal guidance, transport machinery, transcription factors, and cofactors. The consensus NRSE/RE1 sequence is conserved between Xenopus, mouse, rat, chicken, sheep, and human.

The maintenance of neuronal gene repression in non-neuronal cells depends on the ability of NRSF/REST to bind the NRSE sequence. NRSF/REST mediates transcriptional repression through the association of the N-terminal repressor domain with the mSin3/histone deacetylase-1/2 (HDAC1/2) complex and through the association of C-terminal repressor domain with the CoREST complex by recruitment of MeCP2 or HDACs as described by Andres et al., Proc Natl Acad Sci USA 96:9873-8 (1999); Grimes et al., J Biol Chem 275:9461-7 (2000); Huang et al., supra, 1999; Lunyak et al., supra, 2002; Naruse et al., Proc Natl Acad Sci USA 96:13691-6 (1999); Roopra et al., Mol Cell Biol 20:2147-57 (2000), each of which is incorporated herein by referewnce in its entirety.

As described herein, the NRSF is a flexible mediator of NRSE regulatory elements. An NRSE smRNA of the invention can interact with the NRSF and exploit the role of the NRSF as a mediator of gene rexpression by causing the NRSF to switch co-factors from repressors to activators. In certain embodiments, the NRSF represents a vertebrate silencer protein that regulates a large battery of cell type-specific genes, and therefore can function as a master negative regulator of neurogenesis. Several of the genes containing the NRSE are essential for establishment and maintenance of the neuronal phenotype; these include neuron-specific cytoskeletal proteins, neurotransmitters and their biosynthetic enzymes, synaptic vesicle components, neurotrophins, and cell adhesion molecules. Perturbation of REST expression and function results in cellular apoptosis, aberrant differentiation and morphogenesis, and lethality, a further indication that NRSF function is essential for normal embryogenesis.

In one embodiment, the invention provides an isolated double-stranded nucleic acid molecule encompassing a first ribonucleic acid molecule having a sequence set forth as SEQ ID NO: 1 and a second ribonucleic acid molecule having a sequence corresponding to the complement of said first nucleic acid molecule. The nucleic acid molecule designated SEQ ID NO: 1 corresponds to the 5' to 3' strand of the NRSE dsRNA (see FIG. 3C (see Original Patent)) and has the sequence 5'-UUCAGCACCACGGACAGCGCC-3'. In this embodiment of the invention, the nucleic acid complex corresponds to an intermolecular NRSE/RE1 dsRNA that is formed from two distinct ribonucleic acid molecules having complementary sequences. The dsRNA can be formed within a cell upon introduction, or the dsRNA can be introduced into a cell as a preformed complex.

The term "nucleic acid molecule," as used herein, refers to an oligonucleotide or polynucleotide of natural or synthetic origin, including analogs such as peptide nucleic acids. A nucleic acid molecule can be single- or double-stranded genomic DNA, cDNA or RNA, and can represent the sense strand, the antisense strand, or both. A "nucleic acid complex," as used herein refers more specifically to a double-stranded nucleic acid molecule formed by intermolecular bonds between complementary bases. A nucleic acid molecule can include one or more non-native nucleotides, having, for example, modifications to the base, the sugar, or the phosphate portion, or having a modified phosphodiester linkage. Such modifications can be advantageous in increasing the stability of the nucleic acid molecule. Furthermore, a nucleic acid molecule can include, for example, a detectable moiety, such as a radiolabel, a fluorochrome, a ferromagnetic substance, a luminescent tag or a detectable binding agent such as biotin. Such modifications can be advantageous in applications where detection of a nucleic acid molecule is desired, for example, where a nucleic acid molecule of the invention is used to screen for promoter responsive elements.

The term "double-stranded ribonucleic acid molecule" or "dsRNA" refers specifically to a nucleic acid complex consisting of two complementary strands corresponding to a first and a second ribonucleic acid sequence that can have, for example, between 14 and 26 contiguous nucleotides per strand, between 15 and 25, between 16 and 24, between 17 and 23, between 18 and 22, between 19 and 21 contiguous nucleotides per strand. The term encompasses both partially or completely double-stranded molecules. A nucleic acid molecule of the invention can be a double-stranded ribonucleic acid molecule that includes a portion of sequence substantially identical to a portion of a non-coding, nucleic acid regulatory element corresponding to one or more genes, except for possessing Uracil instead of Thymine. The nucleic acid regulatory element can embedded at any location with in the genome, for example, in the promoter, intron, exon, 3' UTR, or 6' UTR.

A nucleic acid molecule comprising a double-stranded ribonucleic acid can be formed from a single nucleic acid molecule that contains the first and second ribonucleic acid sequences separated by a spacer sequence. In this embodiment of the invention, the transcription of the two ribonucleic acid sequences can be driven by a single promoter and the nucleic acid sequences are separated by a short spacer sequence. The spacer sequence can be of any length desired by the user, and can have for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more nucleotides. The sequence of the spacer can be selected based on the sequence and length of the dsRNA. For a smRNA of the invention a spacer sequence can have any length and sequence that facilitates formation of a functional dsRNA including, for example, seven nucleotides having the sequence TCAAGAG, or four nucleotides having the sequence TAAA. It is understood that depending on the sequence of dsRNA and the possible structure, the both the length and sequence of the spacer can be adjusted. If desired, the spacer sequence can further modified to include regulatory elements useful for expression. Upon expression, the resulting transcript folds back on itself to form a dsRNA stem-loop, also referred to as hairpin, structure based upon complementary base pairing of the first ribonucleic acid sequence and the second ribonucleic acid sequence, which is its reverse complement.

In a related embodiment, the two ribonucleic acid sequences are expressed off separate promoters rather than a single promoter and the term double-stranded ribonucleic acid molecule refers to a short nucleic acid complex consisting of the first ribonucleic acid sequence and a second ribonucleic acid sequence, which is its complement.

The term "isolated" in reference to a nucleic acid molecule is intended to mean that the molecule is substantially removed or separated from components with which it is naturally associated, or otherwise modified by a human hand, thereby excluding nucleic acid molecules as they exist in nature. An isolated nucleic acid molecule of the invention can be in solution or suspension, or immobilized on a filter, glass slide, chip, culture plate or other solid support. The degree of purification of the nucleic acid molecule, and its physical form, can be determined by those skilled in the art depending on the intended use of the molecule.

The term "comprising" or "containing" in reference to a nucleic acid molecule of the invention, is intended to mean that the nucleic acid molecule can contain additional nucleic acid sequences at either the 5' or 3' end of the recited sequence, or branching from an internal position within the recited sequence. The additional nucleotide sequences can, if desired, correspond to naturally occurring sequences, including promoter sequences, coding or non-coding sequences, or other untranslated regions. Alternatively, the additional nucleotide sequence can correspond to a spacer sequence that allows the first and second nucleic acid sequences to fold over in a hairpin structure so as to align by complementary base pairing. The additional sequences also can correspond to sites useful in cloning applications; to vector sequences or other nucleic acid sequences that can be useful in gene expression. Those skilled in the art can determine appropriate sequences flanking the recited nucleotide sequences for a particular application of the method.

In a further embodiment, the invention provides an isolated nucleic acid molecule encompassing a ribonucleic acid sequence set forth as SEQ ID NO: 1. The nucleic acid molecule designated as SEQ ID NO: 1 corresponds to the 5' to 3' strand of the double-stranded NRSE/RE1 ribonucleic acid molecule provided by the invention.

In a further related embodiment, the invention provides an isolated nucleic acid molecule encompassing a first ribonucleic acid sequence set forth as SEQ ID NO: 1 and a second ribonucleic acid sequence corresponding to the reverse complement of said first ribonucleic acid sequence, wherein the first and second ribonucleic acid sequences are separated by a short spacer sequence. In this embodiment, an intramolecular NRSE/RE1 dsRNA is formed from a single, linear ribonucleic acid molecule that corresponds to the isolated nucleic acid molecule. Thus, the nucleic acid molecule of this embodiment can be depicted as follows: UUCAGCACCACGGACAGCGCC-SPACER-AAGUCGUGGUGCCUGUCGCGG.

As used herein, the term "reverse complement" when used in reference to a first nucleic acid sequence refers to the complementary sequence of the first nucleic acid sequence as dictated by base-pairing, but in reverse orientation so as to result in complementarity upon fold-over into a hairpin structure. The term encompasses partial complementarity where only some of the bases are matched according to base pairing rules as well as total complementarity between the two nucleic acid sequences. The degree of complementarity between the first and second nucleic acid sequences can have significant effects on the stability and efficiency of the dsRNA.

In contrast, a complement of a first nucleic acid molecule in embodiments where an intermolecular dsRNA is formed from two distinct ribonucleic acid refers to the complementary sequence as dictated by base-pairing. A second nucleic acid sequence corresponding to the complement of the first nucleic acid sequence is present in those embodiments of the invention where the nucleic acid sequences are not expressed from a single transcriptional unit and, consequently, do not fold over into a hairpin structure. Rather, the two physically distinct molecules align and form intermolecular bonds as dictated by base complementarity.

The terms "double-stranded ribonucleic acid" and "dsRNA" can be used interchangeably and both refer to a short duplex consisting of a first and a second nucleic acid sequence. The term includes intramolecular dsRNAs formed from a single, linear ribonucleic acid molecule as well as intermolecular dsRNAs formed from two disctinct molecules. Stated differently, the double-stranded molecule can be formed by a single self-complementary RNA strand where one sequence is the reverse complement of the second sequence strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. The nucleic acid molecule may be introduced in an amount that allows delivery of at least one copy per cell. Higher doses, for example, at least 5, 10, 100, 500 or 1000 copies per cell of double-stranded material can also be introduced and can allow for more efficient modulation of gene expression, while lower doses can be useful for specific applications.

Modulation of gene expression is sequence-specific in that the dsRNA sequence corresponds to a particular nucleic acid regulatory element, for example, a promoter regulatory element, that regulates expression of a specific gene. In particular, a NRSE/RE1 dsRNA molecule provided by the present invention recognizes and binds to a sequence-specific motif or region located in the promoter region of particular genes and modulates gene expression by interfering with the suppression of transcription. A smRNA of the invention can associate with a promoter regulatory element, for example, the NRSE/RE1. Modulation of gene expression by a dsRNA of invention can occur in several distinct ways. In particular, a smRNA of the invention can modulate gene expression by altering the interaction between a trans-acting protein and a nucleic acid regulatory element resulting, for example, in a shift from transcriptional repression to de-repression.

As shown in FIG. 4 (see Original Patent), the NRSF/REST trans-acting protein remains stably associated with the NRSE/RE1 nucleic acid regulatory element in both the repressed stem cell state, as well as during differentiated states that require dsRNA dependent de-repression. The NRSE chromatin changes from a repressed state characterized by the association between the NRSF with co-factor modulatory proteins including, HDACs, MBD1 and MeCP2 to an activated state where the NRSF associates with a distinct set of modulatory proteins, in particular, acetylated histones, does not involve a change in the association of the NRSF/REST protein itself. Thus, the change in NRSF/REST function is not necessarily a consequence of physical severance from the NRSE/RE1, but rather can be a consequence of the association, in this particular embodiment through physical interaction, between the NRSE smRNA of the invention and the NRSF/REST trans-acting regulatory protein. As a result of the interaction between a dsRNA and the NRSF/REST trans-acting regulatory protein, the NRSF/REST can switch cofactors from repressors to activators or vice versa. Thus, a smRNA of the invention can be useful to exploit the intrinsic ability of the NRSF/REST to function as a flexible mediator of NRSE regulatory elements and indirectly serve as an activator as well as an inhibitor of gene expression.

In the absence of an NRSE/smRNA of the invention, NRSE/RE1-containing neuronal genes are actively repressed by the NRSF/REST machinery through the association of HDACs and MBD-binding proteins. At the onset of neuronal induction, the dsRNA interacts directly with the NRSE/RE1/NRSF/REST regulatory machinery on the genome, and triggers an organizational change in transcriptional activation (FIG. 8 (see Original Patent)). A smRNA of the invention also can act through physical association with the NRSF/REST and alter NRSF/REST function by inducing a conformational change. Through both modes of modulation, a NRSE smRNA of the invention alleviates transcriptional repression that results from the association between the NRSF/REST trans-acting protein with co-factor modulatory proteins, including, for example, HDACs, MeCP2, and MBD1.

Generally, a dsRNA encompasses fragments of at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more nucleotides per strand, with characteristic 3' overhangs of at least 1, at least 2, at least 3, or at least 4 nucleotides. As set forth above, a double-stranded RNA molecule can be of any length desired by the user as long as the ability to modulate gene expression is preserved. The ribonucleic acid molecules can correspond to strands that are polyadenylated.

A nucleic acid molecule or complex can be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell can mediate transcription in vivo; or cloned RNA polymerase can be used for transcription in vivo or in vitro. For transcription from a transgene in vivo or an expression construct, a regulatory region such as, for example, a promoter, enhancer, silencer, splice donor and acceptor can be used to transcribe the ribonucleic acid strand or strands. Modulation can be targeted by specific expression in an organ, tissue, or cell type; stimulation of an environmental condition, for example, infection, stress, temperature, chemical inducers; and/or engineering transcription at a developmental stage or age.

A nucleic acid molecule of the invention can be chemically or enzymatically synthesized by manual or automated reactions. An NRSE/RE1 dsRNA can be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase, for example, T3, T7, SP6. The use and production of an expression construct are known in the art as described, for example, by Goeddel, Gene Expression Technology, Academic Press, 1990; Kriegler, Gene Transfer and Expression, Stockton Press, 1990; Murray, Gene Transfer and Expression Protocols, Humana Press, 1991; WO 97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693; and the references cited therein.

If synthesized chemically or by in vitro enzymatic synthesis, the RNA can be purified prior to introduction into the cell. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the RNA can be used with no or a minimum of purification to avoid losses due to sample processing. The RNA can be dried for storage or dissolved in an aqueous solution. The solution can contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.

A nucleic acid molecule of the invention can be directly introduced into the cell (intracellularly); or can be introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or can be introduced by immersing an organism in a solution containing the nucleic acid.

Physical methods of introducing a nucleic acid molecule, for example, injection directly into the cell or extracellular injection into the organism, can also be used. Vascular or extravascular circulation, the blood or lymph system, the phloem, the roots, the embryonic fluid and the cerebrospinal fluid are sites where the RNA can be introduced. Furthemore, a transgenic organism that expresses a NRSE/RE1 dsRNA from a recombinant construct can be produced by introducing the construct into a zygote, an embryonic stem cell, or another multipotent cell derived from the appropriate organism.

A promoter useful in the present invention can comprise a promoter of eukaryotic or prokaryotic origin that can provide high levels of constitutive expression across a variety of cell types and will be sufficient to direct the transcription of a distally located sequence, which is a sequence linked to the 5' end of the promoter sequence in a cell. The promoter region can also include control elements for the enhancement or repression of transcription and can be modified as desired by the user and depending on the context. Suitable promoters include, for example, RNA polymerase (pol) III promoters including, but not limited to, the human and murine U6 pol III promoters as well as the human and murine H1 RNA pol III promoters; RNA polymerase (pol) II promoters; cytomegalovirus immediate early promoter (pCMV), the Rous Sarcoma virus long terminal repeat promoter (pRSV), and the SP6, T3, and T7 promoters. In addition, a hybrid promoter also can be prepared that contains elements derived from, for example, both a RNA polymerase (pol) III promoter and an RNA polymerase (pol) II promoter. Modified promoters that contain sequence elements derived from two or more naturally occurring promoter sequences can be combined by the skilled person to effect transcription under a desired set of conditions or in a specific context.

Enhancer sequences upstream from the promoter or terminator sequences downstream of the coding region can optionally be included in a vector for expression of a dsRNA of the present invention to facilitate expression. Vectors useful for expression of a dsRNA of the present invention can also contain additional nucleic acid sequences, such as a polyadenylation sequence or a localization sequence. Such additional sequences can be inserted into the vector such that they are operably linked with the promoter sequence, if transcription is desired. Alternatively, the inserted sequences can be placed at any position in the vector.

An inducible promoter also can be useful for expressing a smRNA of the invention. An inducible promoter is transcriptionally active when bound to a transcriptional activator, which in turn is activated under a specific set of conditions, for example, in the presence of a particular combination of chemical signals that affect binding of the transcriptional activator to the inducible promoter and/or affect function of the transcriptional activator itself. Thus, an inducible promoter is a promoter that, either in the absence of an inducer, does not direct expression, or directs low levels of expression, of a nucleic acid sequence to which the inducible promoter is operably linked; or exhibits a low level of expression in the presence of a regulating factor that, when removed, allows high-level expression from the promoter, for example, the tet system. In the presence of an inducer, an inducible promoter directs transcription at an increased level.

The function of a promoter can be further modified, if desired, to include appropriate regulatory elements to provide for the desired level of expression or replication in the host cell. For example, appropriate promoter and enhancer elements can be chosen to provide for constitutive, inducible or cell type-specific expression. Useful constitutive promoter and enhancer elements for expression of a smRNA of the invention can include, for example, RSV, CMV, CAG, SV40 and IgH elements. Other constitutive, inducible and cell type-specific regulatory elements are well known in the art.

A promoter that is particularly useful in a lentiviral vector is compatible with mammalian genes and, further, can be compatible with expression of genes from a wide variety of species. For example, a promoter useful for practicing the invention can be a promoter of the eukaryotic RNA polymerases pol II and pol III, or a hybrid thereof. The RNA polymerase III promoters have a transcription machinery that is compatible with a wide variety of species, a high basal transcription rate and recognize termination sites with a high level of accuracy. For example, the human and murine U6 RNA polymerase (pol) III and H1 RNA pol III promoters are well characterized and useful for practicing the invention. As exemplified below, because the activities of these two promoters as well as the localization of expressed nucleic acid sequences can vary from cell type to cell type, if desired, U6 and H1 lentiviral vectors can be prepared and targeted to the desired cells for modulation of the expression of one or more genes. One skilled in the art will be able to select and/or modify the promoter that is most effective for the desired application and cell type so as to optimize modulation of the expression of one or more genes.

Thus, promoters that are useful in the invention include those promoters that are sufficient to render promoter-dependent gene expression controllable for cell-type specificity, cell-stage specificity, or tissue-specificity, and those promoters that are inducible by external signals or agents. The promoter sequence can be one that does not occur in nature, so long as it functions in a mammalian cell.

As used herein, the term "in vivo" means an environment within a living organism or living cell. Such a living organism can be, for example, a multi-cellular organism such as a rodent, mammal, primate or human or another animal such as an insect, worm, frog or fish, or a uni-cellular organism such as a single-celled protozoan, bacterium or yeast. The cell can be in an in utero animal, or in an ex utero animal. Both living cells derived from an organism and used directly (primary cells) as well as cells grown for multiple generations or indefinitely in culture are encompassed within the term "in vivo" as used herein. As an example, an oocyte removed from an organism such as a mouse or a frog and used directly or grown in a tissue culture dish constitutes an in vivo environment. In vivo applications of the invention include applications in which a ribonucleic acid molecule of the invention is introduced, for example, into a mammalian, primate, human, murine, porcine, bovine, yeast or bacterial cell, as well as into a mammalian fertilized oocyte, a mammalian embryonic or neuronal stem cell.

As used herein, the term "in vitro" means an environment outside of a living organism or cell. Applications performed, for example, in a microfuge tube, or a 96, 384 or 1536 well plate, or another assay format with purified or partially purified proteins or cellular extracts outside of a living organism are in vitro applications. Thus, applications performed using whole-cell or fractionated extracts derived from lysed cells, or performed with reconstituted systems, are encompassed within the term "in vitro" as used herein.

As used herein, the term "vector" refers to one or more nucleic acid molecules capable of transporting another nucleic acid sequence, for example, a ribonucleic acid sequence encompassing a first and second nucleic acid sequence, to which it has been linked. The term is intended to include any vehicle for delivery of a nucleic acid, for example, a virus, plasmid, cosmid or transposon. The term also encompasses vector systems of one or more physically separate vectors, for example, third-generation retroviral vector systems where the nucleic acid sequences encoding polypeptides having virus packaging functions necessary for generation of a retroviral vector of the invention can be divided onto separate expression plasmids that are independently transfected into the packaging cells.

The invention also provides a method for modulating gene expression by contacting a cellular system with a double-stranded ribonucleic acid molecule, wherein the double-stranded ribonucleic acid molecule is capable of associating with a regulatory machinery that controls transcription of one or more genes, and wherein the association results in altered expression of these one or more genes.

The cellular system in which the modulation of gene expression is effected using a smRNA of the invention can be an in vitro or in vivo. For example, modulation of gene expression by a smRNA of the invention can be assayed in vitro, for example, to confirm or maximize modulatory activity and efficiency of the dsRNA. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of modulation in the presence of a smRNA of the invention, which can be an increase or decrease in gene expression of greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not contacted. Lower doses of injected and longer times after administration of dsRNA can result in modulation of gene expression in a smaller fraction of cells, for, at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells.

Quantitation of gene expression in a cell can be measured by detection of a gene product produced by the modulated gene as well as, indirectly, by measuring phenotypic changes associated with expression of the gene product. For example, the amount of gene product in the cell can be detected with a hybridization probe having a nucleotide sequence, or translated polypeptide can be detected with an antibody raised against a polypeptide epitope. In addition, a phenotypic change associated with expression of the gene can be measured, for example, cell type differentiation. In order to measure modulation of gene expression with a NRSE/RE1 ds RNA it can be useful to detect the differentiation of neuronal stem cells into neuronal cells. Differentiation into neuronal cells can be detected by a variety of techniques and assays known in the art and exemplified herein, for example, by ascertaining the presence of certain characteristics associated with neuronal cells including morphology as well as the presence of particular neuronal cell surface markers.

The one or more genes whose expression is modulated can be genes that have a pleiotropic effect. As used herein, the phrase "pleiotropic effect" refers to downstream regulation by one gene product of two or more genes so as to effect a number of features, characteristics or phenotypes. Significantly, two neuronal transcription factor genes contain the NRSE in their regulatory region, demonstrating the indirect effect on downstream genes. Neuronal cell fate determination involves the activation of a number of genes NRSE-containing genes that are essential for establishment and maintenance of the neuronal phenotype including, for example, neuron specific cytoskeletal proteins, neurotransmitters and their biosynthetic enzymes, synaptic vesicle components, neurotrophins, and cell adhesion molecules. Global regulation of neuronal gene expression, which depends on the interplay within a network of neuron-specific genes, is thus orchestrated by the NRSE/NRSF regulatory machinery and can be modulated with a NRSE/RE1 smRNA of the invention.

A variety of vectors can be utilized to deliver a double-stranded nucleic acid molecule to a cellular system. As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring another nucleic acid sequence to which it has been linked. The term is intended to include any vehicle for delivery of a nucleic acid, for example, a virus, plasmid, cosmid or transposon.

Modulation of gene expression with a smRNA of the invention capitalizes on the discovery disclosed herein that presence of a dsRNA can affect the nature of the interaction of a nucleic acid regulatory element with a trans-acting protein. As described herein, a smRNA of the invention can be an inhibitor as well as an activator of gene expression based on the particular regulatory element and co-factor modulatory proteins recruited. The association between the dsRNA and the components of regulatory machinery can be a direct physical association as well as an indirect association, for example, through an intermediate molecule. While exemplified herein with regard to the promoter regulatory element (PRE) known as NRSE/REST, which is bound by the trans-acting protein NRSF, resulting in repression of neuronal gene transcription, the skilled person will appreciate the disclosure of a global dsRNA-mediated mechanism of regulating gene transcription that has broad applicability to cellular processes. In particular, the ability of dsRNA molecules of the invention to recognize specific regulatory sequences or motifs and associate with such regulatory sequences and their corresponding trans-acting proteins to globally regulate gene expression during cell fate determination is not spefific to neuronal cell differentiation as evidence by the presence of functional NRSEs in non-neuronal genes as described by Schoenherr et al., Procl. Natl. Acad. Sci. USA 93(18): 9881-9886 (1996). In this regard, the differences in responses among neuronal genes described herein (FIG. 4 (see Original Patent)) further demonstrate the diversity among the REST/NRSF regulatory machinery and the responses, despite being generated by a common elemental-directed regulator (NRSE/NRSF), can vary based on expression timing, tissue and age dependency, maturation and/or each selective subtype of neurons.

In the absence of the smRNA of the invention, a lack of activation of neuron-specific genes occurs in neuronal stem cells as a result of NRSF binding to the NRSE and, ultimately, neuronal differentiation potency is lost, resulting in the neuronal stem cells remaining at the progenitor stage. This lack of activation of neuron-specific genes in the absence of an invention dsRNA can be attributed to the recruitment by the NRSE-bound NRSF of histone deacetylases (HDACs) and particular co-factor modulatory proteins, including, methyl-CpG binding proteins (MeCPs) and methyl-CpG binding domain proteins (MBDs), which mediate transcriptional repression by binding methylated nucleic acid sequences in a sequence specific manner.

In the absence of an smRNA of the invention, co-factor modulating proteins including, methyl-CpG binding proteins (MeCPs) and methyl-CpG binding domain proteins (MBDs), mediate transcriptional repression by associating with additional modulating proteins, for example, members of the Sin3 and histone deactylase protein families. As a consequence of the particular set of co-factor modulatory proteins that is thus recruited, transcription of one or more genes specific to expression in the nervous system is repressed. In the presence of an invention dsRNA, transcriptional repression can be relieved through several mechanisms. First, the presence of an invention dsRNA can effect a conformational change in the NRSF/NRSE complex that prevents association with the HDACs and associated binding proteins or other modulatory proteins that are part of the NRSE/REST machinery. Second, the presence of an invention dsRNA can result in a switch in the function of the NRSE/REST complex from transcritional repressor to enhancer by affecting the choice of co-factors or modulatory proteins that make up the NRSE/NRSF regulatory machinery, thereby alleviating transcriptional repression and effecting de-repression. Thus, by virtue of its association with the NRSE, the dsRNA can switch the function of the NRSF/REST trans-acting protein from transcriptional repressor to transcriptional activator. As a consequence, the neuron-specific genes are transcribed resulting in the differentiation of neuronal stem cells into neurons.

As described herein, several of the genes containing the NRSE are essential for establishment and maintenance of the neuronal phenotype; these include neuron-specific cytoskeletal proteins, neurotransmitters and their biosynthetic enzymes, synaptic vesicle components, neurotrophins, and cell adhesion molecules. An NRSE motif is present in the regulatory region of over thirty neuron-specific genes, including for example, synapsin I, sodium channel type II, brain derived neurotrophic factor, Ng-CAM and L1. FIG. 9 (see Original Patent) provides a table listing further genes that contain the NRSE motif and can be modulated via the invention methods.

Thus, the invention provides a method for modulating gene expression by contacting a cellular system with a double-stranded ribonucleic acid molecule, wherein the double-stranded ribonucleic acid molecule is capable of associating with a regulatory element that controls transcription of one or more genes, and wherein the association results in altered expression of these one or more genes. The nucleic acid molecules and compositions described above are particularly useful in the methods disclosed for mediating gene expression.

In a further embodiment, the invention provides a method for treating a condition associated with reduced expression of one or more genes by administering to a subject an effective amount of a double-stranded ribonucleic acid molecule capable of associating with a regulatory element that controls transcription of the one or more genes. The association between the double-stranded ribonucleic acid molecule and the regulatory element results in increased expression of the one or more genes.

Also provided is a method for controlling a cellular process by modulating the expression of one or more genes involved in cell-type differentiation through administration to a subject of an effective amount of a double-stranded ribonucleic acid molecule capable of associating with a regulatory machinery that controls transcription of the one or more genes involved in cell-type differentiation. In a particular embodiment, the invention is directed to a method for controlling cell-type differentiation by modulating the expression of one or more genes involved in cell-type differentiation through administration to a subject of an effective amount of a double-stranded ribonucleic acid molecule capable of associating with a regulatory machinery that controls transcription of the one or more genes involved in cell-type differentiation.

In a related embodiment provided by the invention cell-type differentiation is modulated in a subject by administering an effective amount of an agent that interferes a function of a double-stranded ribonucleic acid molecule that associats with a regulatory machinery so as to increase the transcription of one or more genes. In this embodiment, an agent can be administered that interferes with the in vivo function of an endogenous dsRNA and prevents the alleviation of transcriptional repression by the dsRNA. For example, for subjects suffering from or predisposed for a condition characterized by hyperproliferation of the nervous system, an agent that interferes with an endogenous NRSE/RE1 dsRNA can prevent the activation of neuron specific genes.

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents can include ribozymes and other small molecules. Agents are evaluated for potential biological activity by inclusion in screening assays.

Thus, the invention also provides a method of identifying an agent that interferes with an activity of a double-stranded ribonucleic acid molecule that associates with a regulatory machinery of one or more genes by contacting a cellular system comprising the double-stranded ribonucleic acid molecule and the one or more genes with an agent, and identifying an agent that interferes with an activity of said double-stranded ribonucleic acid molecule. An activity of a double-stranded ribonucleic acid molecule actvity can be modulation of gene expression. The interference with an activity of a double-stranded ribonucleic acid molecule can be a result of preventing or reducing the association between the double-stranded ribonucleic acid molecule and the regulatory machinery of one or more genes and can result in modulation of a cellular process, for example, inhibition with cellular differentiation such as neuronal differentiation from stem cells.

The ability to introduce RNA into an intact cell or organism containing a gene that has a corresponding regulatory element, for example, a NRSE, allows applications of the invention in high throughput screening (HTS). For example, an NRSE/RE1 dsRNA can be produced as described herein and added to, for example, a multi-well microtiter plate containing cell samples. Solutions containing dsRNAs that are capable of modulating gene expression can be placed into individual wells positioned on a microtiter plate as an ordered array, and intact cells or organisms in each well can be assayed for any changes or modifications in behavior or development due to modulation of gene expression. An NRSE/RE1 dsRNA also can be fed directly to, injected into, the cell/organism containing the gene or genes whose expression can be modulated by virtue of having an NRSE in tis promoter region. Alternatively, the dsRNA can be produced by in vivo or in vitro transcription from an expression construct. The modulation of gene expression can be assayed from the effects on the cell/organism, for example, a change in differentiative state.

Numerous embodiments for the method described above are included within the scope of the invention. For example, a screening method of the invention can be used to identify an agent that has the ability to interfere with activation of neuron-speficic gene expression by an endogenous NRSE/RE1 dsRNA and therefore can prevent the activation of a neuron specific gene if the agent is added to an appropriate cell line or introduced into a transgenic non-human mammal or into a cell line in which the expression of a target gene is inhibited. Transgenic animals in which the expression of one or more genes is modulated as well as cell lines generated according to this invention can be used in these methods.

Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection. Agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of naturally-occurring agents in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and can be used to produce combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, to produce structural analogs.

The present invention therefore encompasses introduction of dsRNA into a cell for the treatment or prevention of a condition. In particular, the therapeutic applications of the present invention include delivery of the smRNA of the invention into somatic, nonreproductive cells as well as into reproductive, germ line cells of host mammals. Mammals carrying foreign exogenous genes in their germ line, generally referred to as transgenic animals, presently include, for example, mice, rats, rabbits, and some domestic livestock.

A smRNA of the invention can be introduced into a progenitor or stem cell and thereby modulate the expression of one or more genes invovled in cell fate determination, for example, neuronal differentiation. An NRSE/RE1 smRNA of the invention is useful for the treatment of neurological and neurodegenerative disorders. Treatment includes amelioration of any symptom associated with the condition or clinical indication associated with the condition. In particlar, a smRNA of the invention can be administered to a subject having a condition characterized by either a deficiency in the number of neuronal cells or degeneration of neuronal cells.

It is understood that the therapeutic embodiments of the present invention can be practiced with a variety of delivery vector systems known in the art and able to introduce relatively high levels of nucleic acid sequences into a variety of cells. Suitable viral vectors include yet are not limited to Herpes simplex virus vectors (Geller et al., Science 241:1667-1669 (1988)); vaccinia virus vectors (Piccini et al., Meth. Enzymology 153:545-563 (1987)); cytomegalovirus vectors (Mocarski et al., Viral Vectors 78-84 (1988)); Moloney murine leukemia virus vectors (Danos et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988); Blaese et al., Science 270:475-479 (1995); Onodera et al., J. Viol. 72:1769-1774 (1998)); adenovirus vectors (Berkner, Biotechniques 6:616-626 (1988); Cotten et al., Proc. Natl. Acad. Sci., USA 89:6094-6098 (1992); Graham et al., Meth. Mol. Biol. 7:109-127 (1991); Li et al., Human Gene Therapy 4:403-409 (1993); Zabner et al., Nature Genetics 6:75-83 (1994)); adeno-associated virus vectors (Goldman et al., Human Gene Therapy 10:2261-2268 (1997); Greelish et al., Nature Med. 5:439-443 (1999); Wang et al., Proc. Natl. Acad. Sci., USA 96:3906-3910 (1999); Snyder et al., Nature Med. 5:64-70 (1999); Herzog et al., Nature Med. 5:56-63 (1999)); retrovirus vectors (Donahue et al., Nature Med. 4:181-186 (1998); Shackleford et al., Proc. Natl. Acad. Sci., USA 85:9655-9659 (1988); U.S. Pat. Nos. 4,405,712, 4,650,764 and 5,252,479, and WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829.

A viral vector useful for practicing the invention methods, in particular, the therapeutic and prophylactic applications, can be derived from a retrovirus. Retroviridae encompass a large family of RNA viruses that is, in part, characterized by its replicative strategy, which includes as essential steps reverse transcription of the virion RNA into linear double-stranded DNA and the subsequent integration of this DNA into the genome of the cell. A retroviral vector useful in the invention can be a modified lentivirus, for example, an HIV-1, that is used to introduce a nucleic acid sequence into a cell. Lentiviruses are diploid positive-strand RNA viruses of the family Retroviridae that replicate through an integrated DNA intermediate. In particular, upon infection by the RNA virus, the lentiviral genome is reverse-transcribed into DNA by a virally encoded reverse transcriptase that is carried as a protein in each lentivirus. The viral DNA is then integrated pseudo-randomly into the host cell genome of the infecting cell, forming a provirus that is inherited by daughter cells. Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC"; 10801 University Blvd., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.

Lentiviral vectors based on human immunodeficiency virus (HIV) have been developed that can transduce nondividing cells both in vitro and in vivo. Such vectors are useful in the methods of the invention for administration of a NRSE/RE1 dsRNA to a subject or cellular system because these vectors can stably integrate into the host cell genome to effect long-term expression of the transgene and are free from significant cellular or humoral immune responses demonstrating their utility as delivery vehicles for genes in vivo or in vitro. As the etiological agent of acquired immunodeficiency syndrome (AIDS), safety in the use of lentiviral vectors has been enhanced through a variety of molecular engineering designs that inhibit the generation of replication-competent vectors through fortuitous recombination of vector gene components. Such engineering designs include removal of accessory genes which play a role in virulence, removal of trans-acting factors for transcription, the generation of self-inactivating vectors, separating the packaging signals from the functions required for vector production and splitting the packaging functions into multiple components.

For in vivo gene therapy, cells to be transducted are not removed from the subject. Rather, the dsRNA is introduced into cells of the recipient organism in situ that is, within the recipient. In vivo gene therapy has been reported in several animal models and the methods described herein are specifically contemplated for human gene therapy. For a description of viral vectors and their uses in gene therapy, see, for example, Gene Therapy: Principles and Applications (T. Blankenstein, et., 1999, Springer-Verlag, Inc.) and Understanding Gene Therapy (N. Lemoine, ed., 2000, R-G Vile), both of which are incorporated herein in their entirety.

Furthermore, in vivo applications encompass transduction of a mammalian cell in utero, more specifically, into the somatic cells of a mid-trimester fetus. In utero gene therapy allows for the correction of some types of genetic diseases before the appearance of any clinical manifestations; in addition, introduction of a therapeutic dsRNA into the fetus offers a number of potential advantages over postnatal gene transfer. For neurologic genetic diseases that appear to produce irreversible damage during gestation, treatment before birth, if desired early in pregnancy, can be useful to allow the birth of a normal baby. If desired, a smRNA of the invention can be incorporated into a gene therapy vector, for example, a lentiviral vector, that can intergrate efficiently into the target cell's genome and therefore insert the therapeutic dsRNA into the genetic make-up of the cell. Successful early treatment with a smRNA of the invention can preempt the appearance of clinical manifestations of a condition by, for example, alleviating the repression of transcription of neuron-specific genes and causing neuronal cell differentiation of neuronal stem cells. Therapeutic intervention with the invention methods is particularly useful during embryonic development since neuronal stem cells still have neuronal differentiation potency that can be exploited by introduction of an NRSE/RE1 dsRNA. Furthermore, gene transfer in the fetus can be more efficient than in the more mature organism, so that gene therapy should be easier to accomplish prenatally than postnatally. In addition, the immunological naivete and the permissive environment of the early gestational fetus allow acceptance of cells and lentivectors without the need for immunosuppression or myeloablation because during early immunologic development, before thymic processing of mature lymphocytes, the fetus is largely tolerant of foreign antigens.

If desired, the smRNA of the invention can be introduced into the cell by administering an vector containing the dsRNA to a mammal that carries the cell. For example, the vector carrying the smRNA of the invention can be administered to a mammal by subcutaneous, intravascular, or intraperitoneal injection. If desired, a slow-release device, such as an implantable pump, can be used to facilitate delivery of the vector to cells of the mammal. A particular cell type within a mammal can be targeted by modulating the amount of the vector of the invention administered to the mammal and by controlling the method of delivery. For example, intravascular administration of a vector to the portal, splenic, or mesenteric veins or to the hepatic artery can be used to facilitate targeting the smRNA of the invention to liver cells. Furthermore, a homing molecule that specifically targets a particular cell type, for example, neuronal precursor cells or stem cell can ne used to target the dsRNA to te dsired target cell. In another method, a vector carrying the smRNA of the invention can be administered to cells or organ of a donor individual (human or non-human) prior to transplantation of the cells or organ to a recipient.

In a preferred method of administration, the vector used to introduce a smRNA of the invention is administered to a tissue or organ containing the targeted cells of the mammal. Such administration can be accomplished by injecting a solution containing the lentiviral vector of the invention into a tissue, such as skin, brain (e.g., the olfactory bulb), kidney, bladder, trachea, liver, spleen, muscle, thyroid, thymus, lung, or colon tissue. Alternatively, or in addition, administration can be accomplished by perfusing an organ with a solution containing the vector used to introduce a smRNA of the invention, according to conventional perfusion protocols.

In another therapeutic embodiment, the vector used to introduce a smRNA of the invention is administered intranasally by applying a solution of the vector to the nasal mucosa of a mammal. This method of administration can be used to facilitate transportation of the vector into the brain. This delivery mode provides a means for delivering the vector used to introduce a smRNA of the invention to brain cells, in particular, mitral and granule neuronal cells of the olfactory bulb, without subjecting the mammal to surgery. In an alternative method, a vector intended to express a dsRNA transgene in the brain can be delivered to the brain by osmotic shock according to conventional methods for inducing osmotic shock.
 

Claim 1 of 13 Claims

1. A method for increasing transcription comprising: contacting in vitro a cellular system comprising a regulatory machinery that controls transcription of one or more genes with a small modulatory double-stranded ribonucleic acid (smRNA) molecule, wherein: (i) the regulatory machinery comprises a nucleic acid regulatory element located in a promoter region; (ii) said one or more genes are specific to expression in the nervous system; (iii) said nucleic acid regulatory element comprises a neuron-restrictive silencer element/repressor element 1 (NRSE/RE1); and (iv) the nucleic acid sequence of said smRNA molecule consists of SEQ ID NO:1 and a complementary sequence of SEQ ID NO:1.

 

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