Hildinger, Markus (Boston, MA, US)
Auricchio, Alberto (Napoli, IT)

10/604340

01/27/2005

07/13/2003

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(IPC1-7): A61K048/00; C12N015/86; C12N005/02

Markus, Hildinger (ONE DEVONSHIRE PLACE, APT. 3401, BOSTON, MA, 02109, US)

1. A method of decreasing the expression of a target gene in a cell of a mammalian subject comprising administering to the subject in vivo a therapeutically effective amount of an RNAi expression cassette, comprising: (a) providing a recombinant adeno-associated viral vector, wherein said vector comprises said RNAi expression cassette whose RNA expression product(s) directly or indirectly lead to the decrease of expression of an RNAi target gene, wherein the RNA expression product(s) of the RNAi expression cassette comprise a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the RNAi target gene mRNA transcript (b) delivering said recombinant adeno-associated viral vector to and/or within said mammalian subject wherein transduction of suitable target cells results in expression of said RNAi expression cassette.

2. A method of decreasing the expression of (at least) one target gene in a cell of a mammalian subject comprising administering to the subject in vivo a therapeutically effective amount of (at least) one RNAi expression cassette, comprising: (a) providing (at least) one recombinant adeno-associated viral vector, wherein said vector comprises (at least) one RNAi expression cassette whose RNA expression product(s) directly or indirectly lead to the decrease of expression of an RNAi target gene, wherein the RNA expression product(s) of the RNAi expression cassette comprise a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the RNAi target gene mRNA transcript (b) delivering said recombinant adeno-associated viral vector(s) to and/or within said mammalian subject wherein transduction of suitable target cells results in expression of said RNAi expression cassette.

3. The method of claims 1 and 2, wherein expression of the RNA coding region of the RNAi expression cassette results in the down-regulation of the expression of the RNAi target gene, wherein the target gene comprises a sequence that is at least about 90% identical with the RNA coding region.

4. The method of claims 1 and 2, in which the RNAi target gene expression is inhibited by at least 10%.

5. The method of claims 1 and 2, wherein said RNAi expression cassette(s) encode one or more RNA molecules which are capable of forming an RNA interference inducing double-stranded RNA complex.

6. The method of claims 1 and 2, wherein said RNAi expression cassette encodes (at least) one RNA molecule which is self-complementary.

7. The method of claims 1 and 2, wherein said RNAi expression cassette encodes (at least) two separate complementary single-stranded RNA molecules.

8. The RNA molecule or RNA molecules of claims 6 and 7, wherein said RNA molecule or RNA molecules are capable of forming an RNA interference inducing double-stranded RNA complex.

9. The method of claims 1 and 2, wherein (at least) two recombinant adeno-associated viral vectors are used with each vector comprising its own RNAi expression cassette, and each RNAi expression cassette encoding at least one RNA molecule which is complementary to the RNA molecule expressed by the other RNAi expression cassette.

10. The RNA molecule(s) of claims 5, 6, 7, 8 and 9 having a nucleotide sequence which is substantially identical and/or complementary to at least a part of the RNAi target gene.

11. The RNA molecule(s) of claims 5, 6, 7, 8 and 9 with the RNA molecule(s) being siRNA.

12. The method of claims 1 and 2, wherein said RNAi expression cassette encodes a self-complementary RNA molecule comprising a sense region, a loop region and an antisense region.

13. The method of claim 12, wherein the loop region is about 2 to about 10 nucleotides in length.

14. The method of claim 12, wherein the sense region and the antisense region are each between about 10 and about 30 nucleotides in length.

15. The method of claim 12, wherein the sense region hybridizes under stringent conditions to a nucleotide sequence of the RNAi target gene, and the antisense region, which is a complementary inverted repeat of said sense region, hybridizes to said sense region to form a hairpin structure.

16. The method of claims 1 and 2, wherein said RNAi expression cassette comprises a first promoter and a second promoter, each operably linked to an RNA coding region, such that expression of the RNA coding region from the first promoter results in the synthesis of a first RNA molecule and expression of the RNA coding region from the second promoter results in the synthesis of a second RNA molecule substantially complementary to the first RNA molecule.

17. The method of claims 1 and 2, wherein said RNAi expression cassette comprises two promoters operably linked to the same RNA coding region, such that expression of the RNA coding region from the first promoter results in the synthesis of a first RNA molecule and expression of the RNA coding region from the second promoter results in the synthesis of a second RNA molecule substantially complementary to the first RNA molecule.

18. The method of claims 1 and 2, wherein said RNAi expression cassette encodes (at least) two RNA molecules, wherein (a) one of the (at least) two RNA molecules consists essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the RNAi target gene and another of the (at least) two RNA molecules consists essentially of a ribonucleotide sequence which is complementary to said nucleotide sequence of the RNAi target gene (b) the (at least) two RNA molecules are separate complementary strands that hybridize to each other to form a double-stranded RNA complex, and the double-stranded RNA complex directly or indirectly inhibits expression of the RNAi target gene.

19. The method of claims 1 and 2, wherein said RNAi expression cassette comprises a promoter operably linked to a DNA sequence which, when expressed by a host cell produces one RNA molecule having: (a) homology to at least one target mRNA expressed by the host cell (b) two (internally) complementary RNA regions wherein the expressed RNA reduces the intracellular concentration of the target mRNA or any substantially similar endogenous mRNA either directly or indirectly.

20. The method of claims 1 and 2, wherein said RNAi expression cassette encodes (at least) one RNA molecule for inhibiting expression of a target gene, comprising a first nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the RNAi target gene, and a second nucleotide sequence which is a complementary inverted repeat of said first nucleotide sequence and hybridizes to said first nucleotide sequence to form a hairpin structure.

21. The RNA molecule of claim 19, wherein the two nucleotide sequences are joined by an RNA loop structure.

22. The method of claims 1 and 2, wherein expression of said RNAi expression cassette leads to the generation of a double-stranded RNA complex comprising: (a) a first RNA portion capable of hybridizing under physiological conditions to at least a part of an mRNA molecule encoded by a gene; and (b) a second RNA portion wherein at least a part of the second RNA portion is capable of hybridizing under physiological conditions to the first RNA portion.

23. The RNA complex of claim 22 wherein the first and second portions are separate ribonucleic acid molecules.

24. The RNA complex of claim 22 wherein the first and second portions are comprised within the same RNA molecule.

25. The method of claims 1 and 2, wherein said RNAi expression cassette encodes a linear RNA molecule capable of forming a double-stranded RNA complex wherein the RNA molecule comprises: (a) a first portion that hybridizes under physiologic conditions to at least a portion of an mRNA molecule encoded by a gene; and (b) a second portion wherein at least part of the second portion is capable of hybridizing to the first portion to form a hairpin double-stranded RNA complex.

26. The linear RNA molecule of claim 25 further comprising a third portion of ribonucleic acid interposed between the first and second portions.

27. The linear RNA molecule of claim 26 wherein the third portion promotes hybridization between the first and second portion.

28. The method of claims 1 and 2, wherein said RNAi expression cassette encodes a linear RNA molecule capable of forming a double-stranded RNA complex wherein the RNA molecule comprises: (a) a first portion that comprises a region of RNA that is complementary to at least a portion of an mRNA molecule encoded by a gene (b) a second portion capable of hybridizing to at least part of the first portion (c) a third portion positioned between the first and second portions to facilitate the hybridization of the first and second portions with one another.

29. The linear RNA molecule of claim 22 and 25 wherein the second sequence comprises a transcription termination signal positioned at the 3′ end of the linear RNA molecule.

30. The method of claims 1 and 2, wherein the recombinant adeno-associated viral vector further comprises a gene of interest.

31. The method of claims 1 and 2, wherein the rAAV vector is of serotype 1, 2, 3, 4, 5, 6, 7 or 8 or any homologous serotypes or hybrids thereof.

32. The method of claims 1 and 2, wherein said RNAi expression cassette comprises an RNA Polymerase III promoter.

33. The method of claims 1 and 2, wherein said RNAi expression cassette comprises an RNA Polymerase II promoter.

34. The method of claims 1 and 2, wherein said RNAi expression cassette comprises an RNA Polymerase I promoter.

35. The method of claims 1 and 2, wherein said RNAi target gene causes or is likely to cause disease.

36. The method of claims 1 and 2, wherein said RNAi target genes are the Rhodopsin gene, the CCR5 gene, the CXCR4 gene, the VEGF gene, the HIF gene or any other gene of therapeutic interest.

37. The method of claims 1 and 2, wherein said RNAi target gene is the Rhodopsin gene.

38. The method of claims 1 and 2, wherein said transduced cells are cells of and/or in the eye, retinal cells, retinal pigment epithelial cells, photoreceptor cells, cells of the eye, gut cells, muscle cells, lung cells, intestinal cells, liver cells, pancreatic cells, hematopoietic cells, stem cells, skin cells, endothelial cells, neurons, cells of ectodermal origin, cells of neurodermal original, cells of endodermal original and/or brain cells.

39. The method of claims 1 and 2, wherein said transduced cells are photoreceptor cells.

40. The pharmaceutical preparation comprising a recombinant adeno-associated viral vector comprising an RNAi expression cassette as claimed in claim 1.

41. The pharmaceutical preparation as claimed in claim 40, wherein said preparation is suitable for and/or administered by intravenous administration.

42. The pharmaceutical preparation as claimed in claim 40, wherein said preparation is suitable for and/or administered by intra-arterial administration.

43. The pharmaceutical preparation as claimed in claim 40, wherein said preparation is suitable for and/or administered by intracavity injection.

44. The pharmaceutical preparation as claimed in claim 40, wherein said preparation is suitable for and/or administered by injection into tissue.

45. The pharmaceutical preparation as claimed in claim 40, wherein said preparation is suitable for and/or administered by injection into gaps in tissue.

46. The pharmaceutical preparation as claimed in claim 40, wherein said preparation is suitable for and/or administered by local administration.

47. The pharmaceutical preparation as claimed in claim 40, wherein said preparation is suitable for and/or administered by inhalation and/or nasal instillation.

48. The pharmaceutical preparation as claimed in claim 40, wherein said preparation is suitable for and/or administered by intraocular and/or intravitreal administration.

49. A method for treating a mammalian subject with an autosomal-dominant disorder or other disease including cancer and infectious diseases by administering to the subject an adeno-associated viral vector for initiating decrease of RNAi target gene expression at the mRNA level, wherein the method comprises using RNAi to achieve post-transcriptional gene silencing.

50. The method of claim 49, wherein the mammalian subject is a human patient.

BACKGROUND OF INVENTION

(1) Field of the Invention

The present invention relates generally to methods for altering gene expression in a cell of a mammalian subject using recombinant adeno-associated viral vectors engineered to express one or more RNA molecules that induce RNA interference in said cell. In a more specific aspect, gene expression is decreased or down-regulated by administering in vivo to a mammalian subject a recombinant adeno-associated viral vector with said vector comprising an RNA interference (RNAi) expression cassette whose RNA expression product(s) directly or indirectly lead to a decrease in expression of the corresponding RNAi target gene.

Upon successful transduction with the recombinant adeno-associated viral vector, the RNA expression products of the RNAi expression cassette will decrease the cellular concentration of the mRNA transcript of the RNAi target gene, thus resulting in decreased concentration of the protein encoded by the RNAi target gene.

(2) Background of the Invention

(2.1) General Usefulness of Decreasing Expression of a Specific Gene In Vivo

Decreasing expression of a specific gene in a mammalian subject has multiple utilities in the medical field such as:

• • (1) Treatment of diseases where an endogenous gene is pathologically overexpressed, e.g., Tumor Necrosis Factor alpha in Rheumatoid Arthritis
  • (2) Treatment of genetically inherited diseases where one or more alleles are mutated, and the mutated allele(s) have pathologic effects, e.g., mutations in the Rhodopsin gene in autosomal-dominant Retinitis Pigmentosa
  • (3) Treatment of cancer where overexpression of a gene results into cancer, e.g., overexpression of Ras or the Epidermal Growth Factor Receptor (EGFR).
  • (4) Treatment of infectious diseases, primarily viral diseases, eases, where (a) exogenous (e.g., viral) genes contribute to disease pathogenesis (e.g., viral spread), such as the HIV integrase gene in AIDS; (b) endogenous genes contribute to disease pathogenesis (e.g., viral spread), such as the CCR5 Receptor gene in AIDS.

Thus, a method would be desirable that results in down-regulation of the expression of a specific gene with (1) high versatility/flexibility in terms of genes that can be targeted (i.e., broad potential applications); (2) high specificity for the target gene (i.e., no inadvertent inhibition of other genes); (3) high efficacy in terms of expression down-regulation of the target gene; (4) low/no side effects.

(2.2) Limitations of Prior Approaches

Different strategies have been tried so far to decrease gene expression such as

• • (1) nucleic acid based strategies, such as (a) ribozymes [1]; (b) antisense oligonucleotides [2]; and
  • (2) protein-based approaches, such as (a) artificial transcription factors [3, 4]; (b) intrabodies [5]

Unfortunately, their utility is limited mainly due to several factors:

• • (1) Their efficacy varies depending on the target gene;
  • (2) Their versatility/flexibility is low;
  • (3) Their generation and production is cumbersome and time consuming, especially in case of the protein-based approaches
  • (4) Introducing the therapeutic entity into the target cells is difficult in general and particularly in vivo, e.g., cells normally do not uptake extracellular nucleic acids or proteins
  • (5) In case of protein-based approaches: As these are foreign (non-self) proteins, artificial transcription factors and intrabodies might elicit an immune response, thus limiting a potential therapeutic effect.
  • (6) Their in vivo application meets a major hurdle in terms of delivery to the target cells in amounts high enough to provide a therapeutic benefit.

Antisense technology in particular has been the most commonly described approach in protocols to achieve down-regulation of gene expression. For antisense strategies, stochiometric amounts of single-stranded nucleic acid complementary to the messenger RNA for the gene of interest are introduced into the cell. Some difficulties with antisense-based approaches relate to delivery, stability, and dose requirements. In general, cells do not have an uptake mechanism for single-stranded nucleic acids, hence uptake of unmodified single-stranded material is extremely inefficient (see also point (4) above). Because antisense interference requires that the interfering material accumulate at a relatively high concentration (at or above the concentration of endogenous mRNA), the amount required to be delivered is a major constraint on efficacy (see also point (6) above). The use of antisense for gene therapy or other whole-organism applications has been limited by the large amounts of oligonucleotide that need to be synthesized from non-natural analogs, the cost of such synthesis, and the difficulty even with high doses of maintaining a sufficiently concentrated and uniform pool of interfering material in each cell.

(2.3) Advantages of RNAi Over Prior Approaches in General

The discovery that RNA interference (RNAi) seems to be a ubiquitous mechanism to silence genes suggests an alternative, novel approach to decrease gene expression, which is able to overcome the limitations of the other approaches outlined above. Short interfering RNAs (siRNAs) are at the heart of RNAi. The antisense strand of the siRNA is used by an RNAi silencing complex to guide cleavage of complementary mRNA molecules, thus silencing expression of the corresponding gene [6-10].

The present invention—leveraging RNAi—thus differs from other nucleic acid based strategies (antisense and ribozyme methods) in both approach and effectiveness:

• • (a) Compared to antisense strategies, RNAi leverages a catalytic process, i.e., a small amount of siRNA is capable of decreasing the concentration of the target gene mRNA within the target cell. As antisense is based on a stochiometric process, a much larger concentration of effector molecules is required within the target cell, i.e., a concentration is required that is equal to or greater than the concentration of endogenous mRNA. Thus, as RNAi is a catalytic process, a lower amount of effector molecules (i.e., siRNAs) is sufficient to mediate a therapeutic effect.
  • (b) Compared to ribozymes (which have a catalytic function as well), RNAi seems to be a more flexible strategy, which allows targeting a higher variety of target sequences and thus offers more flexibility in construct design. Moreover, design of RNAi constructs is fast and convenient as the artisan can design those constructs based on the sequence information of the RNAi target gene. With ribozymes, more trial-and-error experiments and more sophisticated design algorithms are required as ribozymes are more complex in nature. Last, RNAi is more efficacious in vivo compared to ribozymes as RNAi leverages ubiquitous, endogenous cell machinery.

The present invention also differs from protein-based strategies, as RNAi does not require the expression of non-endogenous proteins (such as artificial transcription factors), thus lowering the risk of an unintended immune response.

In summary, RNAi-mediated down-regulation of gene expression is a novel mechanism with clear advantages over existing gene expression down-regulation approaches.

(2.4) Advantages of RNAi Induced by rAAV-Mediated RNAi Expression Cassette Gene Transfer In Vivo

However, to decrease gene expression by RNAi, the siRNA molecules have to be within the target cell. Several methods have been used so far successfully in vitro such as transfection of in vitro synthesized siRNA molecules. However, these methods (based on in vitro synthesized RNA) are not highly effective in vivo for the following reasons:

• • (1) Due to the presence of RNAses in the extracellular milieu, RNAs have only a short half-life in vivo, which might require large amounts of RNA to be administered to a subject.
  • (2) Cells normally do not uptake naked RNAs or uptake naked RNA only at low rates.
  • (3) Naked nucleic acids outside of cells are assumed to induce autoimmune disorders and impose as such as safety concern (e.g., causing Systemic Lupus Erythematosus).
  • (4) Even if one succeeds in delivering the RNA to the target cell (e.g., by using liposomes), one still has to (a) readminister the RNA frequently as RNA is degraded intracellularly; (b) has to overcome the problems associated with non-viral delivery methods such as low efficiency and low cell tropism.

One first step to overcome these limitations partially, was the development of RNAi expression cassettes to mediate the expression of siRNA molecules in vivo. In that context, a gene transfer system is desirable that

• • (1) allows flexible targeting of a broad range of cells
  • (2) targets the intended target cells with (a) high specificity (e.g., through use of different serotypes), (b) high efficacy
  • (3) offers long-term gene expression
  • (4) is non-immunogenic (e.g., virus particles do not evoke an immune response)
  • (5) has an acceptable safety profile (e.g., non-integrating system).

Gene transfer vectors based on recombinant adeno-associated viruses (AAVs) meet all of these criteria and show great promise for in vivo gene transfer: rAAV virions can infect a broad spectrum of non-dividing cells with high efficacy and specificity (including cells of the CNS such as photoreceptor cells), are safe (replication defective, lack viral coding sequences) and induce no significant immune response to transgene products. This allows for long-term and stable gene expression [11-13].

The inventors are the first to describe the utility of AAV-mediated RNA interference in a mammalian subject in vivo by administering in vivo a recombinant adeno-associated viral gene transfer vector comprising an RNAi expression cassette. The inventors are also the first to show the usefulness of RNA Polymerase I promoters in that context. AAV-mediated RNA interference has clear advantages over other approaches for in vivo applications:

• • (1) AAV-mediated gene transfer allows the flexible, yet specific targeting of a broad range of cells by using alternative serotypes. More than eight AAV serotypes have been discovered so far, with each serotype having a distinct tropism. This is a clear advantage of AAV over all non-viral methods and also over retroviral gene transfer (as retroviral vectors can only transduce dividing cells).
  • (2) AAV-mediated gene transfer is more specific and more efficacious compared to non-viral approaches, i.e., a specific cell type can be targeted (without inadvertently transducing neighbouring cells), and transduction efficiency of the intended cell type is high.
  • (3) AAV offers long-term gene expression and does not induce an immune response—as compared to e.g., adenoviral vectors, which still harbor viral genes and induce an immune response.
  • (4) AAV vectors are relatively safe compared to retroviral or lentiviral constructs as they do not (or only to a limited extent) integrate into the host genome.

Thus, AAV-mediated RNA interference in a mammalian subject in vivo will provide useful and novel applications in at least 4 areas:

• • (1) Cancer therapy: siRNAs might be used to silence oncogenes [14-16]
  • (2) Anti-infective Therapy: siRNAs might inhibit the expression of essential viral genes or silence the expression of non-essential viral receptors [17-19], which could be used to treat infectious diseases such as virus infections (e.g., HIV) or bacterial infections.
  • (3) Treatment of (autosomal dominant) inherited disorders: siRNAs should be able to specifically silence mutated alleles (also in the context of gene therapy). To cure autosomal dominant diseases by gene therapy, the primary goal is not to introduce an intact copy of the mutated gene into the cells affected, but to inactivate the endogenous mutated copy, which causes the observed, undesired phenotype. Introduction of an intact copy in case of autosomal dominant mutations is only required if the patient is homozygous for the mutation, if the amount of correctly expressed protein is too low, or if the method chosen to inactivate the mutated copy also inactivates the second, non-mutated endogenous copy [20].
  • (4) Diseases caused by abnormal gene expression: Many diseases (such as endocrine disorders, immune disorders and so on) arise from the abnormal expression of a particular gene or group of genes within a mammal. The inhibition of the gene or group can therefore be used to treat these conditions.

(3) Description of Prior Art

(3.1) RNA Interference

Double-stranded RNA (dsRNA) can induce many different epigenetic gene-silencing processes in eukaryotes, including the degradation of homologous mRNAs—a process called RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants. RNA interference (RNAi) has first been discovered in 1998 by Andrew Fire and Craig Mello in C. elegans, confirming former studies of PGTS in plants [21]. It now seems to be a ubiquitous mechanism—also applicable to humans [6, 7, 17, 22-29].

In both plants and animals, one key function of RNAi is to maintain genome integrity by suppressing the mobilization of transposons and the accumulation of repetitive DNA in the germ line. In plants, and perhaps also in animals, the RNAi machinery also defends cells against pathogens with double-stranded RNA genomes as part of an inborn antiviral immune response. Last, RNAi seems to regulate the expression of endogenous genes in developmental contexts [7, 29].

(3.1.1) Small RNA Species

Components of the RNAi and PTGS machinery are involved in the processing and function of different small RNA species: small interfering RNAs (siRNAs), short temporal RNAs (stRNAs) and microRNAs (miRNAs) [6, 28].

The generation of siRNAs is catalyzed by the enyzme complex Dicer [30]. Dicer recognizes the presence of dsRNA in the cytosol and catalyzes the degradation of dsRNA into 21-23 base pair (bp) dsRNA fragments (=siRNAs) with two or three 3′ overlaps on each side [31, 32]. These siRNAs subsequently function as substrates for the degradation of complementary mRNA species (RNA interference) [29].

In contrast to siRNAs, which are double-stranded and direct destruction of their target mRNAs, stRNAs are single-stranded and repress translation of their target mRNAs by binding to partially complementary sequences in the 3′-untranslated regions of their mRNA targets. stRNAs are synthesized as branch of an imperfect 70-nt RNA stemloop structure and released by the enzyme Dicer [33]. Two examples for stRNAs are lin-4 and let-7, which regulate the timing of development in C. elegans. Whereas lin-4 seems to be restricted to worms, let-7 is found more widely among animals with bilateral symmetry (including humans) [28, 34, 35].

miRNAs also exist single-stranded in vivo in many animals (from nematodes to humans). Their synthesis equals the one of stRNAs with the difference of being part of a perfect 70-nt RNA stem-loop structure. miRNAs silence genes through mRNA degradation (analogous to siRNAs; different from stRNAs) and might play a role in early development [28, 36-38].

Several groups have been reported successful siRNA-mediated knock-down of mammalian genes in tissue culture [6, 32, 39-44]. The genes targeted range from structural (e.g., lamin [32, 42]) to exogenous reporter genes (e.g., GFP [39]), confirming the hypothesis that RNAi is a ubiquitous mechanism and generally applicable to potentially any desired gene. Only recently, there have been reports of in vivo use of RNAi in mammals [39, 45, 46]: McCafrey et al. [45] were able to make a proof-of-principle in mice by co-transfecting a luciferase reporter construct together with an RNAi construct into mouse liver by hydrodynamic transfection. Similar results were obtained by Lewis et al. [46]. This group was able to silence an endogenous gene (GFP in a GFP-transgenic mouse) by hydrodynamic transfection of complementary siRNA oligonucleotides. Last, Xia et al. [39] were able to apply RNA interference in vivo in the context of a disease model (polyglutamine diseases).

(3.1.2) Mechanism of RNAi

The mechanism of RNA interference has been studied best in D. melanogaster. Researchers identified two phases: An initiation phase and an effector phase [29]:In the initiation phase, the RNAi enzyme complex Dicer recognizes dsRNA in the cytosol and catalyzes the degradation of dsRNA into 21-23 bp dsRNA fragments (=siRNAs). Structurally, Dicer contains two RNase III motifs, an RNA helicase domain, a dsRNA-binding domain (dsRBD), and a PAZ domain (PAZ: Piwi/Argonaute/Zwille). After processing, the siRNAs are integrated into the RNA-induced silencing complex (RISC), a 500 kD multiprotein complex, comprising the proteins EIF2C2, GEMIN3 and GEMIN4 in humans [6]. Within the RISC, the double-stranded siRNA is unwound, giving rise to a single-stranded guide RNA. This guide RNA is then used as a template for targeting of homologous mRNA sequences.

In the effector phase, if a complementary mRNA sequence has been found, the target mRNA will be cleaved in the middle of the annealed sequence through the RNase activity of RISC. The cleaved mRNA fragments are then released and degraded by cellular exonucleases.

It has been reported that in several lower eukaryotes an RNA-dependent polymerase amplifies the introduced dsRNA, possibly leading to a higher concentration of siRNAs [47]. Mammalian cells most likely lack this amplification mechanism. Thus, gene silencing in mammalian cells might require a much higher dosage of dsRNA compared to lower eukaryotes.

(3.1.3) Applications of RNAi

RNA interference has been successfully used to silence endogenous genes by introducing dsRNA with homology to the cellular gene transcript of interest [6, 32, 39-44]. This makes RNAi applicable in the context of basic science (reverse genetics) and medical therapy (including gene therapy) [48].

In forward genetics, one first isolates a mutant with a specific phenotype and then tries to identify the gene(s) involved; with reverse genetics, one starts with a specific gene of interest, downregulates its expression, and looks for phenotypic changes. Before the discovery of RNAi, downregulation was primarily achieved by knocking-out genes of interest through homologous recombination, a tedious process, which is difficult to upscale. With (inducible) RNAi as a new tool, this process can be standardized and even industrialized, allowing to downregulate each potential gene identified through the sequencing of the human genome for functional studies—without the need for homologous recombination or potentially even germ-line transmission [48].

The focus of the present invention is on medical applications of RNAi via AAV-mediated RNAi expression cassette transfer to a mammalian subject in vivo. Here, four areas might prove ideal candidates for leveraging RNAi:

• • (1) Cancer therapy: siRNAs might be used to silence oncogenes [14-16]
  • (2) Anti-infective Therapy: siRNAs might inhibit the expression of essential viral genes or silence the expression of non-essential viral receptors [17-19], which could be used to treat infectious diseases such as virus infections (e.g., HIV) or bacterial infections.
  • (3) Treatment of autosomal dominant inherited disorders: siRNAs should be able to specifically silence mutated alleles (also in the context of gene therapy). To cure autosomal dominant diseases by gene therapy, the primary goal is not to introduce an intact copy of the mutated gene into the cells affected, but to inactivate the endogenous mutated copy, which causes the observed, undesired phenotype. Introduction of an intact copy in case of autosomal dominanmutations is only required if the patient is homozygous for the mutation, if the amount of correctly expressed protein is too low, or if the method chosen to inactivate the mutated copy also inactivates the second, non-mutated endogenous copy [20].
  • (4) Diseases caused by abnormal gene expression: Many diseases (such as endocrine disorders, immune disorders and so on) arise from the abnormal expression of a particular gene or group of genes within a mammal. The inhibition of the gene or group can therefore be used to treat these conditions.

(3.2) Gene Transfer

Gene transfer systems can be classified along different dimensions

(A) Nature or origin of the system

(B) Delivery mechanism

(C) Site of gene transfer (e.g., ex vivo, in vitro, in vivo)

to (A): Based on the nature or origin of the gene transfer system, existing delivery systems for nucleic acid compositions can be subdivided into three groups: (1) viral vectors, (2) non-viral vectors, and (3) naked nucleic acids. Regarding vector targeting (specificity) and efficiency, viral vector systems are superior to conventional non-viral vectors and naked nucleic acids. On the other hand, non-viral vectors and naked nucleic acids are safer, easier to upscale in production and allow for the delivery of modified nucleic acids compared to viral vectors.

to (B): Alternatively, based on the delivery mechanism, gene transfer methods fall into the following three broad categories: (1) physical (e.g., electroporation, direct gene transfer and particle bombardment), (2) chemical (e.g. lipid-based carriers and other non-viral vectors) and (3) biological (e.g. virus or bacterium derived vectors).

to (C): Gene therapy methodologies can also be described by delivery site. Fundamental ways to deliver genes include ex vivo gene transfer, in vivo gene transfer, and in vitro gene transfer. In ex vivo gene transfer, cells are taken from the subject and grown in cell culture. The nucleic acid composition is introduced into the cells, the transduced or transfected cells are (in some instances) expanded in number and then reimplanted in the subject. In in vitro gene transfer, the transformed cells are cells growing in culture, such as tissue culture cells, and not particular cells from a particular subject. These “laboratory cells” are transfected or transduced; the transfected or transduced cells are then in some instances selected and/or expanded for either implantation into a subject or for other uses. In vivo gene transfer involves introducing the nucleic acid composition into the cells of the subject when the cells are within the subject.

Several delivery mechanisms may be used to achieve gene transfer in vivo, ex vivo, and/or in vitro.

Mechanical (i.e. physical) methods of DNA delivery can be achieved by direct injection of DNA, such as microinjection of DNA into germ or somatic cells, pneumatically deivered DNA-coated particles, such as the gold particles used in a “gene gun,” and inorganic chemical approaches such as calcium phosphate transfection. It has been found that physical injection of plasmid DNA into muscle cells yields a high percentage of cells that are transfected and have a sustained expression of marker genes. The plasmid DNA may or may not integrate into the genome of the cells. Non-integration of the transfected DNA would allow the transfection and expression of gene product proteins in terminally differentiated, non-proliferative tissues for a prolonged period of time without fear of mutational insertions, deletions, or alterations in the cellular or mitochondrial genome. Long-term, but not necessarily permanent, transfer of therapeutic genes into specific cells may provide treatments for genetic diseases or for prophylactic use. The DNA could be reinjected periodically to maintain the gene product level without mutations occurring in the genomes of the recipient cells. Non-integration of exogenous DNAs may allow for the presence of several different exogenous DNA constructs within one cell with all of the constructs expressing various gene products. Particle-mediated gene transfer may also be employed for injecting DNA into cells, tissues and organs. With a particle bombardment device, or “gene gun,” a motive force is generated to accelerate DNA coated high-density particles (such as gold or tungsten) to a high velocity that allows penetration of the target organs, tissues or cells. Electroporation for gene transfer uses an electrical current to make cells or tissues susceptible to electroporation-mediated gene transfer. A brief electric impulse with a given field strength is used to increase the permeability of a membrane in such a way that DNA molecules can penetrate into the cells. The techniques of particle-mediated gene transfer and electroporation are well known to those of ordinary skill in the art.

Chemical methods of gene therapy involve carrier mediated gene transfer through the use of fusogenic lipid vesicles such as liposomes or other vesicles for membrane fusion. A carrier harboring a DNA of interest can be conveniently introduced into body fluids or the bloodstream and then site specifically directed to the target organ or tissue in the body. Liposomes, for example, can be developed which are cell specific or organ specific. The foreign DNA carried by the liposome thus will be taken up by those specific cells. Injection of immunoliposomes that are targeted to a specific receptor on certain cells can be used as a convenient method of inserting the DNA into the cells bearing the receptor. Another carrier system that has been used is the asialoglycoprotein/polylysine conjugate system for carrying DNA to hepatocytes for in vivo gene transfer. Transfected DNA may also be complexed with other kinds of carriers so that the DNA is carried to the recipient cell and then resides in the cytoplasm or in the nucleoplasm of the recipient cell. DNA can be coupled to carrier nuclear proteins in specifically engineered vesicle complexes and carried directly into the nucleus. Carrier mediated gene transfer may also involve the use of lipid-based proteins which are not liposomes. For example, lipofectins and cytofectins are lipid-based positive ions that bind to negatively charged DNA, forming a complex that can ferry the DNA across a cell membrane. Another method of carrier mediated gene transfer involves receptor-based endocytosis. In this method, a ligand (specific to a cell surface receptor) is made to form a complex with a gene of interest and then injected into the bloodstream; target cells that have the cell surface receptor will specifically bind the ligand and transport the ligand-DNA complex into the cell.

Biological gene therapy methodologies usually employ viral vectors to insert genes into cells. The transduced cells may be cells derived from the patient's normal tissue, the patient's diseased tissue, or may be non-patient cells. Viral vectors that have been used for gene therapy protocols include but are not limited to, retroviruses, lentivruses, other RNA viruses such as pol1ovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, simian virus 40, vaccinia and other DNA viruses.

Replication-defective murine retroviral vectors are commonly utilized gene transfer vectors. Murine leukemia retroviruses are composed of a single strand RNA complex with a nuclear core protein and polymerase (pol) enzymes, encased by a protein core (gag) and surrounded by a glycoprotein envelope (env) that determines host range. The genomic structure of retroviruses include the gag, pol, and env genes flanked by 5′ and 3′ long terminal repeats (LTR). Retroviral vector systems exploit the fact that a minimal vector containing the 5′ and 3′ LTRs and the packaging signal are sufficient to allow vector packaging, infection and integration into target cells providing that the viral structural proteins are supplied in trans in the packaging cell line. Fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most dividing cell types, precise single copy vector integration into target cell chromosomal DNA, and ease of manipulation of the retroviral genome. For example, altered retrovirus vectors have been used in ex vivo and in vitro methods to introduce genes into peripheral and tumor-infiltrating lymphocytes, hepatocytes, epidermal cells, myocytes, or other somatic cells (which may then be introduced into the patient to provide the gene product from the inserted DNA). For descriptions of various retroviral systems, see, e.g., U.S. Pat. No. 5,219,740; [49-53]. The main disadvantage of retroviral systems is that retroviral vectors can only infect dividing cells. Lentiviral vectors overcome this limitation. Nevertheless, production of retro- and lentiviral vectors is complex, and the virions are not very stable compared to other viruses. More recently, the danger of inducing cancer through insertional mutagenesis has been raised as a major safety concern [54] [55].

A number of adenovirus based gene delivery systems have also been developed. Human adenoviruses are double stranded, linear DNA viruses with a protein capsid that enter cells by receptor-mediated endocytosis. Adenoviral vectors have a broad host range and are highly infectious, even at low virus titers. Moreover, adenoviral vectors can accommodate relatively long transgenes compared to other systems. A number of adenovirus based gene delivery systems have also been described [56-62]. The main limitation of adenoviral vectors is their high degree of immunogenicity, which limits their use in respect to applications that require long-term gene expression.

For many applications, long-term gene expression (over several years) will have to be achieved. This is also the case for the present invention. So far, primarily adeno-associated virus based vectors allow for this. Most other viral vectors are limited by expression of viral genes so that transduced cells will be eliminated by the immune system (e.g., adenoviral vectors), gene silencing (retroviral vectors or lentiviral vectors) or questionable safety profile (e.g., retroviral vectors or adenoviral vectors).

(3.2.1) Adeno-Associated Viral Vectors

The present invention uses adeno-associated virus-based vectors [63] [64] [65] for the transfer of an RNAi expression cassette into the appropriate target cells of a mammalian subject in vivo.

Adeno associated virus (AAV) is a small nonpathogenic virus of the parvoviridae family. AAV is distinct from the other members of this family by its dependence on a helper virus for replication. The approximately 5 kb genome of AAV consists of single stranded DNA of either plus or minus polarity. The ends of the genome are short inverted terminal repeats (ITRs), which can fold into hairpin structures and serve as the origin of viral DNA replication. Physically, the parvovirus virion is non-enveloped and its icosohedral capsid is approximately 20 nm in diameter. To date, at least 8 serologically distinct AAVs have been identified and isolated from humans or primates and are referred to as AAV types 1-8. The most extensively studied of these isolates are AAV type 2 (AAV2) and AAV type 5 (AAV5).

The genome of AAV2 is 4680 nucleotides in length and contains two open reading frames (ORFs). The left ORF encodes the non-structural Rep proteins, Rep40, Rep52, Rep68 and Rep78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Furthermore, two of the Rep proteins have been associated with the preferential integration of AAV2 genomes into a region of the q arm of human chromosome 19. Rep68/78 have also been shown to possess NTP binding activity as well as DNA and RNA helicase activities. The Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. Mutation of one of these kinase sites resulted in a loss of replication activity. The ends of the genome are short inverted terminal repeats, which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Within the ITR region two elements have been described which are central to the function of the ITR, a GAGC repeat motif and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding serves to position Rep68/78 for cleavage at the trs, which occurs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent trs. These elements have been shown to be functional and necessary for locus specific integration.

The right ORF of AAV2 encodes related capsid proteins referred to as VP1, 2 and 3. These capsid proteins form the icosahedral, non-enveloped virion particle of ˜20 nm diameter. VP1, 2 and 3 are found in a ratio of 1:1:10. The capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis has shown that removal or alteration of VP1, which is translated from an alternatively spliced message, results in a reduced yield of infectious particles. Mutations within the VP3 coding region result in the failure to produce any single-stranded progeny DNA or infectious particles.

The findings described in the context of AAV2 are generally applicable to other AAV serotypes as well.

The following features of AAV have made it an attractive vector for gene transfer. AAV vectors possess a broad host range [66], transduce both dividing and non dividing cells in vitro and in vivo and maintain high levels of expression of the transduced genes in the absence of a significant immune response to the transgene product in general. Moreover, as wild-type AAV is non-pathogenic, AAV vector particles are assumed to be non-pathogenic as well (in contrast to adenoviral vectors). Viral particles are heat stable, resistant to solvents, detergents, changes in pH and temperature. The ITRs have been shown to be the only cis elements required for replication and packaging and may contain some promoter activities. Thus, AAV vectors encode no viral genes.

SUMMARY OF INVENTION

(1) Substance or General Idea of the Claimed Invention

The present invention provides a method for decreasing or down-regulating gene expression at the mRNA level in a cell of a mammalian subject in vivo. The method involves administering to a (cell of a) mammalian subject in vivo a recombinant adeno-associated viral vector with said vector comprising an RNA interference (RNAi) expression cassette whose RNA expression product(s) directly or indirectly lead to a decrease in expression of the corresponding RNAi target gene by forming a double-stranded RNA complex which induces “RNA mediated interference” or “RNA interference” (“RNAi”), a post-transcriptional gene silencing mechanism. The dsRNA complex comprises a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript of the gene to be down-regulated (i.e., the RNAi target gene). In particular, the RNA expression products of the RNAi expression cassette will decrease the cellular concentration of the mRNA transcript of the RNAi target gene, thus resulting in decreased concentration of the protein encoded by the RNAi target gene in the mammalian subject. Down-regulation of gene expression is specific in that a nucleotide sequence from a portion of the RNAi target gene is chosen in designing the sequence properties of the RNA coding region of the RNAi expression cassette to be transferred via rAAV-mediated gene transfer into the cells of a mammalian subject in vivo; or alternatively said: Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the double-stranded RNA complex are targeted for RNA interference.

We disclose that the method of the present invention (1) Is effective in decreasing or down-regulating gene expression in a mammalian subject in vivo; (2) allows decreasing of gene expression of many different types of RNAi target genes; (3) allows decreasing of gene expression in many different cell types, tissues, and organs of a mammalian subject in vivo; (4)Allows decreasing of gene expression via rAAV-mediated RNAi expression cassette gene transfer to a mammalian subject in vivo using a multitude of RNAi expression cassette designs.

A significant aspect of the present invention relates to the demonstration that RNAi can in fact be accomplished in vivo in mammalian subjects by AAV-mediated gene transfer of an RNAi expression cassette. This had not been previously described in the art. Thus, the present invention provides, for the first time, a demonstration of the application of the RNAi technique in a mammalian subject in vivo using adeno-associated viral vectors: Upon successful in vivo transduction with the recombinant adeno-associated viral vector, the RNA expression products of the RNAi expression cassette will decrease the cellular concentration of the mRNA transcript of the RNAi target gene, thus resulting in decreased concentration of the protein encoded by the RNAi target gene in the mammalian subject.

Also disclosed are pharmaceutical kits containing the rAAV vector in a suitable pharmaceutical suspension for administration. In this aspect, the invention provides a pharmaceutical kit for delivery of said recombinant adeno-associated viral vector or virion. The kit may contain a container for administration of a predetermined dose. The kit further may contain a suspension containing the gene transfer vector or virion for delivery of a predetermined dose, said suspension comprising (a)the rAAV gene transfer vector or virion comprising an RNAi expression cassette(b)a physiologically compatible carrier.

In another aspect, the present invention relates to methods of controlling the expression of known genes or known nucleic acid sequences in mammalian cells in vivo by expressing sense and antisense RNA sequences (with respect to the gene or nucleic acid sequence) capable of forming double-stranded RNA complexes and inducing RNAi. In that context, the RNA molecules are expressed by administering in vivo a recombinant adeno-associated viral vector comprising an RNAi expression cassette encoding said RNA molecule(s). Thus, the invention also relates to rAAV-mediated expression of RNA molecules for forming dsRNA complexes, to DNA molecules (e.g., RNAi expression cassettes) encoding the RNA molecules for forming dsRNA complexes, to rAAV vectors and cells comprising such molecules, to rAAV virions comprising such rAAV vectors, to compositions comprising said rAAV virions, and to prophylactic and therapeutic methods for administering said rAAV vectors or virions.

The invention also provides RNAi expression cassettes that encode the RNA molecule(s) capable of forming a double-stranded RNA complex and thus capable of inducing RNA interference. Such RNAi expression cassettes may be a single DNA molecule as part of a rAAV genome which, when introduced into a cell, gives rise to a single RNA molecule capable of forming intramolecularly a dsRNA complex. However it will be understood from the following description that more than one rAAV genomes or RNAi expression cassettes or RNA coding regions may be introduced into a cell, either simultaneously or sequentially, to give rise to two or more RNA molecules capable of forming intermolecularly a dsRNA complex. Typically, the two RNA moieties capable of forming a dsRNA complex, whether intra- or intermolecularly, are at least in part sense and at least in part antisense sequences of a gene or nucleic acid sequence whose expression is to be down-regulated or decreased.

The design of the RNAi expression cassette does not limit the scope of the invention. Different strategies to design an RNAi expression cassette can be applied, and RNAi expression cassettes based on different designs will be able to induce RNA interference in vivo. (Although the design of the RNAi expression cassette does not limit the scope of the invention, some RNAi expression cassette designs are included in the detailed description of this invention and below. ) Features common to all RNAi expression cassettes are that they comprise an RNA coding region which encodes an RNA molecule which is capable of inducing RNA interference either alone or in combination with another RNA molecule by forming a double-stranded RNA complex either intramolecularly or intermolecularly.

Different design principles can be used to achieve that same goal and are known to those of skill in the art. For example, the RNAi expression cassette may encode one or more RNA molecules. After or during RNA expression from the RNAi expression cassette, a double-stranded RNA complex may be formed by either a single, self-complementary RNA molecule or two complementary RNA molecules. Formation of the dsRNA complex may be initiated either inside or outside the nucleus.

In one aspect there is provided a double-stranded RNA complex, which comprises, a first RNA portion capable of hybridizing under physiological conditions to at least a portion of an mRNA molecule, and a second RNA portion wherein at least a part of the second RNA portion is capable of hybridizing under physiological conditions to the first portion. Preferably the first and second portions are part of the same RNA molecule and are capable of hybridization at physiological conditions, such as those existing within a cell, and upon hybridization the first and second portions form a double-stranded RNA complex.

In another aspect there is provided a linear RNA molecule for forming a double-stranded RNA complex, which RNA comprises a first portion capable of hybridizing to at least a portion of an mRNA molecule, preferably within a cell and a second portion wherein at least part of the second portion is capable of hybridizing to the first portion to form a hairpin dsRNA complex.

In yet another aspect, the method comprises AAV-mediated expression of RNA with partial or fully double-stranded character in vivo.

A dsRNA complex containing a nucleotide sequence identical to a portion of the RNAi target gene is preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the RNAi target sequence have also been found to be effective for inhibition. Thus, sequence identity may be optimized by alignment algorithms known in the art and calculating the percent difference between the nucleotide sequences. Alternatively, the duplex region of the dsRNA complex may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.

In the preferred embodiment the RNAi expression cassette comprises at least one RNA coding region. Preferably the RNA coding region is a DNA sequence that can serve as a template for the expression of a desired RNA molecule in the host cell. In one embodiment, the RNAi expression cassette comprises two or more RNA coding regions. The RNAi expression cassette also preferably comprises at least one RNA Polymerase III promoter. The RNA Polymerase III promoter is operably linked to the RNA coding region, and the RNA coding region can also be linked to a terminator sequence. In addition, more than one RNA Polymerase III promoters may be incorporated.

In certain embodiments the invention employs ribozyme-containing RNA molecules to generate dsRNA complexes, thereby overcoming certain known difficulties associated with generating dsRNA. For example, the ribozyme functionality might be used to remove polyadenylation signals, thus preventing or minimizing release of the RNA molecule from the nucleus of a cell. In other embodiments the invention is based on the ability of a portion of the RNA molecule to encode an RNA or protein that enhances specific activity of dsRNA. One example of this specific activity-enhancing portion of the RNA molecule is a portion of the molecule encoding the HIV Tat protein to inhibit the cellular breakdown of dsRNA complexes. Such a portion is additionally useful in treating disorders such as HIV infection.

In another aspect of the invention, expression of the RNA coding region results in the down regulation of a target gene. Preferably the target gene comprises a sequence that is at least about 90% identical with the RNA coding region, more preferably at least about 95% identical, and even more preferably at least about 99% identical.

The RNAi target gene does not limit the scope of this invention and may be any gene derived from the cell: an endogenous gene, a transgene, or a gene of a pathogen that is present in the cell after infection thereof. Thus, the choice of the RNAi target gene is not limiting for the present invention: The artisan will know how to design an RNAi expression cassette to down-regulate the gene expression of any RNAi target gene of interest. Depending on the particular RNAi target gene and the dose of rAAV virions delivered, the procedure may provide partial or complete loss of function for the RNAi target gene.

Additionally, the RNAi target cell to be transduced in vivo does not limit the scope of this invention and may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The RNAi target cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands. The RNAi target cell might be a muscle cell, a liver cell, a lung cell or a brain cell. In its preferred embodiment, the RNAi target cell is a photoreceptor cell of the retina.

Moreover, the use of a specific AAV serotype does not limit the scope of this invention. Different AAV serotypes can be used to transduce different types of cells, and the tissue tropism of different AAV serotypes are known to those of skill in the art or can be determined by the artisan without undue effort. Thus, the artisan will choose the most appropriate AAV serotype for the transfer of an RNAi expression cassette into the corresponding RNAi target cell type.

According to a further aspect of the invention, the rAAV vector may also comprise a nucleotide sequence encoding a gene of interest. The gene of interest is preferably operably linked to a Polymerase II promoter. Such a construct also can contain, for example, an enhancer sequence operably linked with the Polymerase II promoter. The gene of interest is not limited in any way and includes any gene that the skilled practitioner desires to have expressed. For example, the gene of interest may be one that encodes a protein that serves as a marker to identify transduced cells. In other embodiments the gene of interest encodes a protein that has a therapeutic or palliative effect on the mammalian subject. In addition, more than one gene of interest may be included in the rAAV vector. For example a gene encoding a marker protein may be placed after the primary gene of interest to allow for identification of cells that are expressing the desired protein.

The gene of interest could encode any variety of proteins including, but not limited to viral proteins capable of modulating the global mammalian cell response to dsRNA, and would include but not be restricted to, mammalian viral proteins (vaccinia virus early protein E3L, reovirus p3 protein, vaccinia virus pK3, HIV-1 Tat) or cellular proteins (PKR dominant negative proteins, p58, and oncogenes such as v-erbB, sos or activated ras). In addition the gene of interest could encode any enzyme component of the host protein complex that acts specifically on dsRNA to enhance the efficacy of the dsRNA in controlling specific gene expression. In a preferred embodiment the protein that enhances the specific activity of dsRNA would be the HIV Tat protein. Moreover, the gene of interest might encode proteins involved in RNA interference within a cell, e.g., Argonaut proteins or Dicer proteins.

In one embodiment, the RNAi target gene is the Rhodopsin gene and the gene of interest is a version of the Rhodopsin transgene (cDNA) with silent point mutations in the RNAi target sequence so that this Rhodopsin gene version with silent point mutations will not be subject to the RNA interference induced by rAAV-mediated transfer of such an RNAi expression cassette.

In another embodiment a fluorescent marker protein, preferably green fluorescent protein (GFP), is incorporated into the construct along with the gene of interest. If a second reporter gene is included, an internal ribosomal entry site (IRES) sequence is also preferably included.

In yet another embodiment, the gene of interest is a gene included for safety concerns to allow for the selective killing of the transduced RNAi target cells within a heterogeneous population, for example within a mammal, or more particularly within a human patient. In one such embodiment, the gene of interest is a thymidine kinase gene (TK) the expression of which renders a target cell susceptible to the action of the drug gancyclovir.

Practice of the present invention will provide useful medical applications as described below under “Utility Of The Present Invention”. Moreover, this discovery of the value of AAV-gene transfer mediated RNAi for down-regulating, decreasing or inhibiting mammalian gene expression offers a tool for developing new strategies for blocking gene function, and for producing AAV-based RNAi vectors to treat human disease. The invention provides the method, wherein the cells are mammalian cells, and in one embodiment the cells are human. In the present invention, the double-stranded RNA complex expressed by the RNAi expression cassette transferred into the target cell via a rAAV vector can be used to inhibit a target gene which causes or is likely to cause disease, i.e. it can be used for the treatment or prevention of disease. In the prevention of disease, the RNAi target gene may be one which is required for initiation or maintenance of the disease, or which has been identified as being associated with a higher risk of contracting the disease. Thus, the invention provides a method for treating a mammalian subject with a genetic disorder or disease caused by overexpression of a gene or by expression of a mutated gene by administering to the mammalian subject in vivo a rAAV vector comprising an RNAi expression cassette for initiating down-regulation of the RNAi target gene expression at the mRNA level, wherein the method comprises using RNAi to achieve post-transcriptional gene silencing. In this embodiment, the preferred mammalian subject is a human patient. An embodied target cell in the method of the invention is a pathogen infected cell or a tumor cell, and the tumor cell may be malignant. In another preferred embodiment, the target cell is a photoreceptor cell, and the RNAi target gene is the Rhodopsin gene. Moreover, in these embodiments, as in the method above, the method further comprises initiating RNAi, wherein the dsRNA complex is specific for the intended RNAi target gene.

In another aspect the invention provides a mammalian cell in which a specified gene or a specified nucleic acid sequence has been suppressed by a method of the present invention.

In yet another aspect the invention provides a method of modulating expression of a gene or a nucleic acid sequence in mammalian cells including exposing said cells to recombinant adeno-associated viral vectors in vivo.

In another aspect the present invention provides a method of modulating a cellular response wherein said response is due either directly or indirectly to the expression of a gene or nucleic acid sequence and wherein expression of said gene or nucleic acid sequence is suppressed by a method of the present invention.

In a further aspect the present invention provides a method of treating a disorder resulting either directly or indirectly from expression of a gene or nucleic acid sequence wherein expression of said gene or nucleic acid sequence is suppressed by a method of the present invention.

This invention also provides a method of treating a subject having a disorder ameliorated by inhibiting the expression of a known gene in the subject's cells, comprising administering to the subject a therapeutically effective amount of the instant pharmaceutical compositions comprising rAAV virions comprising RNAi expression cassette(s) encoding (at least) one RNA molecule which is capable of forming a dsRNA complex wherein, under hybridizing conditions, the a portion of the dsRNA complex is able to hybridize to at least a portion of an mRNA encoded by the gene whose expression is to be inhibited.

This invention also provides a method of inhibiting in a subject the onset of a disorder ameliorated by inhibiting the expression of a known gene in the subject's cells, comprising administering to the subject a prophylactically effective amount of the instant pharmaceutical composition comprising rAAV virions comprising RNAi expression cassette(s) encoding (at least) one RNA molecule which is capable of forming a dsRNA complex wherein, under hybridizing conditions, the a portion of the dsRNA complex is able to hybridize to at least a portion of an mRNA encoded by the gene whose expression is to be inhibited.

(2) Advantages of the Invention Over Prior Approaches

AAV-mediated transfer of RNAi expression cassettes in vivo represents a useful, novel and non-obvious advancement. Prior approaches to induce RNAi in vivo include (1) direct transfection of RNA; (2) transfection with plasmids or generally DNA comprising an RNAi expression cassette; (3) use of lentiviral or adenoviral vectors. However, all these prior approaches reveal significant shortcomings when compared to rAAV-mediated transfer of RNAi expression cassettes.

to (1): Direct in vivo transfection of in vitro synthesized RNA is not highly effective in vivo for the following reasons:

• • (a) Due to the presence of RNAses in the extracellular milieu, RNAs have only a short half-life in vivo, which might require large amounts of RNA to be administered to a subject.
  • (b) Cells normally do not uptake naked RNAs or uptake naked RNA only at low rates.
  • (c) Even if one succeeds in delivering the RNA to the target cell (e.g., by using liposomes), one still has to readminister the RNA frequently as RNA is degraded intracellularly and to overcome the problems associated with non-viral delivery methods such as low efficiency and low cell tropism.

One first step to overcome these limitations partially, was the development of RNAi expression cassettes to mediate the expression of siRNA molecules in vivo. In that context, a gene transfer system is desirable that (1) allows flexible targeting of a broad range of cells; (2) targets the intended target cells with (a) high specificity (e.g., through use of different serotypes), (b) high efficacy; (3) offers long-term gene expression; (4) is non-immunogenic (e.g., virus particles do not evoke an immune response); (5) has an acceptable safety profile (e.g., non-integrating system).

Gene transfer vectors based on recombinant adeno-associated viruses (AAVs) meet all of these criteria and show great promise for in vivo gene transfer: rAAV vectors can infect a broad spectrum of non-dividing cells with high efficacy and specificity (including cells of the CNS such as photoreceptor cells), are safe (replication defective, lack viral coding sequences) and induce no significant immune response to transgene products. This allows for long-term and stable siRNA expression [11-13].

The inventors are the first to describe the utility of AAV-mediated RNA interference in a mammalian subject in vivo by administering in vivo a recombinant adeno-associated viral gene transfer vector comprising an RNAi expression cassette. AAV-mediated RNA interference has clear advantages over other approaches for in vivo applications:

• • (1) AAV-mediated gene transfer allows the flexible, yet specific targeting of a broad range of cells by using alternative serotypes. More than eight AAV serotypes have been discovered so far, with each serotype having a distinct tropism. This is a clear advantage of AAV over all non-viral methods and also over retroviral gene transfer (as retroviral vectors can only transduce dividing cells).
  • (2) AAV-mediated gene transfer is more specific and more efficacious compared to non-viral approaches, i.e., a specific cell type can be targeted (without inadvertently transducing neighbouring cells), and transduction efficiency of the intended cell type is high.
  • (3) AAV offers long-term gene expression and does not induce an immune response—as compared to e.g., adenoviral vectors, which still harbor viral genes and induce an immune response.
  • (4) AAV vectors are relatively safe compared to retroviral or lentiviral constructs as they do not (or only to a limited extent) integrate into the host genome.

(3.) Utility of the Present Invention

AAV-mediated RNA interference in a mammalian subject in vivo will provide useful and novel applications in at least 4 areas:

• • (1) Cancer therapy: siRNAs might be used to silence oncogenes [14-16]
  • (2) Anti-infective Therapy: siRNAs might inhibit the expression of essential viral genes or silence the expression of non-essential viral receptors [17-19], which could be used to treat infectious diseases such as virus infections (e.g., HIV) or bacterial infections.
  • (3) Treatment of (autosomal dominant) inherited disorders: siRNAs should be able to specifically silence mutated alleles (also in the context of gene therapy), an area, we would like to pursue with our grant application. To cure autosomal dominant diseases by gene therapy, the primary goal is not to introduce an intact copy of the mutated gene into the cells affected, but to inactivate the endogenous mutated copy, which causes the observed, undesired phenotype. Introduction of an intact copy in case of autosomal dominant mutations is only required if the patient is homozygous for the mutation, if the amount of correctly expressed protein is too low, or if the method chosen to inactivate the mutated copy also inactivates the second, non-mutated endogenous copy [20].
  • (4) Diseases caused by abnormal gene expression: Many diseases (such as endocrine disorders, immune disorders and so on) arise from the abnormal expression of a particular gene or group of genes within a mammal. The inhibition of the gene or group can therefore be used to treat these conditions.

In one aspect, the methods of the invention relate to the treatment or prevention of infection through the rAAV-mediated expression of one or more RNA molecules that inhibit one or more aspects of the life cycle of a pathogen through RNA interference with a target nucleic acid, such as a viral genome, a viral transcript or a host cell gene that is necessary for viral replication. The RNA coding region preferably comprises a sequence that is at least about 90% identical to a target sequence within the target nucleic acid. Preferably the target nucleic is necessary for the life cycle of a pathogen, for example, part of a pathogenic virus RNA genome or genome transcript, or part of a target cell gene involved in the life cycle of a pathogenic virus. In a particular embodiment the methods are used to disrupt the life cycle of a virus having an RNA genome, for example a retrovirus or lentivirus, by targeting the RNA genome directly. In another embodiment a viral genome transcript is targeted, including transcripts of individual viral genes. The methods also can be used to down-regulate a gene in a host cell, where the gene is involved in the viral life cycle, for example, a receptor or coreceptor necessary for viral entry into the host cell. According to the invention, one of skill in the art can target a cellular component, either an RNA or an RNA encoding a cellular protein necessary for the pathogen life cycle, particularly a viral life cycle. In a preferred embodiment, the cellular target chosen will not be a protein or RNA that is necessary for normal cell growth and viability. Suitable proteins for disrupting the viral life cycle include, for example, cell surface receptors involved in viral entry, including both primary receptors and secondary receptors, and transcription factors involved in the transcription of a viral genome, proteins involved in integration into a host chromosome, and proteins involved in translational or other regulation of viral gene expression.

A number of cellular proteins are known to be receptors for viral entry into cells. Some such receptors are listed in an article by E. Baranowski, C. M. Ruiz-Jarabo, and E. Domingo, “Evolution of Cell Recognition by Viruses,” Science 292: 1102-1105, which is hereby incorporated by reference in its entirety. Some cellular receptors that are involved in recognition by viruses are listed below: Adenoviruses: CAR, Integrins, MHC I, Heparan sulfate glycoaminoglycan, Siliac Acid; Cytomegalovirus: Heparan sulfate glycoami noglycan; Coxsackieviruses: Integrins, ICAM-1, CAR, MHC I; Hepatitis A: murine-like class I integral membrane clycoprotein; Hepatitis C: CD81, Low density lipoprotein receptor; HIV (Retroviridae): CD4, CXCR4, Heparan sulfate glycoaminoglycan; HSV: Heparan sulfate glycoaminoglycan, PVR, HveB, HveC; Influenza Virus: Sialic acid; Measles: CD46, CD55; Pol1ovirus,: PVR, HveB, HveC; Human papillomavirus: Integrins. One of skill in the art will recognize that the invention is not limited to use with receptors that are currently known. As new cellular receptors and coreceptors are discovered, the methods of the invention can be applied to such sequences.

The methods of the invention can be used to treat a variety of viral diseases, including, for example, human immunodeficiency virus (HIV-1 and HIV-2), hepatitis A, hepatitis B, hepatitis C. The invention also includes methods of treating a patient having a viral infection. In one embodiment the method comprises administering to the patient an effective amount of a recombinant AAV particle (or particles) encoding at least one double stranded RNA having at least 90% homology and preferably identical to a region of at least about 15 to 25 nucleotides in a nucleotide that is important for normal viral replication. For example, the dsRNA complex may have homology to a nucleic acid in a viral genome, a viral gene transcript or in a gene for a patient's cellular receptor that is necessary for the life cycle of the virus.

Other aspects and advantages of the invention will be readily apparent to one of skill in the art from the detailed description of the invention. Additional objects, advantages and novel features of the invention will be set forth in part in the description, examples and embodiments which follow, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1

Design principle 1a: An rAAV vector comprising an RNAi expression cassette encoding a single RNA molecule capable of forming an RNAi inducing dsRNA complex intramolecularly (based on pol III promoter).

Design principle 1b: An rAAV vector comprising an RNAi expression cassette encoding a single RNA molecule capable of forming an RNAi inducing dsRNA complex intramolecularly (based on pol II promoter)

Design principle 2: An rAAV vector comprising an RNAi expression cassette comprising two RNA coding regions with each region encoding one RNA molecule with the two RNA molecules (encoded by the two different RNA coding regions) combined capable of forming an RNAi inducing dsRNA complex intermolecularly.

Design principle 3: An rAAV vector comprising an RNAi expression cassette comprising one RNA coding region transcribed into a sense and antisense RNA molecule with the sense and antisense RNA molecule combined capable of forming an RNAi inducing dsRNA complex intermolecularly.

Design principle 4: Two rAAV vectors each comprising an RNAi expression cassette with the first rAAV vector encoding a sense RNA molecule, the second rAAV vector encoding a (complementary) antisense RNA molecule with both RNA molecules combined (when expressed in same cell) capable of forming an RNAi inducing dsRNA complex intermolecularly.

DETAILED DESCRIPTION

The present invention provides a method for decreasing or down-regulating gene expression at the mRNA level in a cell of a mammalian subject in vivo. The method involves administering to a (cell of a) mammalian subject in-vivo a recombinant adeno-associated viral vector with said vector comprising an RNA interference (RNAi) expression cassette whose RNA expression product(s) directly or indirectly lead to a decrease in expression of the corresponding RNAi target gene by forming a double-stranded RNA complex which induces “RNA mediated interference” or “RNA interference” (“RNAi”), a post-transcriptional gene silencing mechanism. The dsRNA complex comprises a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript of the gene to be down-regulated (i.e., the RNAi target gene). In particular, the RNA expression products of the RNAi expression cassette will decrease the cellular concentration of the mRNA transcript of the RNAi target gene, thus resulting in decreased concentration of the protein encoded by the RNAi target gene in the mammalian subject. Down-regulation of gene expression is specific in that a nucleotide sequence from a portion of the RNAi target gene is chosen in designing the sequence properties of the RNA coding region of the RNAi expression cassette to be transferred via rAAV-mediated gene transfer into the cells of a mammalian subject in vivo; or alternatively said: Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the double-stranded RNA complex are targeted for RNA interference.

Quantization of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention. Quantization of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell: mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.

In another aspect, the present invention relates to methods of controlling the expression of known genes or known nucleic acid sequences in mammalian cells in vivo by expressing sense and antisense RNA sequences (with respect to the gene or nucleic acid sequence of the RNAi target) capable of forming double-stranded RNA complexes and inducing RNAi. In that context, the RNA molecules are expressed by administering in vivo a recombinant adeno-associated viral vector comprising an RNAi expression cassette encoding said RNA molecule(s). Thus, the invention also relates to rAAV-mediated expression of RNA molecules for forming dsRNA complexes, to DNA molecules (e.g., RNAi expression cassettes) encoding the RNA molecules for forming dsRNA complexes, to rAAV vectors/virions and cells comprising such molecules, to compositions comprising said rAAV vectors/virions, and to prophylactic and therapeutic methods for administering said rAAV vectors/virions.

The invention also provides RNAi expression cassettes that encode the RNA molecule(s) capable of forming a double-stranded RNA complex and thus capable of inducing RNA interference. Such RNAi expression cassettes may be a single DNA molecule as part of a rAAV genome which, when introduced into a cell, gives rise to a single RNA molecule capable of forming intramolecularly a dsRNA complex. However it will be understood from the following description that more than one rAAV genome or RNAi expression cassette or RNA coding region may be introduced into a cell, either simultaneously or sequentially, to give rise to two or more RNA molecules capable of forming intermolecularly a dsRNA complex. Typically, the two RNA moieties capable of forming a dsRNA complex, whether intra- or intermolecularly, are at least in part sense and at least in part antisense sequences of a gene or nucleic acid sequence whose expression is to be down-regulated or decreased. The transcribed RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.

The design of the RNAi expression cassette does not limit the scope of the present invention. Different strategies to design an RNAi expression cassette can be applied, and RNAi expression cassettes based on different designs will be able to induce RNA interference in vivo. (Although the design of the RNAi expression cassette does not limit the scope of the invention, some RNAi expression cassette designs are included in the detailed description of this invention and below. ) The RNAi expression cassette may use a regulatory region (e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation) to transcribe an RNA coding region. Down-regulation of gene expression may be targeted by specific transcription in an organ, tissue, or cell type, stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age.

Features common to all RNAi expression cassettes are that they comprise an RNA coding region which encodes an RNA molecule which is capable of inducing RNA interference either alone or in combination with (an)other RNA molecule(s) by forming a double-stranded RNA complex either intramolecularly or intermolecularly.

Different design principles can be used to achieve that same goal and are known to those of skill in the art. For example, the RNAi expression cassette may encode one or more RNA molecules. After or during RNA expression from the RNAi expression cassette, a double-stranded RNA complex may be formed by either a single, self-complementary RNA molecule or two complementary RNA molecules. Formation of the dsRNA complex may be initiated either inside or outside the nucleus.

In one aspect there is provided a double-stranded RNA complex, which comprises, a first RNA portion capable of hybridizing under physiological conditions to at least a portion of an mRNA molecule, and a second RNA portion wherein at least a part of the second RNA portion is capable of hybridizing under physiological conditions to the first portion. Preferably the first and second portions are part of the same RNA molecule and are capable of hybridization at physiological conditions, such as those existing within a cell, and upon hybridization the first and second portions form a double-stranded RNA complex.

In another aspect there is provided a linear RNA molecule for forming a double-stranded RNA complex, which RNA comprises a first portion capable of hybridizing to at least a portion of an mRNA molecule, preferably within a cell and a second portion wherein at least part of the second portion is capable of hybridizing to the first portion to form a hairpin dsRNA complex.

In yet another aspect, the method comprises AAV-mediated expression of RNA with partial or fully double-stranded character in vivo.

A dsRNA complex containing a nucleotide sequence identical to a portion of the RNAi target gene is preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the RNAi target sequence have also been found to be effective for inhibition. Thus, sequence identity may be optimized by alignment algorithms known in the art and calculating the percent difference between the nucleotide sequences. Alternatively, the duplex region of the dsRNA complex may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the RNAi target gene transcript.

In the preferred embodiment, the RNAi expression cassette comprises at least one RNA coding region. Preferably the RNA coding region is a DNA sequence that can serve as a template for the expression of a desired RNA molecule in the host cell. In one embodiment, the RNAi expression cassette comprises two or more RNA coding regions. The RNAi expression cassette also preferably comprises at least one RNA Polymerase III promoter. The RNA Polymerase III promoter is operably linked to the RNA coding region, and the RNA coding region can also be linked to a terminator sequence. In addition, more than one RNA Polymerase III promoters may be incorporated.

In certain embodiments the invention employs ribozyme-containing RNA molecules to generate dsRNA complexes, thereby overcoming certain known difficulties associated with generating dsRNA, such as for example the removal polyadenylation signals, thus preventing or minimizing release of the RNA molecule from the nucleus of a cell. In other embodiments the invention is based on the ability of a portion of the RNA molecule to encode an RNA or protein that enhances specific activity of dsRNA. One example of this specific activity-enhancing portion of the RNA molecule is a portion of the molecule encoding the HIV Tat protein to inhibit the cellular breakdown of dsRNA complexes. Such a portion is additionally useful in treating disorders such as HIV infection.

In another aspect of the present invention, the RNA expression products of the RNAi expression cassette lead to the generation of a double-stranded RNA complex for inducing RNA interference and thus down-regulating or decreasing expression of a mammalian gene. The dsRNA complex comprises a first nucleotide sequence that hybridizes under stringent conditions, including a wash step of 0.2×SSC at 65° C., to a nucleotide sequence of at least one mammalian gene and a second nucleotide sequence which is complementary to the first nucleotide sequence. The first nucleotide sequence might be linked to the second nucleotide sequence by a third nucleotide sequence (e.g., an RNA loop) so that the first nucleotide sequence and the second nucleotide sequence are part of the same RNA molecule (scenario 1); alternatively, the first nucleotide sequence might be part of one RNA molecule and the second nucleotide sequence might be part of another RNA molecule (scenario 2). Thus, in scenario 1, the dsRNA complex is formed by intramolecular hybridization or annealing whereas in scenario 2, the ds RNA complex is formed by intermolecular hybridization or annealing.

In one embodiment, the first nucleotide sequence of said ds RNA complex is at least 17, 18, 19, 20, 21, 22, 25, 50, 100, 200, 300, 400, 500, 800 nucleotides in length.

In another embodiment, the first nucleotide sequence of said ds RNA complex is identical to at least one mammalian gene.

In another embodiment, the first nucleotide sequence of said ds RNA complex is identical to (at least) one mammalian gene.

In yet another embodiment, the first nucleotide sequence of said ds RNA complex hybridizes under stringent conditions to at least one human gene.

In still another embodiment, the first nucleotide sequence of said ds RNA complex is identical to at least one human gene.

In still another embodiment, the first nucleotide sequence of said ds RNA complex is identical to one human gene.

In one embodiment, said double-stranded RNA complex is a hairpin comprising a first nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of at least one mammalian gene, and a second nucleotide sequence which is a complementary inverted repeat of said first nucleotide sequence and hybridizes to said first nucleotide sequence to form a hairpin structure. The first nucleotide sequence of said double-stranded RNA complex can hybridize to either coding or non-coding sequence of at least one mammalian gene.

In another aspect of the invention, expression of the RNA coding region results in the down regulation of an RNAi target gene. Preferably the target gene comprises a sequence that is at least about 90% identical with the RNA coding region, more preferably at least about 95% identical, and even more preferably at least about 99% identical.

The RNAi target gene does not limit the scope of this invention and may be any gene derived from the cell, an endogenous gene, a transgene, or a gene of a pathogen which is present in the cell after infection thereof. Thus, the choice of the RNAi target gene is not limiting for the present invention: The artisan will know how to design an RNAi expression cassette to down-regulate the gene expression of any RNAi target gene of interest. Depending on the particular target gene and the dose of rAAV virions delivered, the procedure may provide partial or complete loss of function for the target gene.

Additionally, the RNAi target cell to be transduced in vivo does not limit the scope of this invention and may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands. The RNAi target cell might be a muscle cell, a liver cell, a lung cell or a brain cell. In its preferred embodiment, the RNAi target cell is a photoreceptor cell.

Moreover, the use of a specific AAV serotype does not limit the scope of this invention. Different AAV serotypes can be used to transduce different types of cells, and the tissue tropism of different AAV serotypes are known to those of skill in the art or can be determined by the artisan without undue effort. Thus, the artisan will choose the most appropriate AAV serotype for the transfer of an RNAi expression cassette into the corresponding RNAi target cell type.

According to a further aspect of the invention, the rAAV vector may also comprise a nucleotide sequence encoding a gene of interest. The gene of interest is preferably operably linked to a Polymerase II promoter. Such a construct also can contain, for example, an enhancer sequence operably linked with the Polymerase II promoter. The gene of interest is not limited in any way and includes any gene that the skilled practitioner desires to have expressed. For example, the gene of interest may be one that encodes a protein that serves as a marker to identify transduced cells. In other embodiments the gene of interest encodes a protein that has a therapeutic or palliative effect on the mammalian subject. In addition, more than one gene of interest may be included in the rAAV vector. For example a gene encoding a marker protein may be placed after the primary gene of interest to allow for identification of cells that are expressing the desired protein.

In one embodiment, the RNAi target gene is the Rhodopsin gene and the gene of interest is a version of the Rhodopsin transgene (cDNA) with silent point mutations in the RNAi target sequence so that this Rhodopsin gene version with silent point mutations will not be subject to the RNA interference induced by rAAV-mediated transfer of such an RNAi expression cassette.

In another embodiment a fluorescent marker protein, preferably green fluorescent protein (GFP), is incorporated into the construct along with the gene of interest. If a second reporter gene is included, an internal ribosomal entry site (IRES) sequence is also preferably included.

In yet another embodiment, the gene of interest is a gene included for safety concerns to allow for the selective killing of the transduced RNAi target cells within a heterogeneous population, for example within a mammal, or more particularly within a human patient. In one such embodiment, the gene of interest is a thymidine kinase gene (TK) the expression of which renders a target cell susceptible to the action of the drug gancyclovir.

Practice of the present invention will provide useful medical applications as described below under “Utility Of The Present Invention”. Moreover, this discovery of the value of rAAV-gene transfer mediated RNAi for down-regulating, decreasing or inhibiting mammalian gene expression/offers a tool for developing new strategies for blocking gene function, and for producing AAV-based RNAi vectors to treat human disease. The invention provides the method, wherein the cells are mammalian cells, and in one embodiment the cells are human. In the present invention, the double-stranded RNA complex expressed by the RNAi expression cassette transferred into the RNAi target cell via a rAAV vector can be used to inhibit an RNAi target gene which causes or is likely to cause disease, i.e. it can be used for the treatment or prevention of disease. In the prevention of disease, the RNAi target gene may be one which is required for initiation or maintenance of the disease, or which has been identified as being associated with a higher risk of contracting the disease.

Thus, the invention provides a method for treating a mammalian subject with a genetic disorder or disease caused by overexpression of a gene or by expression of a mutated gene by administering to the mammalian subject in vivo a rAAV vector comprising an RNAi expression cassette for initiating down-regulation of the RNAi target gene expression at the mRNA level, wherein the method comprises using RNAi to achieve post-transcriptional gene silencing. In this embodiment, the preferred mammalian subject is a human patient. An embodied RNAi target cell in the method of the invention is a pathogen infected cell or a tumor cell, and the tumor cell may be malignant. In another preferred embodiment, the RNAi target cell is a photoreceptor cell, and the RNAi target gene is the Rhodopsin gene. Moreover, in these embodiments, as in the method above, the method further comprises initiating RNAi, wherein the dsRNA complex is specific for the intended RNAi target gene.

Thus, the present invention may be used for the treatment or prevention of disease by administering to a mammalian subject in vivo a recombinant adeno-associated viral vector comprising an RNAi expression cassette. For example, an RNAi expression cassette may be introduced into a cancerous cell or tumor via rAAV gene transfer and—upon expression of the RNAi cassette—inhibit gene expression of a gene required for maintenance of the carcinogenic/tumorigenic phenotype. To prevent a disease or other pathology, an RNAi target gene may be selected which is required for initiation or maintenance of the disease/pathology. Treatment would include amelioration of any symptom associated with the disease or clinical indication associated with the pathology.

A gene derived from any pathogen may be targeted for inhibition. For example, the gene could cause immuno-suppression of the host directly or be essential for replication of the pathogen, transmission of the pathogen, or maintenance of the infection. For example, cells at risk for infection by a pathogen or already infected cells, particularly human immunodeficiency virus (HIV) infections, may be targeted for treatment by introduction of an RNAi expression cassette via rAAV-mediated gene transfer according to the invention. The RNAi target gene might be a pathogen or host gene responsible for entry of a pathogen into its host, drug metabolism by the pathogen or host, replication or integration of the pathogen's genome, establishment or spread of an infection in the host, or assembly of the next generation of pathogen. Methods of prophylaxis (i.e., prevention or decreased risk of infection), as well as reduction in the frequency or severity of symptoms associated with infection, can be envisioned.

The present invention could be used for treatment or development of treatments for cancers of any type, including solid tumors and leukemias, including: apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukemia (e.g., B cell, mixed cell, null cell, T cell, T-cell chronic, HTLV-II-associated, lymphocytic acute, lymphocytic chronic, mast cell, and myeloid), histiocytosis malignant, Hodgkin disease, immunoproliferative small, non-Hodgkin lymphoma, plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelibma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, cranio-pharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma, carcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyl lodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing, experimental, Kaposi, and mast cell), neoplasms (e.g., bone, breast, digestive system, colorectal, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis, and cervical dysplasia, and for treatment of other conditions in which cells have become immortalized or transformed. The invention could be used in combination with other treatment modalities, such as chemotherapy, cryotherapy, hyperthernia, radiation therapy, and the like.

As disclosed herein, the present invention is not limited to any type of RNAi target gene or nucleotide sequence. The following classes of possible RNAi target genes are listed for illustrative purposes: developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, angiogenic factors and their receptors such as VEGF, HIF, VEGFR, antiangiogenic factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, and WT1); and enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hemicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, topoisomerases, and xylanases). In one preferred embodiment, the RNAi target gene is the Rhodopsin gene, either in its non-mutated (non-pathogenic) form or its mutated (pathogenic) form.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature; see, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.)It must be noted that as used herein and in the appended claims, the singular forms “a” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” or “the cell” includes a plurality (“cells” or “the cells”), and so forth. Moreover, the word “or” can either be exclusive in nature (i.e., either A or B, but not A and B together), or inclusive in nature (A or B, including A alone, B alone, but also A and B together) unless the context clearly dictates otherwise. One of skill in the art will realize which interpretation is the most appropriate unless it is detailed by reference in the text as “either A or B” (exclusive “or”) or “and/or” (inclusive “or”).

(1) Definitions

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

For purposes of this invention, the term “gene therapy” means the transfer of nucleic acid compositions into cells of a multicellular eukaryotic organism, be it in vivo, ex vivo or in vitro (see also [67] [68]). The term “gene therapy” should not be limited to the purpose of correcting metabolic disorders, but be interpreted more as a technical term for the transfer of nucleic acid compositions for therapeutic purposes in general, independent of a specific therapeutic purpose. Therefore, the term “gene therapy” would include without limitation correction of metabolic disorders, cancer therapy, vaccination, monitoring of cell populations, cell expansion, stem cell manipulation etc. by means of transfer of nucleic acid compositions.

For purposes of this invention, “transfection” is used to refer to the uptake of nucleic acid compositions by a cell. A cell has been “transfected” when an exogenous nucleic acid composition has crossed the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., [69, 70], Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and [71]. Such techniques can be used to introduce one or more nucleic acid compositions, such as a plasmid vector and other nucleic acid molecules, into suitable host cells. The term refers to both stable and transient uptake of the genetic material. For purposes of this invention, “transduction” is a special form of “transfection” via a viral vector.

For purposes of this invention, “transduction” denotes the delivery of a nucleic acid composition to, into or within a recipient cell either in vivo, in vitro or ex vivo, via a virus or viral vector, such as via a recombinant AAV virion. Transduction is a special form of transfection, i.e., the term transfection includes the term transduction.

For purposes of this invention, “nucleic acid composition transfer”, “nucleic acid composition delivery”, “gene transfer” or “gene delivery” refers to methods or systems for transferring nucleic acid compositions into host cells. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Nucleic acid composition transfer provides a unique approach for the treatment of inherited and acquired diseases including cancer. A number of systems and methods have been developed for nucleic acids composition transfer into mammalian cells. The transfer of an RNAi expression cassette is one example of a nucleic acid composition transfer.

For purposes of this invention, by “vector”, “transfer vector”, “gene transfer vector” or “nucleic acid composition transfer vector” is meant any element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of transferr