WO2011103394A2 - Methods for gene inhibition - Google Patents

Abstract

A Method of gene inhibition by circular, circular-like, or bidirectional interfering nucleotide (iNA) duplexes. The method can silence one or more gene(s) through RNAi mechanism.

Description

METHODS FOR GENE INHIBITION

BACKGROUND All publications, references, patents, patent publications and patent applications cited herein are each hereby specifically incorporated by reference in its entirety.

RNA interference (RNAi) is a form of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) induces the enzymatic degradation of homologous messenger RNA (mRNA). When a long dsRNA enters a cell, an enzyme called Dicer binds and cleaves long, dsRNA. Cleavage by Dicer results in the production of a small interfering RNA (siRNA) that is generally 20 - 25 base pairs in length and has a 2-nucleotide-long 3' overhang on each strand. Generically, an interfering RNA is also called an interfering nucleic acid (iNA), because non-RNA nucleotides can be incorporated into the construct. One of the two strands of each iNA, generally the antisense strand, is then incorporated into an RNA- induced silencing complex (RISC), and pairs with complementary sequences. RISC first mediates the unwinding of the iNA duplex. A single-stranded iNA that is coupled to RISC, then binds to a target mRNA in a sequence-specific manner. The binding mediates target mRNA cleavage by slicer, an argonaute protein that is the catalytic component of RISC. The cleavage of the mRNA prevents translation from occurring, which prevents the ultimate expression of the gene from which the mRNA is transcribed.

As the fragments produced by Dicer are double-stranded, each strand could in theory plays as the guide strand. However, only one of the two strands binds the argonaute protein as the guide strand and directs gene silencing. The other strand as the passenger strand is degraded during RISC activation. The strand selected as the antisense tends to be that with a less stable 5' end.

RNA interference has a tremendous potential in medicinal therapeutics, such as in anti-viral, anti-oncogenic and anti-inflammatory applications. The double -stranded iNA may be a long double-strand designed to be cleaved by Dicer, called Dicer substrate. Or the iNA may be short and designed to bypass Dicer serve directly as a

RISC substrate. The dsRNAs are synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, RNA interference can cause a drastic decrease in the expression of a targeted gene.

Medicine

RNAi interference can be used to develop a whole new class of therapeutics. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA mimics has been more successful. Among the first group of applications to reach clinical trials were in the treatment of age-related macular degeneration, and respiratory syncytial virus. Other proposed clinical uses center on antiviral therapies, including the inhibition of viral gene expression in cancerous cells, knockdown of host receptors and co-receptors for HIV, knockdown of gene of various kinds of virus including hepatitis A, hepatitis B and hepatitis C genes, influenza, and measles. Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the

polyglutamine diseases such as Huntington's disease. RNA interference is also often seen as a promising way to treat cancer by silencing genes differentially up -regulated in tumor cells or genes involved in cell division. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method.

Despite the proliferation of promising cell culture studies for RNAi -based drugs, some concern has been raised regarding the safety of RNA interference, especially the potential for "off-target" effects in which a gene with a coincidentally similar sequence to the targeted gene is also repressed. A computational genomics study estimated that the error rate of off-target interactions is about 10%. In mammalian cells, however, the use of RNAi for targeted gene silencing has been limited due to nonspecific effects induced by long dsRNAs, which result in interferon response. Therefore, for applications in mammals, iNAs have to be designed to be less than 30 based pairs in length to avoid the interferon response.

However, in developing a therapeutic drug for a mammal, it would be desirable to create an iNA for use in RNAi without interferon response. An example of this is an iNA or siRNA that contains one or more than one therapeutic targets in bidirectional, circular, or circular-like configurations which will be different from traditional double- stranded siRNA.

DESCRIPTION The disclosed compounds and processes fill this need by providing for interfering nucleic acid (iNA) having antisense sequences that can target or hybridize to targeted RNA(s), microRNA (miRNA), or promoter of targeted gene(s) in a sequence specific manner. The present invention provides for iNAs having a bidirectional double-stranded linear structure (Fig. 1). Two or more complementary strands of RNA or RNA-like oligonucleotides form double-stranded RNA or RNA-like oligo. At least one strand is intact and is bidirectional that some region(s) of the oligo is 5 ' to 3 ' direction and some region(s) of the same oligo is 3 ' to 5 ' direction. In addition, one or more RNA or RNA- like oligonucleotides is/are complementary to different regions of the intact

bidirectional strand to form one or more double-stranded segmented region(s).

Although the intact strand is bidirectional, any double-stranded region is formed between the intact strand and any other strand(s) as normal double stranded nucleic acid which means one strand is 5 ' to 3 ' direction and another strand is 3 ' to 5 ' direction. The length of the intact bidirectional strand can be as short as 10 nucleotides and as long as thousand nucleotides or whole length of any mR A or whole length of more than one mRNA combined, the length of any region of continuous nucleotides in one direction (either 5' to 3' or 3' to 5') should be any number of nucleotides from 10 to 200 nucleotides, preferable from 15 to 29 nucleotides. The other strand(s)

complementary to different section(s) of the intact bidirectional strand can be any length from 10 to 200 nucleotides, but formed double-stranded region should be from 10 to 200 nucleotides in length, preferable 15 to 29 nucleotides in length. The gap between double-stranded regions can be as short as 0 nucleotide (called nick) and as long as thousand nucleotides. The short strand in the double-stranded region can be fully or partially complementary to the long strand. If only partially complementary between two strands in a region, one or more than one nucleotides of the strands may not be paired (complementary). The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends, or anywhere between 5' end and 3 '-end. One strand of the one double -stranded region is complementary to targeted RNA(s) resulting in the degradation of the targeted RNA(s) through RNAi mechanisms. One bidirectional iNA or siRNA can have one or more double-stranded region(s). Therefore, one bidirectional iNA or siRNA is able to silence one or more genes through RNAi mechanisms or one bidirectional siRNA is able to complement to one or more region(s) of same RNA. The strand of the double- stranded oligo complementary to the targeted RNA can be fully or partially

complementary to target region of a RNA. If only portion of the strand is

complementary to the targeted RNA, some nucleotides of the strand will not be paired with the targeted RNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends, or anywhere between 5' end and 3 '-end.

The present invention also provides for iNAs having stem-loop like bidirectional structure (Fig. 2 and Fig. 3). One strand of RNA or RNA-like unidirectional or bidirectional [some region(s) of the oligo is 5 ' to 3 ' direction and some region(s) of the same oligo is 3 ' to 5 ' directionl] oligonucleotides can form one or more than one loop(s) with one or more than one stem(s). Each loop region can be as short as 3 nucleotides and as long as several thousand nucleotides or whole length of any mRNA or whole length of more than one mRNA combined. The length of stem region can be as short as 10 nucleotides and as long as 200 nucleotides, preferable 15 to 29

nucleotides. One or more strand RNA or RNA-like oligo(s) can be complementary to the whole or part of loop region to form one or more double-stranded region(s). The each double-stranded region can be as short as 10 nucleotides and as long as 200 nucleotides, preferable 15 to 29 nucleotides. Furthermore, one or more than one RNA or RNA-like oligo(s) can be complementary to a single strand region(s) which is not in loop to form double-strand. Any formed double-stranded region can be from 10 to 200 nucleotides in length, preferable 15 to 29 nucleotides in length. Any gap between each double-stranded region (either the double-stranded region on loop or on stem) can be as short as 0 nucleotide (called nick) and as long as several thousand nucleotides. Two strands of any double-stranded region can be fully or partially complementary to each other. If only partially complementary between two strands, one or more than one nucleotides of the strands may not be paired. The unpaired nucleotides can be on 5'- end, or 3 '-end, or both ends, or anywhere between 5' -end and 3 '-end. One strand of one double-stranded region (either the double-stranded region is on loop region or stem region) is complementary to targeted R A(s) resulting in the degradation of the targeted R A(s) through RNAi mechanisms. One unidirectional or bidirectional iNA or siRNA can have one or more double-stranded region(s). Therefore, one unidirectional or bidirectional iNA or siRNA is able to silence one or more genes through RNAi mechanisms or one unidirectional or bidirectional iNA or siRNA is able to complement to more than one regions of the targeting RNA. The strand of the double-stranded oligo complementary to the targeted mRNA can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted mRNA, some nucleotides of the strand will not be paired with the targeted mRNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends, or anywhere between 5' end and 3 '-end.

The present invention further provides for an interfering nucleic acid (iNA) duplex comprised of one strand of RNA or RNA-like bidirectional oligonucleotides forming one or more than one circular loop-like structure (Fig. 4). Some region(s) of the oligo is 5 ' to 3 ' direction and some region(s) of the same oligo is 3 ' to 5 ' direction. In addition, some regions of the strand are complementary each other and can form self- assembled double-stranded region according to the law of base-pair formation of nucleic acid. Preferably, the 5 '-end region and 3 '-end region of the same strand are complementary each other to form double-stranded region, the other region between the 5 '-end region and 3 '-end region will form a circular-like loop. The each double- stranded region can function either as a siRNA or as a binder to bind different regions together. If the double-stranded region functions as a siRNA, the length of the double- stranded region is preferable 15 to 29 nucleotides. If the double -stranded region functions as a binder, the length of the double-stranded region can be as long as several thousand nucleotides and as short as 3 nucleotides. As long as the number of paired nucleotides or paired modified nucleotides is enough to form stable double-stranded region, any number of nucleotides is fine. The length of any circular-like loop can be as short as 10 nucleotides and as long as several thousand nucleotides or whole length of any mRNA or whole length of more than one mRNA combined. One or more than one strand RNA or RNA-like oligo(s) can be complementary to the whole or part of the loop region to form one or more than one double-stranded region(s). The each double- stranded region can be as short as 10 nucleotides and as long as 200 nucleotides, preferable 15 to 29 nucleotides. Any gap between each double-stranded region can be as short as 0 (called nick) nucleotide and as long as several thousand nucleotides. Two strand of any double-stranded region are fully or partially complementary to each other. If only partially complementary between two strands, one or more than one nucleotides of the strands may not be paired. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends, or anywhere between 5' end and 3 '-end. One strand of any one double- stranded region is complementary to targeted RNA(s) resulting in the degradation of the targeted RNA(s) through RNAi mechanisms. One bidirectional circular loop-like siRNA can have one or more than one double-stranded region(s). Therefore, one bidirectional circular loop-like iNA or siRNA is able to silence one or more than one genes through RNAi mechanisms or one bidirectional iNA or siRNA is able to complement to different region of the one RNA. The strand of the double-stranded oligo complementary to the targeted mRNA can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted mR A, some nucleotides of the strand will not be paired with the targeted mRNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends, or anywhere between 5' end and 3 '-end. The present invention further provides for an iNA having a circular or circularlike double stranded structure containing an antisense strand and a sense strand. In an alternative embodiment, one or more sense strands can be discontinuous and

complementing to the antisense strand which is intact and the antisense strand can target one or more sequences of different RNA, or one or more places of one RNA. In another alternative embodiment, one or more antisense strands can be discontinuous and the sense strand which is intact and the antisense strands can target one or more RNAs, or one or more region on same RNA.

In another alternative embodiment, both strands can be discontinuous. The antisense strands can target one or more than one RNAs, or one or more than one different sequences on same RNA.

Generally, the iNA is comprised of a sense strand and an antisense strand in which the total length of the duplex is at least 15 nucleotides in length and one or both of the strands of the iNA duplex has a nick or a gap in the nucleotide strand in a circular or circular-like configuration. The use of such circular or circular-like iNA having segmented sense or antisense strand unexpectedly results in lowering interferon response that would be predicted when a double-stranded RNA having length of longer than 30 nucleotides is introduced into a mammalian cell. The circular-like iNA structures also less likely induce interferon response since the circular configuration iNA is different from conventional double stranded linear siRNA which can cause cell innate immune response.

The present invention further provides for an interfering nucleic acid (iNA) duplex comprised of complementary strands of RNA or RNA-like oligonucleotides form double stranded RNA or RNA-like oligo. Sense strand and anti-sense strand of the double-stranded oligo are complementary to each other to form a circular or circularlike iNA with a nick or a gap, or both (Figure 5). The antisense strand of the double- stranded circular or circular-like oligo is complementary to one or more than one targeted RNA resulting in the degradation of the targeted RNA(s) through RNAi mechanisms. Also, the antisense strand of the double-stranded circular or circular-like oligo is able to complementary to one or more regions of a targeted RNA resulting in the degradation of the targeted RNA through RNAi mechanisms. The strand of the double-stranded oligo complementary to the targeted RNA can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted RNA, some nucleotides of the strand will not pair with the targeted RNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends. Although, the length of any strand of the circular or circular-like iNA can be as short as 10 nucleotides and as long as several thousands of nucleotides or full length of targeting RNA or full length of more than one RNA combine, each double -stranded region can be 7 to 100 nucleotides, preferable 15 to 29 nucleotides. The gap between each double -stranded region can be as short as 1 nucleotide and as long as several thousand nucleotides.

The present invention further provides for an interfering nucleic acid (iNA) duplex comprised of complementary strands of RNA or RNA-like oligonucleotides form double stranded RNA or RNA-like oligo. One strand of the double-stranded oligo has circular formation. The other strand is complementary to the circular strand with a nick (Figure 6). The circular strand can be antisense and the strand with nick can be sense strand, verse versa. One strand of the double-stranded oligo (which can be the strand with or without nick) is complementary to targeted RNA resulting in the degradation of targeted RNA through RNAi mechanisms. The strand of the double- stranded oligo complementary to the targeted RNA can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted RNA, some nucleotides of the strand will not be paired with the targeted RNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends. The length of the double-stranded RNA oligo can be 10 to 100 nucleotides, preferable 15 to 29 nucleotides.

The present invention further provides for an interfering nucleic acid (iNA) duplex comprised of complementary strands of RNA or RNA-like oligonucleotides form double stranded RNA or RNA-like oligo. One strand of the double-stranded oligo has circular formation. The other strand is complementary to the circular strand with a gap (Figure 7). The circular strand can be antisense and gap stranded can be sense strand, verse versa. Any strand of the double-stranded oligo is complementary to targeted RNA (can be any RNA) and the siRNA is able to degrade the targeted RNA through RNAi mechanisms. The strand of the double-stranded oligo complementary to the targeted RNA can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted RNA, some nucleotides of the strand will not be paired with the targeted RNA. The unpaired nucleotides can be on 5 ' - end, or 3 '-end, or both ends. The length of the double-stranded RNA oligo can be 10 to 100 nucleotides, preferable 15 to 29 nucleotides.

The present invention further provides for an interfering nucleic acid (iNA) duplex comprised of two complementary strands of RNA or RNA-like oligonucleotides form double stranded RNA or RNA-like oligo. Some of the RNA nucleotides can be chemically modified. Also, some of the nucleotides can be DNA nucleotides or any non-nucleotides. One strand of double-stranded oligo has circular formation. The other strand is complementary to the circular strand with a gap (Figure 7). The circular strand can be antisense and gap stranded can be sense strand, verse versa. The length of the strand with closed circular configuration can be as short as 10 nucleotides and as long as several thousands of nucleotides or whole length of targeting RNA. However, the length of the other strand(s) which is complementary to the circular strand is from 10 to 100 nucleotides, preferable 15 to 29 nucleotides. Therefore, the length of any double- stranded region is 10 to 100 nucleotides, preferable 15 to 29 nucleotides. The gap can be as short as 0 nucleotide (called nick) and as long as several thousand nucleotides. Any one strand of the double-stranded oligo can be complement to one or more targeted RNA(s) and resulting in the degradation of the targeted RNA(s) through RNAi mechanisms. The strand of the double-stranded oligo complementary to the targeted RNA can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted RNA, some nucleotides of the strand will not be paired with the targeted RNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends.

The present invention further provides for an interfering nucleic acid (iNA) duplex comprised of one strand of RNA or RNA-like oligonucleotides has a circular structure with one or more strand(s) complementary to different regions of the circular stranded (Figure 8). Therefore, it can also be described that the iNA duplex comprised of one strand of RNA or RNA-like oligonucleotides has a circular structure with one or more complementary strands with nick or gap (Figure 4 and Figure 8). The number of short strand(s) and the gap(s) or nick(s) in the structure can be one or more than one. Although, the length of the circular strand can be as short as 10 nucleotides and as long as several thousands of nucleotides or full length of targeted RNA or full length of more than one targeted RNAs combined, any other stand(s) which are complementary to the circular strand and form double-stranded region with the circular strand is 7 to 100 nucleotides, preferable 15 to 29 nucleotides. Therefore, although it can be one, more than one double-stranded regions, each double-stranded region will be 7 to 100 nucleotides, preferable 15 to 29 nucleotides. The gap between each double-stranded region can be a nick or gap between 1 nucleotide and several thousand nucleotides. One strand of the any double-stranded region is complementary to targeted RNA(s) resulting in the degradation of the targeted RNA(s) through RNAi mechanisms. Each double stranded region can target one or more than one RNA. Therefore, one circular-like iNA is able to silence one or more genes through RNAi mechanisms or complement to one than one regions of the same RNA. The strand of the double-stranded oligo

complementary to the targeted RNA can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted RNA, some nucleotides of the strand will not be paired with the targeted RNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends.

The disclosed compounds and processes further fill this need by providing an interfering nucleic acid (iNA) duplexe comprised of multiple strands of RNA or RNA- like oligonucleotides form circular-like double-stranded oligo with one or more gaps or nick (Figure 9). Although the length of the circular-like formation can be as short as 10 nucleotides and as long as several thousand nucleotides or full length of any RNA or full length of more than one RNA combined, any double -stranded region is from 7 to 100 nucleotides, preferable from 15 to 29 nucleotides. The gap can be as short as 1 nucleotide and as long as several thousand nucleotides. One strand of the double - stranded region is complementary to targeted RNA resulting in the degradation of the targeted RNA through RNAi mechanisms. If there are more than one double strand region in the structure, there are more than one RNAs can be targeted or more than one regions of same RNA can be targeted. The strand of the double-stranded oligo complementary to the targeted RNA can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted RNA, some nucleotides of the strand will not be paired with the targeted RNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends.

The disclosed compounds and processes further fill this need by providing a circular or circular like interfering nucleic acid (iNA) duplexes in which some double strand region of RNA or RNA-like oligonucleotides are partially complementary to each other (Figure 10) at any position(s) and any number(s) of nucleotides. The basic structures and circular duplex characteristics are similar to ones in Figure 4, 5, 6, 7, and 8. The disclosed compounds and processes further fill this need by providing an interfering nucleic acid (iNA) duplexes comprised of two or more strands of RNA or RNA-like oligonucleotides form a circular or broken circular conformation (fully or partially) as mentioned in Figure 6, 7, and 8 with sticky ends which are not complement to the opposite strand (Figure 11).

The present invention further provides an interfering nucleic acid (iNA) duplex comprised of one strand of RNA or RNA-like oligonucleotides having a stem-loop structure and another strand is complementary to region of the loop (Figure 3). The length of the stem can be as long as several hundred nucleotides and as short as 3 nucleotides. One strand of RNA or RNA-like oligonucleotides of double stranded loop region is complementary to targeted RNA. The length of double -stranded region can be from 10 to 100 nucleotides, preferable 15 to 29 nucleotides. One strand (antisense) of RNA or RNA-like oligonucleotides of double stranded loop region is complementary to targeted RNA(s), resulting in the degradation of the targeted RNA(s) through RNAi mechanisms. The length of double stranded loop can be full-length of the strand, or portion of the strand. If only portion of the strand is complementary to the targeted RNA, some nucleotides of the strand will not be paired with the targeted RNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends. The present invention further provides for an interfering nucleic acid (iNA) duplex comprised of one strand of RNA or RNA-like oligonucleotides having a stem- loop structure and another strand is complementary to region of the loop (Figure 3). The length of the stem can be as long as several hundred nucleotides and as short as 3 nucleotides. One strand of RNA or RNA-like oligonucleotides of double stranded loop region is complementary to targeted RNA. The length of double-stranded region can be from 10 to 100 nucleotides, preferable 15 to 29 nucleotides. One or more strands of RNA or RNA-like oligonucleotides with gap (s) or nick (s) or both are complementary or partially complementary to the region of the loop to form one or more double- stranded regions in the double stranded stem loop structure. Although the length of the loop region can be as short as 10 nucleotides and as long as several thousands of nucleotides or full length of targeting RNA or full length of more than one RNA combined, the strands complementary to the loop region is 7 to 100 nucleotides, preferable 15 to 27 nucleotides. The gap between each double -stranded regions can be from 0 (nick) to several thousand nucleotides. One strand of the any double-stranded region can be complementary to targeted RNA(s), resulting in the degradation of the targeted R A(s) through RNAi mechanisms. Also, the strands can be complementary to different RNAs to target different RNAs or target different regions of same RNA to degrade the RNA through RNAi mechanism. The strands of the double -stranded oligo complementary to the targeted RNA can be full-length of the strands, or portion of the strands. If only portion of the strands is complementary to the targeted RNA, some nucleotides of the strand will not be paired with the targeted RNA. The unpaired nucleotides can be on 5 '-end, or 3 '-end, or both ends.

Preferably each partial sense or antisense strand should be at least 7 nucleotides long to properly anneal to its complementary antisense or sense sequence. If it is desired that a particular partial strand be an available target to a RNA, then the length of the partial sequence should be at least 14 nucleotides or more in length.

In an alternative embodiment, any of the above described circular, circular-like or bidirectional iNA or siRAN can be used to up-regulate gene expression if one strand of the any double-stranded regions is complementary to a promoter region of any targeted gene(s) through RNAa mechanism. Also, the circular, circular-like or bidirectional iNA or siRNA can be designed as such that certain double-stranded region(s) of one circular, circular-like or bidirectional iNA or siRNA can be silence one or more than one gene through RNAi mechanisms and other regions of the same iNA or siRNA can up-regulate one or more than one gene(s) through RNAa mechanisms.

Generally, the use of such circular, circular-like or bidirectional iNA or siRNA results in lowering interferon response that would be predicted when a double-stranded RNA having length of longer than 30 nucleotides is introduced into a mammalian cell. The circular, circular-like or bidirectional iNA structures also less likely induce interferon response since the iNA configuration is different from conventional double- stranded siRNA which can cause cell innate immune response.

This disclosure provides for pharmaceutically acceptable nucleic acid compositions useful for therapeutic delivery of nucleic acids and gene -silencing iNAs. In particular, this invention provides compositions and methods for in vitro and in vivo delivery of iNAs applicable for decreasing, down regulating, or silencing the translation of a target nucleic acid sequence or expression of a gene. These compositions and methods may be used for prevention and/or treatment of diseases in a mammal.

A therapeutic strategy based on RNAi can be used to treat a wide range of diseases by shutting down the growth or function of a virus or microorganism, as well as by shutting down the function of an endogenous gene product in pathways of diseases.

In some embodiments, this invention provides novel compositions and methods for delivery of RNAi -inducing entities as circular, circular-like or bidirectional iNA or siRNA molecules having one or more than one double-stranded region wherein one or more than one of the segmented double strands can be formed. In particular, this invention further provides for compositions containing an RNAi -inducing entity that is to target to one, two, three, four, five, or more transcripts of a cell, tissue, and/or organ of a subject.

In some embodiments, this invention provides novel compositions and methods for delivery of RNAi -inducing entities as a circular, circular-like or bidirectional iNA or siRNA molecules having one or more than one double-stranded region wherein the biological immune response of cell to traditional double-stranded RNA molecules can be avoided.

The iNAs or siRNAs can mediate specific gene silencing in the mammalian system. Circular, circular-like or bidirectional iNAs or siRNAs having one or more than one double-strand region with gap or nick between each double-strand region enter cells. Dicer in cells including mammalian cells can convert these kinds of iNAs into iNA or siRNA to mediate gene silencing of specific gene(s). It has been surprisingly discovered that when such an iNA duplex having a length of more than 30 nucleotides with segmented sense or antisense strand, is transfected into a mammalian cell, the expected interferon response is greatly reduced or undetectable. This allows for the use of circular, circular-like or bidirectional iNA duplexes that are 30 - 200 or more nucleotides in length to silence gene in mammalian cells.

This disclosure provides pharmaceutically acceptable nucleic acid compositions useful for therapeutic delivery of nucleic acids and gene -silencing iNAs. In particular, this invention provides compositions and methods for in vitro and in vivo delivery of iNAs decreasing, down -regulating, or silencing the translation of a target nucleic acid sequence or expression of a gene. These compositions and methods may be used for prevention and/or treatment of diseases in a mammal. A therapeutic strategy based on RNAi can be used to treat a wide range of diseases by shutting down the growth or function of a virus or microorganism, as well as by shutting down the function of an gene product in pathway(s) of diseases.

In some embodiments, this invention provides novel compositions and methods for delivering RNAi -inducing entities such as circular, circular-like or bidirectional interfering oligonucleotide molecules having one or more segmented strands, and precursors thereof. In particular, this invention further provides for compositions containing an RNAi -inducing entity that is targeted to one or more transcripts of a cell, tissue, and/or organ of a subject.

By having the ability to transfect a mammalian cell with a circular, circular-like or bidirectional iNA duplex that is 30 nucleotides in length or longer, one can design iNA duplexes that can be processed to target more than one RNA. For example, one segment of the antisense strand could target the RNA for a ligand while a second segment of the same antisense strand could target the RNA of a receptor or another ligand. Each segment can be 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides in length or longer, thus the length of each iNA duplex will be preferably 15 to 200 nucleotides in length or longer depending on how many RNAs intend to be targeted. In another embodiment, both the sense and antisense strands can be designed so that one or more segments of both the sense and antisense strands target one or more than one RNAs. The iNAs can be delivered as single or multiple transcription products expressed by a polynucleotide vector encoding the single or multiple iNAs and directing their expression within target cells. Typically, the iNA will target a gene that is expressed at an elevated level as a causal or contributing factor associated with the subject disease state or adverse condition. In this context, the iNA will effectively down -regulate expression of the gene to the levels that prevent, alleviate, or reduce the severity or recurrence of one or more associated disease symptoms. Alternatively, for various distinct diseases where expression of the target gene is not necessarily elevated as a consequence or sequel of disease or other adverse condition, down-regulation of the target gene will nonetheless result in a therapeutic result by lowering gene expression (i.e., to reduce levels of a selected RNA and/or protein product of the target gene).

Alternatively, iNAs of the invention may target to lower expression of one gene, which can result in up-regulation of a "downstream" gene whose expression is negatively regulated by a product or activity of the targeted gene. The circular, circular-like or bidirectional iNAs or siRNAs can be delivered in any cells including mammalian cells to target any transcripts including RNA, none code RNA, rRNA, tRNA, and miRNA. In addition, the circular, circular-like or bidirectional iNAs or siRNAs can be delivered in any cells including mammalian cells to target any promoter of gene to up-regulate its gene expression by RNAa mechanisms.

The present invention further provides molecules as drug to treat a variety of disease by silencing disease gene expression, including, but not limited to heart disease, kidney disease, diabetes, genetic diseases, infectious diseases, hypertension, cancer by targeting genes such as, alpha- 1 -Antitrypsin, transthyretin, hepatitis B viral genes, hepatitis C viral gene, HIV, ApoB, PCSK9, SIP, SREBP2, SCAP, SREBP2, DGAT2, SCD1, PTP-1B, ACC1, Glycogen phosphorylase, Glucagon receptor, angiotensiogen, ATI, ACE, endothelin (ET-1), ETA receptor, Factor II, Factor V, Factor VII, Factor VIII, FactorIX, Factor X, Factor XI, hepcidin, 5-a reductase, ABCB1, ABL1, AHA1 , Angiopoietin-2, ATM, BAG1, BAX, BCL2, BCR, BCR/ABL, BRCA1,CAV1, CAV2, CD 10, CD 19, CD40, CDC2, CDK2, CDK4, CHK-1, CREBl, CREBBP, Cyclin Dl, DAXX, ECGF1, EGFR, EGR-1, EPHA2, ERBB2, ERBB3, ERBB4, EZH2, FGF2, FIGF, FLOT1, FLT1, FLT4, GADD45A, GSK3B, HER1, HER2, HER3, HER4, Heregulin-βΐ, HSP70, HSP90AA, HSP90AB, HSPB1, IL-6, KDR, KIF11, LIMK1, LIMK2, MAP2K2, MAP2K4, MAP2K7, MAPK14, MAPK3, MAPK8, MDM2, MDR, MMP13, MMP19, MMP26, MMP9, MT1-MMP, Myc, NFKB1, N-Ras, p38, MAPK, PCNA, PDGF, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGFR, PDPKl, PGF, PGF-1, PKC-a, Plk-1, PRDX3, PTEN, PTPN11 (Shp2), RAB4A, RAF1, RBI, SDF-1, SMAC, SRC, SRD5A1, STAT3, Telomerase, TGFB1, TGFBR1, TLR3, TLR7, TLR8, TLR9, TOMM20, TOMM70A, TOPI , topoisomerase II alpha, Type 1 IGFR, UBE2C, UBE2H, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR,2 VEGFR3, WNT3A.

DESCRIPTION OF THE DRAWINGS Fig. 1. The diagrams show some forms of linear bidirectional iNA. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 2. The diagrams show some forms of stem-loop like bidirectional iNA. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 3. The diagrams show some forms of stem-loop like bidirectional iNA with one or more strand(s) complementary to the loop region. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 4. The diagrams show some forms of circular-like bidirectional iNA. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 5. Diagrams of circular or circular-like iNA with a nick(s) or a gap on both strands. The figure also shows the diagram of circular or circular-like with or without unpaired nucleotides. The arrows represent direction from 5' to 3' end of

oligonucleotides.

Fig. 6. Diagrams of circular or circular-like iNA with a nick on either one of the two strands. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 7. Diagrams of circular or circular-like iNA with a gap on either one of the two strands. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 8. Diagrams of circular or circular-like iNA with multiple gaps or nicks or both on one of the two strands. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 9. Diagrams of circular or circular-like iNA with multiple gaps or nicks or both on either one or both of the two strands. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 10. Diagrams of circular or circular-like iNA with unpaired nucleotide regions, with or without gap(s) or nick(s) or both on one of the two strands. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 11. Diagrams of circular or circular-like iNA with unpaired nucleotides, with or without gap(s) or nick(s) or both on one of the two strands. The arrows represent direction from 5' to 3' end of oligonucleotides.

Fig. 12 shows knockdown of LacZ gene expression in 9L/LacZ cells with the siRNAs disclosed in the Example section (siRNA number 1 and 4-7).

Fig. 13 shows knockdown of ApoB and Lamin gene expression in HepG2 cells with the siRNAs disclosed in the Example section (siRNA number 2, 3 and 8-13).

Fig. 14 shows the level of interferon βΐ (ΤΕΝβ Ι) when siRNA constructs disclosed in the Example section (siRNA number 1-13) are transfected into HepG2 cells. Poly I:C is the positive control for inducing interferon β 1.

DEFINITIONS Definitions of technical terms provided herein should be construed to include without recitation those meanings associated with these terms known to those skilled in the art, and are not intended to limit the scope of the invention.

The use herein of the terms "a," "an," "the," and similar terms in describing the invention, and in the claims, are to be construed to include both the singular and the plural. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms which mean, for example, "including, but not limited to." Recitation of a range of values herein refers individually to each and any separate value falling within the range as if it were individually recited herein, whether or not some of the values within the range are expressly recited. Specific values employed herein will be understood as exemplary and not to limit the scope of the invention.

As used herein, the term interfering nucleic acid (iNA) refers to a nucleic acid duplexes having strands complementary each other, which when entered into a RISC complex induces enzymatic degradation of RNA through RNAi mechanisms. Also, the iNA can up-regulate gene expression by targeting promoter of genes through RNAa mechanisms. Generally each strand contains predominantly RNA nucleotides but the strands can contain RNA analogs, RNA and RNA analogs, modified nucleotide(s), RNA and DNA, RNA analogs and DNA, non-nucleotide(s), or one strand that is completely DNA and one strand that is RNA as long as the iNA construct induces enzymatic degradation of a homologous RNA.

As used herein, the term "bidirectional iNA duplex" or "bidirectional siRNA duplex" or "bidirectional iNA(s)" is a generic term used throughout the specification to include interfering nucleic acids (iNAs) with a bidirectional configuration, which can be cleaved in cells to form iNAs. The bidirectional iNA duplexes has at least one strand having one or more region(s) in 5' to 3' direction and one or more different region(s) of the same strand in 3 ' to 5 ' direction. One or more other strand(s) are complementary to different region(s) of the strand having bidirectional configuration to form one or more than one double-stranded region(s). Between each double-stranded region, there can be one or more than one nick(s) and/or gap(s) on one of the strands. Bidirectional iNA can be liner, stem-loop, or circular-like configuration. The terminal structure of iNA may be either blunt or cohesive (overhanging) as long as the iNA can silence the target RNA. The cohesive (overhanging) end structure is not limited only to the 3' overhang, as the 5' overhanging structure may be included as long as it is capable of inducing the RNAi effect. In addition, the number of overhanging nucleotides is not limited to the reported 2 or 3, but can be any number as long as the iNA is capable of inducing the RNAi effect. For example, the overhang may be 1 to 8 or longer, or 2 to 4 nucleotides or longer.

As used herein, the term "circular or circular-like iNA duplex" is a generic term used throughout the specification to include interfering nucleic acids (iNAs) with a circular or circular-like configuration, which can be cleaved in cells to form iNAs. The circular or circular-like iNA duplexes herein also include expression vectors (also referred to as iNA expression vectors) capable of giving rise to transcripts which form iNA duplexes or in cells, and/or transcripts which can produce iNAs in vivo.

Optionally, the circular or circular-like iNA includes single strand that form a duplex by double strands of iNA. The circular or circular-like iNA is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target ribonucleic acid molecule for down regulating expression, or a portion thereof. The sense strand or antisense strand has one or more than one nicks or nucleotide. The terminal structure of iNA may be either blunt or cohesive (overhanging) as long as the iNA can silence the target RNA. The cohesive (overhanging) end structure is not limited only to the 3' overhang, as the 5' overhanging structure may be included as long as the iNA is capable of inducing the RNAi effect. In addition, the number of overhanging nucleotides is not limited to the reported 2 or 3, but can be any number as long as the the iNA is capable of inducing the RNAi effect. For example, the overhang may be 1 to 8 or longer, or 2 to 4 nucleotides or longer.

The Stem-loop structure refers to intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The double stranded stem-loop structure refers to the loop region of the stem-loop oligonucleotide are double stranded by complementary to a single or multiple strands. Also, there can be one or more strands complementary to any single strand region on stem. The terminal structure of stem region may be either blunt or cohesive (overhanging). There can be one or more strands complement to the loop region with or without nick or gap and with or without overhangs at either 3 ' or 5 ' end. There are either gaps or nick at the junction between the ends oligo(s) and the very beginning of the stem.

As used herein the length of circular, circular-like, or bidirectional iNA duplex is determined by counting the number of nucleotides in the duplex starting at the first base-pair at the 5' end of the sense strand and ending at the last base-pair at the 3' end of the sense strand.

In genetics, microRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem- loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA molecules, and their main function is to down-regulate gene expression.

Modified nucleotides in a circular, circular-like, or bidirectional iNA molecule can be in any of the strands. For example, modified nucleotides can have a Northern conformation (e.g., Northern pseudorotation cycle, see, for example, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Examples of nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2'-0, 4'- C-methylene-(D-ribofuranosyl) nucleotides), 2'-methoxyethoxy (MOE) nucleotides, 2'- methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy-2'-chloro nucleotides, 2'- azido nucleotides, and 2'-0-methyl nucleotides. Chemically modified nucleotides can be resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. A conjugate molecule attached to a chemically-modified iNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically -modified iNA molecules are described in Vargeese, et al, U.S. Patent Publication No. 20030130186 and U.S. Patent Publication No. 20040110296, which are each hereby incorporated by reference in their entirety.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-0-methyl, 2'- O-allyl, 2'-H, nucleotide base modifications. For a review see Usman and Cedergren, TIBS 17:34, 1992; Usman, et al, Nucleic Acids Symp. Ser. 31 :163, 1994; Burgin, et al, Biochemistry 35: 14090, 1996. Sugar modification of nucleic acid molecules have been extensively described in the art. See Eckstein et al., International Publication PCT No. WO 92/07065; Perrault, et al. Nature 344:565-568, 1990; Pieken, et al. Science 253 :314-317, 1991; Usman and Cedergren, Trends in Biochem. Sci. 17:334-339, 1992; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman, et al., J. Biol. Chem. 270:25702, 1995; Beigelman, et al., International PCT Publication No. WO 97/26270; Beigelman, et al, U.S. Pat. No. 5,716,824; Usman, et al, U.S. Pat. No. 5,627,053; Woolf, et al, International PCT Publication No. WO 98/13526; Thompson, et al, Karpeisky, et al, Tetrahedron Lett. 39:1131, 1998; Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences) 48:39-55, 1998; Verma and Eckstein, Annu. Rev. Biochem. 67:99-134, 1998; and Burlina, et al, Bioorg. Med. Chem. 5 :1999-2010, 1997. Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis. In view of such teachings, similar modifications can be used as described herein to modify the iNA nucleic acid molecules of the claimed duplexes so long as the ability of iNA to promote RNAi in cells is not significantly inhibited.

The circular, circular-like, or bidirectional iNA duplexes may contain modified iNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 1995, pp. 331-417, and Mesmaeker, et al., "Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research," ACS, 1994, pp. 24-39. Examples of chemical modifications that can be made in an iNA include phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. The antisense region of a iNA molecule can include a phosphorothioate internucleotide linkage at the 3 '-end of said antisense region. The antisense region can comprise about one to about five phosphorothioate

internucleotide linkages at the 5'-end of said antisense region. The 3'-terminal nucleotide overhangs of a circular, circular-like, or bidirectional iNA molecule can include ribonucleotides or deoxyribonucleotides that are chemically -modified at a nucleic acid sugar, base, or backbone. The 3 '-terminal nucleotide overhangs can include one or more universal base ribonucleotides. The 3'-terminal nucleotide overhangs can comprise one or more acyclic nucleotides. For example, a chemically -modified iNA can have 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages in one strand, or can have 1 to 8 or more phosphorothioate internucleotide linkages in each strand. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the bidirectional iNA duplex, for example in the sense strand, the antisense strand, or both strands. In some embodiments, a iNA molecule includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or in both strands.

The circular, circular-like, or bidirectional iNA molecules, which can be chemically-modified, can be synthesized by: (a) synthesis of at least two or

complementary strands of the RNA or RNA-like nucleotide oligo molecules; and (b) annealing the two or more complementary strands together under conditions suitable to obtain a iNA molecule. In some embodiments, synthesis of the complementary portions of the circular, circular-like, or bidirectional iNA molecule is by solid phase

oligonucleotide synthesis, or by solid phase tandem oligonucleotide synthesis.

Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers, et al, Methods in Enzymology 211 :3-19, 1992; Thompson, et al, International PCT Publication No. WO 99/54459; Wincott, et al, Nucleic Acids Res. 23 :2677-2684, 1995; Wincott, et al, Methods Mol. Bio. 74:59, 1997; Brennan, et al, Biotechnol Bioeng. 61 :33-45, 1998; and Brennan, U.S. Pat. No. 6,001,31 1. Synthesis of RNA, including certain iNA molecules of the invention, follows general procedures as described, for example, in Usman, et al, J. Am. Chem. Soc. 109:7845, 1987; Scaringe, et al, Nucleic Acids Res. 18:5433, 1990; and Wincott, et al, Nucleic Acids Res. 23 :2677-2684, 1995; Wincott, et al, Methods Mol. Bio. 74:59, 1997. The double-stranded structure may be formed by self-complementary iNA strand such as occurs for any two or more complementary strands or a hairpin RNA or by annealing of distinct complementary circular or circular-like iNA strands.

"Overlapping" refers to when two iNA fragments have sequences which overlap by a plurality of nucleotides on one strand, for example, where the plurality of nucleotides (nt) numbers as few as 2-5 nucleotides or by 5-10 nucleotides or more. "One or more circular, circular-like, or bidirectional iNAs" refers to iNAs that differ from each other on the basis of primary sequence.

By "target site" or "target sequence" or "targeted sequence" is meant a sequence within a target nucleic acid (e.g., RNA) that is "targeted" for cleavage mediated by an iNA or siRNA duplex which contains sequences within its antisense region that are complementary to the target sequence.

A nick in a strand is a break in the phosphodiester bond between two nucleotides in the backbone in one of the strands of the duplex of bidirectional iNA molecule.

A hybrid circular, circular-like, or bidirectional iNA molecule is a circular, circular-like, or bidirectional iNA that is a double-stranded nucleic acid. Instead of a double-stranded RNA molecule, a hybrid iNA is comprised of an RNA strand and a DNA strand. Preferably, the RNA strand is the antisense strand as that is the strand that binds to the target RNA. The hybrid circular, circular-like, or bidirectional iNA created by the hybridization of the DNA and RNA strands have a hybridized complementary portion and preferably at least one 3 Overhanging end.

To "modulate gene expression" as used herein is to up-regulate or down-regulate expression of a target gene, which can include up -regulation or down-regulation of RNA levels present in a cell, or of RNA translation, or of synthesis of protein or protein subunits, encoded by the target gene.

The terms "inhibit," "down-regulate," or "reduce expression," as used herein mean that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or level or activity of one or more proteins or protein subunits encoded by a target gene, is reduced below that observed in the absence of the nucleic acid molecules (e.g., iNA) of the invention.

"Gene silencing" as used herein refers to partial or complete inhibition of gene expression in a cell and may also be referred to as "gene knockdown." The extent of gene silencing may be determined by methods known in the art, some of which are summarized in International Publication No. WO 99/32619.

In some embodiments, circular, circular-like, or bidirectional iNA molecules comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non -nucleotide linker molecules, or are non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions.

The circular, circular-like, or bidirectional iNAs can be assembled from two separate oligonucleotides into a duplex, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure). The antisense strand may comprise a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand may comprise a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.

Alternatively, the bidirectional iNA can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the circular, circular-like, or bidirectional iNA are linked by means of a nucleic acid-based or non-nucleic acid- based linker(s). A circular, circular-like, or bidirectional iNA may be contain a nucleotide, non- nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the iNA to the antisense region of the iNA. In some embodiments, a nucleotide linker can be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the nucleotide linker can be a nucleic acid aptamer. As used herein, the terms "aptamer" or "nucleic acid aptamer" encompass a nucleic acid molecule that binds specifically to a target molecule, wherein the nucleic acid molecule contains a sequence that is recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. For example, the aptamer can be used to bind to a ligand -binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. See, for example, Gold, et al, Annu. Rev. Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical Chemistry 45: 1628, 1999.

A non-nucleotide linker can be an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 18:6353, 1990, and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J. Am. Chem. Soc. 113:6324, 1991; Richardson and Schepartz, J. Am. Chem. Soc. 113 :5109, 1991; Ma, et al, Nucleic Acids Res. 21 :2585, 1993, and Biochemistry 32:1751, 1993; Durand, et al, Nucleic Acids Res. 18:6353, 1990; McCurdy, et al, Nucleosides & Nucleotides 10:287, 1991; Jschke, et al, Tetrahedron Lett. 34:301, 1993; Ono, et al, Biochemistry 30:9914, 1991; Arnold, et al, International Publication No. WO 89/02439; Usman, et al, International Publication No. WO 95/06731; Dudycz, et al., International Publication No. WO 95/11910, and Ferentz and Verdine, J. Am Chem. Soc. 113:4000, 1991. A "non-nucleotide linker" refers to a group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the CI position of the sugar. The term "biodegradable linker" as used herein, refers to a nucleic acid or non- nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a iNA molecule or the sense and antisense strands of a iNA molecule. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be variously modulated, for example, by

combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2'-0-methyl, 2'-fluoro, 2'-amino, 2'-0-amino, 2'-C-allyl, 2'-0-allyl, and other 2'-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

By "antisense nucleic acid", it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA--RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al, U.S. Pat. No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) noncontiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof. "Antisense RNA" is an RNA strand having a sequence complementary to a target gene RNA, that can induce RNAi by binding to the target gene RNA. Antisense RNA" is an RNA strand having a sequence complementary to a target gene RNA, and thought to induce RNAi by binding to the target gene RNA. "Sense RNA" has a sequence complementary to the antisense RNA, and annealed to its complementary antisense RNA to form iNA. These antisense and sense RNAs have been conventionally synthesized with an RNA synthesizer.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral -methyl phosphonates, 2'-0-methyl ribonucleotides, peptide -nucleic acids (PNAs).

By "R A" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a BD-ribo-furanose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the iNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. As used herein, the terms "ribonucleic acid" and "RNA" refer to a molecule containing at least one ribonucleotide residue. A ribonucleotide is a nucleotide with a hydroxyl group at the 2' position of a B-D-ribo- furanose moiety. These terms include double-stranded RNA, single -stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, modification, and/or alteration of one or more nucleotides. Alterations of an RNA can include addition of non-nucleotide material, such as to the end(s) of a iNA or internally, for example at one or more nucleotides of an RNA nucleotides in an RNA molecule include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs.

By the term "non-nucleotide" is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the l'-position. By "nucleotide" as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the Γ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein, et al, International PCT Publication No. WO 92/07065; Usman, et al, International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183, 1994. Some of the non- limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5- alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5- halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6- methyluridine), propyne, and others (Burgin, et al, Biochemistry 35 :14090, 1996;

Uhlman & Peyman, supra). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at Γ position or their equivalents.

As used herein complementary nucleotide bases are a pair of nucleotide bases that form hydrogen bonds with each other. Adenine (A) pairs with thymine (T) or with uracil (U) in R A, and guanine (G) pairs with cytosine (C). Complementary segments or strands of nucleic acid that hybridize (join by hydrogen bonding) with each other. By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence either by traditional Watson-Crick or by other non-traditional modes of binding.

The sense strand of a double stranded iNA molecule may have a terminal cap moiety such as an inverted deoxyabasic moiety, at the 3 '-end, 5 '-end, or both 3' and 5'- ends of the sense strand.

By "cap structure" is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic, et al, U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal

modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5'- terminus (5'-cap) or at the 3'-terminal (3'-cap) or may be present on both termini. In non-limiting examples, the 5'-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4- dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'- inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3 '-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety stillben and pyrene.

Examples of the 3'-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4',5 '-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3- diamino-2 -propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2- aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L- nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo- pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5 '-5 '-inverted nucleotide moiety; 5 '-5 '-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1 ,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or

phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Lyer, Tetrahedron 49:1925 , 1993) and stillben and pyrene.

An "asymmetric hairpin" as used herein is a linear iNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non -nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop.

An "asymmetric duplex" as used herein is an iNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex.

The circular, circular-like, or bidirectional iNA can be chemically synthesized or biologically produced. The methods for chemical synthesis can be found in Nucleic Acids Research, 624-627, 1999 and Nucleic Acids Research, 3547-3553, 1955. The techniques used in molecular cloning techniques can be used for biology production.

As used herein the term small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is used to refer to a class of double-stranded RNA molecules, 16-29 nucleotides in length, that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated.

As used herein, the term RNAa refers to the phenomenon of double-stranded RNA activating gene expression. The phenomenon has been termed "small RNA- induced gene activation" or RNAa. A double-stranded RNA targeting gene promoters induce potent transcriptional activation of associated genes. Studies demonstrate RNAa in human cells using synthetic dsRNAs termed small activating RNAs (saRNAs).

Endogenous miRNA that cause RNAa has also been found in humans. Very recently, RNAa has been demonstrated in several mammalian species other than human including non-human primates, mouse and rat, suggesting that RNAa is a general gene regulation mechanism conserved at least in mammals. It is likely that RNAa also exists in other organisms.

As used herein, the term RNAi refers to an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cell, where they interact with the catalytic RISC component argonaute. When the double-stranded RNA or RNA-like iNA is exogenous (coming from infection by a virus with an RNA genome or from transfected iNA), the RNA or iNA is imported directly into the cytoplasm and cleaved to short fragments by the enzyme dicer. The initiating dsRNA can also be endogenous

(originating in the cell), as in pre-microRNAs expressed from RNA-coding genes in the genome. The primary transcripts from such genes are first processed to form the characteristic stem-loop structure of pre-miRNA in the nucleus, then exported to the cytoplasm to be cleaved by dicer. Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC complex. The active components of an RNA- induced silencing complex (RISC) are endonucleases called argonaute proteins, which cleave the target mRNA strand complementary to their bound siRNA or iNA. As the fragments produced by dicer are double-stranded, they could each in theory produce a functional siRNA. However, only one of the two strands, which is known as the guide strand, binds the argonaute protein and directs gene silencing. The other anti -guide strand or passenger strand is degraded during RISC activation.

FORMULATIONS AND ADMINISTRATION

The RNAi -inducing compound of this invention can be administered in conjunction with other known treatments for a disease condition.

Comparable methods and compositions are provided that target expression of one or more different genes associated with a particular disease condition in a subject, including any of a large number of genes whose expression is known to be aberrantly increased as a causal or contributing factor associated with the selected disease condition.

Supplemental or complementary methods for delivery of nucleic acid molecules for use within then invention are described, for example, in Akhtar et al., Trends Cell Bio. 2:139, 1992; "Delivery Strategies for Antisense Oligonucleotide Therapeutics," ed. Akhtar, 1995, Maurer et al, Mol. Membr. Biol. 16:129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 13 7:165-192, 1999; and Lee et al, ACS Symp. Ser. 752:184- 192, 2000. Sullivan, et al, International PCT Publication No. WO 94/02595, further describes general methods for delivery of enzymatic nucleic acid molecules. Nucleic acid molecules can be administered within formulations that include one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, or preservative.

As used herein, the term "carrier" means a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and/or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. Examples of ingredients of the above categories can be found in the U.S. Pharmacopeia National Formulary, 1990, pp. 1857-1859, as well as in Raymond C. Rowe, et al., Handbook of Pharmaceutical Excipients, 5th ed., 2006, and "Remington: The Science and Practice of Pharmacy," 21st ed., 2006, editor David B. Troy.

Examples of preservatives include phenol, methyl paraben, paraben, m-cresol, thiomersal, benzylalkonium chloride, and mixtures thereof.

Examples of surfactants include oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphotidylcholines, various long chain diglycerides and phospholipids, and mixtures thereof.

Examples of phospholipids include phosphatidylcholine, lecithin,

phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and

phosphatidylethanolamine, and mixtures thereof. Examples of dispersants include ethylenediaminetetraacetic acid.

Examples of gases include nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and mixtures thereof. In certain embodiments, the circular, circular-like, or bidirectional iNA and/or the polypeptide can be encapsulated in liposomes, administered by iontophoresis, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see e.g., O'Hare and Normand, International PCT Publication No. WO 00/53722). Alternatively, a nucleic acid composition can be locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al, Clin. Cancer Res. 5:2330-2337, 1999, and Barry et al, International PCT

Publication No. WO 99/31262.

The compositions of this invention can be effectively employed as

pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence or severity of, or treat (alleviate one or more symptom(s) to a detectable or measurable extent) of a disease state or other adverse condition in a patient.

In some embodiments, this invention provides pharmaceutical compositions and methods featuring the presence or administration of one or more polynucleic acid(s), typically one or more iNAs, combined, complexed, or conjugated with a lipid, which may further be formulated with a pharmaceutically -acceptable carrier, such as a diluent, stabilizer, or buffer.

The circular, circular-like, or bidirectional iNAs of the present invention may be administered in any form, for example transdermally or by local injection (e.g., local injection at sites of psoriatic plaques to treat psoriasis, or into the joints of patients afflicted with psoriatic arthritis or RA). In more detailed embodiments, the invention provides formulations and methods to administer therapeutically effective amounts of iNAs directed against of a mRNA of TNF-. alpha., which effectively down-regulate the TNF-. alpha. R A and thereby reduce or prevent one or more TNF-.alpha.-associated inflammatory condition(s). Comparable methods and compositions are provided that target expression of one or more different genes associated with a selected disease condition in animal subjects, including any of a large number of genes whose expression is known to be aberrantly increased as a causal or contributing factor associated with the selected disease condition. The compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal

administration, sterile solutions, suspensions for injectable administration, and the other forms known in the art. A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, transepithelial, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity.

The circular, circular-like, or bidirectional iNA molecules can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to through injection, infusion pump or stent, with or without their incorporation in biopolymers. In another embodiment, polyethylene glycol (PEG) can be covalently attached to iNA compounds of the present invention, to the polypeptide, or both. The attached PEG can be any molecular weight, preferably from about 2,00 to about 50,000 daltons (Da).

By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.

Examples of agents suitable for formulation with the nucleic acid molecules of this invention include: P -glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet -Riant and Tillement, Fundam. Clin.

Pharmacol. 13:16-26, 1999); biodegradable polymers, such as poly (DL-lactide- coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich, D. F., et al, Cell Transplant 8:47-58, 1999, Alkermes, Inc., Cambridge, Mass.); and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog. Neuropsychopharmacol Biol. Psychiatry 23 :941 -949, 1999). Other examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado, et al, J. Pharm. Sci. 87:1308-1315, 1998; Tyler, et al, FEBS Lett. 421 :280-284, 1999; Pardridge, et al, PNAS USA. 92:5592-5596, 1995; Boado, Adv. Drug Delivey Rev. 15 :73-107, 1995; Aldrian-Herrada et al, Nucleic Acids Res. 26:4910-4916, 1998; and Tyler, et al, PNAS USA. 96:7053-7058, 1999.

The present invention also includes compositions prepared for storage or administration, which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro ed. 1985). For example, preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid. In addition, antioxidants and suspending agents may be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence of, treat, or alleviate a symptom to some extent of a disease state. An amount of from 0.01 mg/kg to 50 mg/kg body weight/day of active nucleic acid should be administered.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl- methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or

condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti -oxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form of oil-in- water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxy ethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The circular, circular-like, or bidirectional iNAs can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non -irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

Methods for the delivery of nucleic acid molecules are described in Akhtar, et al, Trends Cell Bio. 2:139, 1992; "Delivery Strategies for Antisense Oligonucleotide Therapeutics," ed. Akhtar, 1995; Maurer, et al, Mol. Membr. Biol. 16:129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137:165-192, 1999; and Lee, et al., ACS Symp. Ser. 752: 184-192, 2000. Beigelman, et al, U.S. Pat. No. 6,395,713, and Sullivan et al, PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example, Gonzalez, et al, Bioconjugate Chem. 10:1068-1074, 1999; Wang, et al, International PCT Publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)ac-id (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and U.S. Patent

Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand,

International PCT Publication No. WO 00/53722). Alternatively, the nucleic

acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry, et al., Clin. Cancer Res. 5:2330-2337, 1999, and Barry, et al., International PCT

Publication No. WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.

DETERMINING THE LEGTH OF AN CIRCULAR, CIRCULAR-LIKE, OR BIDIRECTIONAL iNA DUPLEX

As stated above, the length of the iNA duplex is determined by counting the number of nucleotides in the duplex starting at the first base-pair at the 5' end of the sense strand and ending at the last base-pair at the 3' end of the sense strand regardless of any nicks or nucleotide gaps between the first and last base pairs.

DESIGNING iNA DUPLEXES TARGETNG MULTIPLE niRNAs

Because the claimed circular, circular-like, or bidirectional iNA duplexes can be more than 30 nucleotides in length and do not induce an interferon response when transfected into mammalian cells, iNA duplexes can be effectively designed that target two or more mRNA transcripts. One segment of the antisense can be complementary to one mRNA transcript and another segment of the antisense strand can be

complementary to another mRNA. Furthermore, the both anti -sense and sense strand can be designed so that one or more segments are long enough to enter RISC and bind to a target mRNA.

The examples given herein, and the exemplary language used herein are solely for the purpose of illustration, and are not intended to limit the scope of the invention.

While this invention has been described in relation to certain embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that this invention includes additional embodiments, and that some of the details described herein may be varied considerably without departing from this invention. This invention includes such additional embodiments, modifications and equivalents. In particular, this invention includes any combination of the features, terms, or elements of the various illustrative components and examples.

Standard iNA or siRNA design usually features a 19-27 base pair contiguous double strand region that is believed to be important for RISC incorporation. Studies have shown that iNAs duplexes longer than 30 base pair double-stranded RNA can cause interferon response. Therefore it was necessary to use iNAs duplexes shorter than 30 base pairs to prevent interferon response. Here we described a novel design in which a circular, circular-like, or bidirectional iNA or siRNA can be as short as 15 nucleotides and as long as several thousand nucleotide or whole length of one mRNA or length of more than one mRNA combined. The circular, circular-like, or bidirectional iNA or siRNA with or without gap or nick or both on either one of the strands or both strands can effectively silence one or more than one genes through RNAi mechanisms. In addition, the circular, circular-like, or bidirectional iNA or siRNA improves silencing property without causing interferon response even the iNA longer than 30 bps. A 30 mer contiguous double strand RNA causes interferon response as indicated by increased expression level of interferon-β. With same sequence and new design in which two or more sense strands or two or more antisense strands separated with either gap or nick and complement with a long contiguous sense or antisense strands in circular, circularlike, or bidirectional configuration, the interferon response was diminished. Moreover, when using the novel circular, circular-like, or bidirectional iNA duplexes of the present invention, one can target more than one site in the same mRNA or more than one different mRNA. The novel iNAs of the present invention have the therapeutic potential to treat viral or bacterial diseases that have a number of mutated forms or diseases wherein the down-regulation of multiple genes would be desirable.

Examples of designed iNA and siRNA: siRNA 1 : Standard 21/21mer LacZ siRNA:

Sense strand: CUACACAAAUCAGCGAUUUdTdT

Anti-sense strand: AAAUCGCUGAUUUGUGUAGdTdC siRNA 2: Standard 21/21 mer Lamin A/C siRNA:

Sense strand: GGUGGUGACGAUCUGGGCUUU

Anti-sense strand: AGCCCAGAUCGUCACCACCUU siRNA 3 : Standard 21/23merApoB siRNA:

Sense strand: GUCAUCACACUGAAUACCAAU

Anti-sense strand: AUUGGUAUUCAGUGUGAUGACAC siRNA 4: Sense strand formed stem-loop like iNA (LacZ iNA-1), the eight nucleotides in italic lower case at 5 '-end of sense strand are complementary to the eight nucleotides in italic lower case at 3 '-end of sense strand.

Sense strand: ccggawcwgauCUACACAAAUCAGCGAUUUccuagawccgg

Anti-sense strand: AAAUCGCUGAUUUGUGUAGdTdC siRNA 5 : Anti-sense strand formed stem-loop like iNA (LacZ iNA-2), the eight nucleotides in italic lower case at 5 '-end of anti-sense strand are complementary to the eight nucleotides in italic lower case at 3 '-end of anti-sense strand.

Sense strand: CUACACAAAUCAGCGAUUUdTdT Anti-sense strand:

ccggawcMgauAAAUCGCUGAUUUGUGUAGUCccuagawccgg siRNA 6: Sense strand formed circular loop-like iNA (LacZ iNA-3), the eight nucleotides in underlined italic lower case at 3 '-end of sense strand are in 3' to 5' direction and are complementary to the eight nucleotides in italic lower case at 5 ' -end of sense.

Sense strand: ccggawcwgauCUACACAAAUCAGCGAUlJLJccuggccMaga Anti-sense strand: AAAUCGCUGAUUUGUGUAGdTdC siRNA 7: Anti-sense strand formed circular loop-like iNA (LacZ iNA-4), the eight nucleotides in underlined italic lower case at 3 ' -end of anti-sense strand are in 3 ' to 5 ' direction and are complementary to the eight nucleotides in italic lower case at 5 ' - end of anti-sense.

Sense strand: CUACACAAAUCAGCGAUUUdTdT

Anti-sense strand:

ccggawcMgauAAAUCGCUGAUUUGUGUAGUCccug^ccwaga siRNA 8 : Sense strand formed stem-loop like iNA targeting both Lamin A/C and ApoB (Lamin- APOB iNA-1), the eight nucleotides in italic lower case at 5 '-end of sense strand are complementary to the eight nucleotides in italic lower case at 3 ' -end of sense strand.

Sense strand:

ccggawcwgauGGUGGUGACGAUCUGGGCUUUaccGUCAUCACACUGAAUACCA AUccuagawccgg

Anti-sense strand 1 (for Lamin A/C): AGCCCAGAUCGUCACCACCUU Anti-sense strand 2 (for ApoB): AUUGGUAUUCAGUGUGAUGACAC siRNA 9: Anti-sense strand formed stem-loop like iNA targeting both Lamin A/C and ApoB (Lamin- APOB iNA-2), the eight nucleotides in italic lower case at 5'- end of anti-sense strand are complementary to the eight nucleotides in italic lower case at 3 '-end of anti-sense strand.

Sense strand 1 (for Lamin A/C): GGUGGUGAC GAUCUGGGCUUU

Sense strand 2 (for ApoB): GUCAUCACACUGAAUACCAAU

Anti-sense strand:

ccggawcwgauAGCCCAGAUCGUCACCACCUUaccAUUGGUAUUCAGUGUGAUG ACACccuagawccgg siRNA 10: Sense strand formed circular loop-like iNA targeting both Lamin A/C and ApoB (Lamin- APOB iNA-3), the eight nucleotides in underlined italic lower case at 3 '-end of sense strand are in 3' to 5' direction and are complementary to the eight nucleotides in italic lower case at 5 '-end of sense.

Sense strand:

ccggawcwgauGGUGGUGACGAUCUGGGCUUUaccGUCAUCACACUGAAUACCA AU ccuggccuaga

Anti-sense strand 1 (for Lamin A/C): AGCCCAGAUCGUCACCACCUU Anti-sense strand 2 (for ApoB): AUUGGUAUUCAGUGUGAUGACAC siRNA 1 1 : Anti-sense strand formed circular loop-like iNA targeting both Lamin A/C and ApoB (Lamin-APOB iNA-4), the eight nucleotides in underlined italic lower case at 3 '-end of anti-sense strand are in 3 ' to 5 ' direction and are complementary to the eight nucleotides in italic lower case at 5 '-end of anti-sense.

Sense strand 1 (for Lamin A/C): GGUGGUGAC GAUCUGGGCUUU

Sense strand 2 (for ApoB): GUCAUCACACUGAAUACCAAU

Anti-sense strand:

ccggawcwgauAGCCCAGAUCGUCACCACCUUaccAUUGGUAUUCAGUGUGAUG ACACccuggccu ga siRNA 12: Linear bidirectional iNA targeting both Lamin A/C and ApoB (Lamin-APOB iNA-5), the 21 nucleotides in underlined italic lower case at 3 '-end of bidirectional strand are in 3 ' to 5 ' direction and are equivalent to the sense strand of standard ApoB siRNA.

Other strand 1 (sense for Lamin A/C): GGUGGUGAC GAUCUGGGCUUU Other strand 2 (anti-sense for ApoB): AUUGGUAUUCAGUGUGAUGACAC Bidirectional strand (Anti-sense of lamin and sense of ApoB):

AGCC C AG AUC GUC ACC AC CXJUaccuaaccauaagucacacuacug siRNA 13 : Linear bidirectional iNA targeting both Lamin A/C and ApoB (Lamin-APOB iNA-6), the 23 nucleotides in underlined italic lower case at 3 '-end of bidirectional strand are in 3 ' to 5 ' direction and are equivalent to the anti -sense strand of standard ApoB siRNA.

Bidirectional strand (sense of lamin and anti-sense of ApoB):

GGUGGUGAC GAXJCXJGGGCXJXJUacccacaguagugugacuuaugguuc

Other strand 1 (anti-sense for Lamin A/C): AGCCCAGAUCGUCACCACCUU Other strand 2 (sense for ApoB): GUCAUCACACUGAAUACCAAU

EXPERIMENTAL METHODS

Transient transfection and cell culture: HepG2 and 9L/LacZ cells are grown in DMEM supplemented with 10% FBS at 37°C in a 5% C0₂ atmosphere. The cells are plated in 96-well plate a day before transfection. For examining gene knockdown, HepG2 and 9L/LacZ cells were transfected with iNAs or siRNAs (final concentration: ΙΟηΜ) by in-house made transfection reagent. For examining interferon induction, HepG2 cells were transfected with iNAs or siRNAs (final concentration: ΙΟΟηΜ) by in- house made transfection reagent. mRNA isolation: Cells are harvested at 48hrs after transfection. Check cell confluency before harvest. Then, remove media and add 90 μΐ TCL cell lysis buffer to each well. Keep at room temperature for 20 min. Transfer 80μ1 lysate into each well of TurboCaptuer plate (Qiagen, Cat#: 72251). Keep in room temperature for 60 min. Then wash three times with TCW buffer with ΙΟΟμΙ for each wash. Add 80μ1 TCE buffer and keep in 65°C for 5 min. Transfer 80μ1 elution solution into new 96-well plate. qRT-PCR: 1 μΐ, of isolated mRNA was used to run RT-PCR with SYBR Green one -step qRT-PCR kit (SensiMix one-step SYBR Green kit, Bio line) by mixing with 13 μΐ_^ master mix containing 7 of 2x master mix (containing reverse transcriptase), 1 μΙ_, forward and reverse primer (6μΜ), 0.3 uL 50X SYBR Green and 4.7 μΙ_, water. The reverse transcription reaction took place at 42°C, 30min; after another 95°C, lOmin for activating Tag enzyme; the thermocyle of reaction of 95°C, 15sec; 60°C, 30sec; 72°C, 20sec; 40-50 cycles was used. β-Galactosidase Assay: Three days after transfection, 9L/LacZ cells were harvested. The cells were washed once with 100 μΙ_, phosphate buffered saline (PBS) and lysed with 70 μί M-PER® Reagent (Pierce). 20 μί of lysate was transferred from each well to new 96-well plate for protein assay with micro BCA kit (Pierce). 30 μί lysate was taken from each well to put in another new plate and add 30μΙ_^ All-in-One™ β-Galactosidase Assay Reagent (Pierce) to each well. Cover plate and incubate for 30 - 40 minutes at 37°C and light absorbance the absorbance was measured at 405 nm.

Micro BCA assay: To measure total protein within the 9L/LacZ cells, 20 μί of cell lysate was transferred to each well of a 96-well plate, 130 μΙ_, of water was added to each well, and 150 μί Micro BCA (Pierce) working solution (25 :24: 1 of Reagent A:B:C) was added to each well and incubated at 37°C for 2 hours and then the light absorbance was measured at 562 nm.

Claims

WHAT IS CLAIMED:

1. A method to silence gene expression by an interfering nucleic acid (iNA).

2. The method of Claim 1 wherein an interfering nucleic acid (iNA), comprising one or more sense strand(s) of nucleotides annealed onto one or more antisense strand of nucleotides.

3. The iNA of Claim 2 wherein one strand of the duplex comprising one or more

fragment(s) in opposite direction wherein the fragments of nucleotides are linked by a chemical bond or a linker; another strand of the duplex comprising one or more fragment(s) wherein the fragments are complementary to fragments in opposite strand and separated by gap(s) or nick(s).

4. The iNA of claim 2 and 3 wherein at one end of double strand duplex there is a single oligonucleotide strand loop

5. The iNA of claim 2, 3, and 4 wherein at each end of double strand duplex there is a single oligonucleotide strand loop.

6. The iNA of claim 2, 3, 4 and 5 wherein either strand or both strand of the duplex may have one or more segments and the duplex results in one or more siRNA(s) in cells.

7. The iNA of claim 6 wherein the duplex can target one or more RNA targets.

8. The method of Claim 1 wherein an interfering nucleic acid (iNA) in a circular or circular-like structure.

9. The structure of Claim 8 wherein the circular iNA duplex comprising one unbroken nucleotide strand and one fragmented strand.

10. The structure of Claim 9 wherein the fragmented strands are complementary to the unbroken strand and separated by gap(s) or nick(s).

11. The structure of Claim 9 wherein the two ends of unbroken strand are complementary each other to form a circular iNA with a double stranded loop structure.

12. The structure of Claim 11 wherein the two ends of the unbroken nucleotide strand forms a partial double strand "stem" and rest of the sequence complement to one or more fragmented strand(s). The length of "stem" can be 5 to 100 nucleotides long.

13. The structure of Claim 11 wherein the two ends of the unbroken nucleotide strand are overlap and form a circular structure through Watson -Crick pair principle by linking a piece of nucleotides to one end of the unbroken strand in the opposite direction.

14. The structure of Claim 8 wherein the circular iNA duplex comprising two fragmented nucleotide strands.

15. The structure of Claim 14 wherein the two fragmented nucleotide strands are

complementary each other with gap(s) and/or nick(s) in between. The gap(s)/nick(s) in one fragmented strand is(are) not at the same position of another fragmented strands.

16. The structure of Claim 15 wherein part of one or both nucleotide strands may not have complementary sequence on opposite strand, resulting in a stick tail at the end of strand or bulge in the middle of the strand.

17. The structure of Claim 8 wherein the circular iNA duplex comprising one closed circular strand and one fragmented nucleotide strand wherein two strands are complementary.

18. The structure of Claim 17 wherein the fragment(s) in fragmented strand are separated either by gap or nick.

19. The structure of Claim 8 wherein the circular iNA duplex comprising one closed circular strand and one open circular strand.

20. The structure of Claim 8 wherein the circular iNA duplex comprising two fragmented strands and each strand with one or more fragment(s). The gap(s)/nick(s) in one fragmented strand is(are) not at the same position of another fragmented strands.

21. The structure of Claim 9, 14, 17, 19, and 20 wherein part of sequences of the circular iNA duplex may not be complementary, resulting in a bulge structure if it is in the middle of the nucleotide fragment or stick end if it is at the end of the nucleotide fragment.

22. The structure of Claim 9, 14, 17, 19, and 20 wherein any strand can be antisense or sense in the reference to target RNA sequences.

23. The iNA of claim 9, 14, 17, 19, and 20 wherein either strand of the duplex may have one or more segments, resulting in one or more siRNA(s) in cells.

24. The method of Claim 2, 9, 14, 17, 19, and 20 wherein an interfering nucleic acid (iNA) has a length of at least 15 nucleotides, preferable 15 to 200 nucleotides, most preferable 19-50 nucleotides.

25. The method of Claim 2, 9, 14, 17, 19, and 20 wherein in an interfering nucleic acid (iNA) there are nick(s)/gap(s) between nucleotide fragments or a open circle strand wherein said nucleotide nick/gap in said strand is at least 0 - 20 nucleotides in length.

26. The method of Claim 2, 9, 14, 17, 19, and 20 wherein in an interfering nucleic acid (iNA) the double-stranded segment(s) is at least 6 nucleotides in length, preferable 15 to 29 nucleotides in length, most preferable 19-27 nucleotides in length.

27. The method of Claim 2, 9, 14, 17, 19, and 20 wherein antisense strand(s) in an

interfering nucleic acid (iNA) target a different mRNA or miRNA.

28. The method of Claim 2, 9, 14, 17, 19, and 20 wherein antisense strand(s) in an

interfering nucleic acid (iNA) target different regions of an mRNA or miRNA.

29. The method of Claim 2, 9, 14, 17, 19, and 20 wherein in an interfering nucleic acid there is at least one nick or one nucleotide gap in either strand.

30. The method of Claim 2, 9, 14, 17, 19, and 20 wherein the nucleotides can be

deoxyribonucleotide, ribonucleotide, nucleotide analog, modified nucleotide, non- natural nucleotide, non-standard nucleotide, and other types of nucleotide, preferable ribonucleotide.

31. The method of Claim 1 wherein a pharmaceutical composition comprised of an

interfering nucleotides (iNA) in Claim 2, 9, 14, 17, and 20, have one or more segments, targeting one or more RNA targets or targeting one or more regions on same RNA target, and a pharmaceutically acceptable excipients.

32. The method of Claim 1 and 31 wherein an interfering nucleic acid (iNA) targeting RNA, said RNA which is mRNA.

33. The method of Claim 1 and 31 wherein an interfering nucleic acid (iNA) targeting RNA, said RNA which is a micro RNA (miRNA).

34. The method of Claim 1 wherein down-regulation of an RNA in a mammal.

35. The method of claim 34 wherein the mammal is human.