Modified polynucleotides for use in rna interference

Abstract

Methods and compositions for performing RNA interference comprising a wide variety of stabilized siRNAs suitable for use in serum-containing media and for in vivo applications, such as therapeutic applications, are provided. These siRNAs permit effective and efficient applications of RNA interference to applications such as diagnostics and therapeutics through the use of one or more modifications including orthoesters, terminal conjugates, modified linkages and 2′ modified nucleotides. Uniquely modified siRNAs have been developed that reduces off-target effects incurred in gene-silencing. The modifications include phosphorylation of the first 5′ terminal antisense nucleotide; 2′ carbon modifications of the first and second or first, second, and third 5′ terminal antisense nucleotides; and optionally 2′ carbon modifications of the first and second or first, second, and third 5′ terminal sense nucleotide. Control and exaequo molecules are also provided. siRNA molecules and related control, trackability and exaequo agents with specific stability modifications were developed.

Description

FIELD OF THE INVENTION [0001] The present invention relates to the field of modified polynucleotides. BACKGROUND [0002] RNA-induced gene silencing in mammalian cells is presently believed to implicate a minimum of three different levels of control: (i) transcription inactivation (siRNA-guided DNA and histone methylation); (ii) small interfering RNA (siRNA)-induced mRNA degradation; and (iii) mRNA-induced transcriptional attenuation. The RNA interference (RNAi) generated by siRNA can be long lasting and effective over multiple cell divisions. Consequently, the ability to assess gene function via siRNA mediated methods, as well as to develop therapies for over-expressed genes, represents an exciting and valuable tool that will accelerate gene function analysis, drug target validation, and genome-wide investigations. Moreover, RNAi has broad potential as a therapeutic tool. [0003] Relatively recent discoveries in the field of RNA metabolism have revealed that the uptake of duplex RNA (...

AI Technical Summary

Benefits of technology

[0260] According to a ninth embodiment, the invention provides a method of performing RNA interference, said method comprising exposing an siRNA to a target nucleic acid, wherein said siRNA is comprised of a sense strand and an antisense strand, and wherein said sense strand is substantially nonfunctional. By “substantially nonfunctional” is meant that the sense strand is incapable of inhibiting expression of any non-targeted gene by 50% or more. Thus, a “substantially nonfunctional” sense strand is one that inhibits expression of non-target mRNAs by less than 50%. An added advantage of the invention is an enhanced stability in serum-containing media and serum.

[0261] According to this embodiment, the sense strand can comprise at least one 2′-O-alkyl modification, at least one cytosine- or uracil-containing nucleotide base, wherein the at least one cytosine- or uracil-containing nucleotide base has a 2′-O-methyl modification. Preferably, the 2′-O-alkyl modification is a 2′-O-methyl modification. More preferably, the 2′O-alkyl modification is a 2′-O-methyl modification is on the first, second, eighteenth and / or nineteenth nucleotide base.

[0262] The sense strand can further comprise a conjugate. Preferably, the conjugate is cholesterol. Preferably, the cholesterol is attached to the 5′ and / or 3′ end of the sense strand. Modification of an siRNA duplex with cholesterol drastically increases the duplex's affinity for albumin and other serum proteins, thus altering the biodistribution of the duplex without any significant toxicity.

[0263] The sense strand can comprise a cap on its 3′ end. Preferably, the cap is an inverted deoxythymidine or two consecutive 2′O-methyl modified bases at the end positions (nucleotides 18 and 19).

[0264] The antisense strand can comprise at least one modified nucleotide. Preferably, the at least one modified nucleotide is a 2′-halogen modified nucleotide. Most preferably, the modified nucleotide is a 2′-fluorine modified nucleotide.

[0265] Where the sense strand comprises one or more cytosine- and / or uracil-containing nucleotide bases, each of the one or more cytosine- and / or uracil-containing nucleotide bases can be 2′-fluorine modified.

Problems solved by technology

Unfortunately, in mammalian cells, the use of long dsRNA to induce RNAi has been met with only limited success.

In large part, this ineffectiveness is due to induction of the interferon response, which results in a general, as opposed to targeted, inhibition of protein synthesis.

For example, when naked siRNA molecules are introduced into blood, serum, or serum-containing media, they are not stable and are almost immediately degraded, which reduces or eliminates their effectiveness.

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