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Multimeric oligonucleotide compounds

a technology of oligonucleotide compounds and oligonucleotide reagents, which is applied in the field of oligonucleotide reagents and oligonucleotide therapeutics, can solve the problems of limiting their effectiveness as therapeutics in some cases, and achieves the effects of reducing clearance, increasing concentration, and prolonging the duration of target knockdown

Inactive Publication Date: 2015-09-03
RANA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a technique called multimers that can knock down multiple targets at once using oligonucleotides. These multimers have advantages such as increased effectiveness in the liver and longer duration of target knockdown compared to monomeric targeting oligonucleotides. The technical effect of this process is a more efficient and effective method for targeting multiple genes simultaneously.

Problems solved by technology

Although natural phosphodiester-backbone oligonucleotides are taken up by cells efficiently, they are highly susceptible to nuclease degradation in plasma, which limits their effectiveness as therapeutics in some cases.

Method used

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  • Multimeric oligonucleotide compounds
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Examples

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example 1

Design of Antisense Oligonucleotides

[0238]Antisense oligonucleotides against ApoC3, ApoB, Hif-1alpha, survivin and B2M were either selected using a series of bioinformatics filters and computational design algorithms or were derived from the literature. They were selected to be 13, 14 or more nucleotides in length and tested using one or multiple chemical modification design patterns (for example, 3LNAs-8DNAs-3LNAs). The list of all targeting oligonucleotide sequences is given in Table 1 and specific chemical modification patterns are explicitly specified when data is presented. Factors taken into account during the design include species homology, alignment to multiple human transcripts, off-target matches, SNPs, exon-exon boundaries, coverage of the transcript, and statistical models of efficacy and polyA regions. For species homology human, rat, mouse and macaque sequences were considered. For off-target matches, putative sequences were searched against the human transcriptome an...

example 2

Synthesis of Antisense Oligonucleotides

(A) General Procedure for Oligomer Synthesis

[0240]All oligonucleotides were synthesized using standard phosphoramidite protocols (Beaucage, S. L.; Caruthers, M. H. “Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis”. Tetrahedron Lett., 1981, 22:1859) on a MerMade 192 oligonucleotide synthesizer (BioAutomation) or Oligopilot 10 synthesizer (GE) at 200 to 1000 nmole scales employing standard CPG supports (BioSearch) or Glen UnySupport (Glen Research). The DNA, 2′-OMe, 2′-F, and G-clamp monomers were obtained from ChemGenes Corporation or Glen Research, and the LNA monomers were obtained from other commercially available sources. All phosphoramidites other than DNA were coupled with extended coupling times (e.g. 8 to 15 min for RNA, LNA, 2′-O-Methyl, 2′-Fluoro, 5-Propynyl and G-Clamps). After the synthesis, the oligonucleotides were cleaved from the support and deprotected using AMA (a 50:50 mixtur...

example 3

Dimer Stability in Plasma and Cleavage in Liver Homogenates

[0274]Stability measurements were performed using 4 different oligonucleotides (including dimers and the monomer, SEQ ID NOs: 1, 2, 3, 4).

[0275]Briefly, oligos were incubated in 95% plasma of mouse or monkey and in 5% liver homogenate at a concentration of 30 μM and at 37° C. Samples for measurement were taken after 0, 7, 24 and 48 h of incubation. Samples were subjected to a phenol / chloroform extraction and analyzed using LC-MS.

[0276]In detail, stock solutions with a final concentration of 600 μM and a final volume of 100 μl have been prepared of all oligonucleotides. Twelve pieces of approximately 50 mg of liver from CD1 mouse (female, Charles River) were added to individual Lysing matrix tubes. A calculated volume of 1×PBS to give a final concentration of 5% liver (W / W) was added to each of the twelve tubes. All samples were homogenized using a BioRad Fast prep System. The resulting homogenate solutions were combined to g...

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Abstract

The disclosure provides multimeric oligonucleotide compounds, comprising two or more target-specific oligonucleotides (e.g., antisense oligonucleotides (ASOs)), each being resistant to cleavage, and linked together by a cleavable linker. In particular, two or more linked target-specific oligonucleotides, each to a different target, allows concomitant inhibition of multiple genes' expression levels, while exhibiting favorable pharmacokinetic and pharmacodynamic properties. Methods of making and uses of the described compounds are also provided

Description

RELATED APPLICATIONS[0001]This application claims priority under 35 U.S.C. §119(e) from U.S. provisional application Ser. No. 61 / 701,351, filed Sep. 14, 2012 and U.S. provisional application Ser. No. 61 / 783,272, filed Mar. 14, 2013, the entire content of both of which are incorporated by reference herein.FIELD OF THE INVENTION[0002]This invention relates to oligonucleotide reagents, oligonucleotide therapeutics, and methods of making and using thereof.BACKGROUND OF THE INVENTION[0003]The development of oligonucleotides into clinical medicines and their use as basic research tools is an ongoing endeavor. For example, the use of antisense oligonucleotides for gene silencing was described as early as 1978. Since this time other oligonucleotide based approaches have emerged for regulating gene expression, including RNA interference, microRNAs, and, recently, targeted inhibition or inactivation of long non-coding RNAs.[0004]Although natural phosphodiester-backbone oligonucleotides are ta...

Claims

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Application Information

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IPC IPC(8): C12N15/113
CPCC12N15/113C12N2310/11C12N2310/3519C12N2330/30C12N2320/30C12N2310/51C12N15/111C12N2310/141C12N2310/3231C12N2310/3341C12N2310/341C12N2310/52C12N2310/315
Inventor UHLMANN, EUGENSUBRAMANIAN, ROMESHKRIEG, ARTHUR M.
Owner RANA THERAPEUTICS INC
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