Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
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Publication Number
US-7939652-B2
Publication Date
2011-05-10
Expiration Date
Abstract
The invention relates to a double-stranded compound, preferably an oligoribonucleotide (siRNA), which down-regulates the expression of a human TGaseII gene at the post-transcriptional level. The invention also relates to a pharmaceutical composition comprising the compound, or a vector capable of expressing the oligoribonucleotide compound, and a pharmaceutically acceptable carrier. The present invention also contemplates a method of treating a patient suffering from a fibrotic disease such as pulmonary, kidney and liver fibrosis or ocular, scarring comprising administering to the patient the pharmaceutical composition in a therapeutically effective dose so as to thereby treat the patient. The invention also relates to treatment of fibrotic and other diseases by use of antibodies to TGaseII polypeptide.
Core Innovation
The described invention identifies transglutaminase II (TGaseII) as a pro-fibrotic target and addresses fibrosis by down-regulating TGaseII post-transcriptionally using RNA interference. The approach uses a double-stranded RNA compound, including double-stranded TGaseII siRNA oligoribonucleotides designed to target TGaseII mRNA and reduce TGaseII activity. TGaseII is further positioned as a therapeutic target across fibrosis-related settings, including lung, kidney, liver, and ocular scarring.
The invention provides specific double-stranded siRNA oligoribonucleotide compounds defined by strand structure, covalent linkage between consecutive ribonucleotides, strand length constraints, and optional 3′ terminal Z/Z′ segments. The antisense and sense strand sequences are defined as comprising fixed sequences (SEQ ID NO: 22 and SEQ ID NO: 4), with each strand having an integer length from 19 to 40, and optional 3′ terminal Z/Z′ segments present in limited length when included. The described dsRNA structural definition includes the ability for ribonucleotides to be modified or unmodified in their sugar residues.
The invention additionally specifies preferred ribose sugar modification patterns for the dsRNA, including 2′-position sugar modifications such as 2′-O-methyl, and defines alternating 2′-O-methyl sugar modified ribonucleotides in both strands with defined terminal modification differences. Alternative TGaseII inhibition is also described via antibodies, including neutralizing monoclonal anti-TGaseII antibodies that inhibit TGaseII crosslinking activity. Documented examples include selection/design of multiple TGaseII siRNAs, in vitro TGaseII protein knockdown, and in vivo kidney fibrosis modeling using systems with TGaseII expression linked to fibrotic phenotype.
Claims Coverage
The provided independent claims cover two distinct aspects: (i) a fully specified double-stranded RNA compound defined by strand structure, lengths, fixed antisense/sense sequences (SEQ ID NO: 22 and SEQ ID NO: 4), optional 3′ terminal Z/Z′ segments, and covalent linkage; and (ii) a further-defined double-stranded RNA compound with explicit ribose modification and terminal modification patterns, both linked by phosphodiester bonds. The claim set therefore defines both the core dsRNA sequence/architecture and an additional chemical modification regime.
Sequence-defined antisense/sense dsRNA structure with length and optional 3′ terminal attachment
A double-stranded RNA compound with a defined structure including an antisense strand (with covalently linked ribonucleotides forming an oligomer of x nucleotides) and a sense strand (with covalently linked ribonucleotides forming an oligomer of y nucleotides), where x and y are integers from 19 to 40, and where optional 3′ terminal Z/Z′ segments (1–5 consecutive nucleotides) may be present as covalently attached at the 3′ terminus of the strand in which it is present; the antisense sequence comprises 5′ UUCCAGAUCAUCUCGCUCU 3′ (SEQ ID NO: 22) and the sense sequence comprises 5′ AGAGCGAGAUGAUCUGGAA 3′ (SEQ ID NO: 4).
Covalent linkage-defined dsRNA backbone
The double-stranded RNA compound wherein each consecutive ribonucleotide in (N)x and (N′)y is joined to the next N or N′ by a covalent bond, with (N)x and (N′)y being oligomers of covalently connected ribonucleotides; ribonucleotides may independently be modified or unmodified in their sugar residues.
Phosphodiester bond dsRNA with defined terminal sugar modification pattern and alternating 2′-O-methyl
A double-stranded RNA compound defined by the antisense strand as 5′ UUCCAGAUCAUCUCGCUCU 3′ (SEQ ID NO: 22) and the sense strand as 3′ AAGGUCUAGUAGAGCGAGA 5′ (SEQ ID NO: 4), wherein each of A, C, U and G is an unmodified or 2′-O-methyl sugar modified ribonucleotide and each consecutive ribonucleotide is joined to the next ribonucleotide by a phosphodiester bond; alternating ribonucleotides in both strands are 2′-O-methyl sugar modified, with a 2′-O-methyl sugar modified ribonucleotide present at both the 5′ and 3′ termini of the antisense strand and an unmodified ribonucleotide present at both the 5′ and 3′ termini of the sense strand.
Across the two independent claims, the coverage is centered on defined TGaseII siRNA dsRNA oligoribonucleotide structures using fixed antisense/sense sequences (SEQ ID NO: 22 and SEQ ID NO: 4) with covalent linkage and length constraints (19–40), optionally including 3′ terminal Z/Z′ segments in the first independent claim. The second independent claim further constrains chemistry by specifying phosphodiester backbone linkage and a defined alternating 2′-O-methyl modification pattern with strand-specific terminal sugar modification differences.
Stated Advantages
Down-regulates TGaseII post-transcriptionally using RNA interference.
Provides a TGaseII-targeted therapy approach for fibrosis, including lung, kidney, liver, and ocular scarring.
Neutralizing monoclonal anti-TGaseII antibodies are described that inhibit TGaseII crosslinking activity.
Documented Applications
RNA interference-based TGaseII down-regulation therapy for fibrosis, including lung fibrosis, kidney fibrosis, liver fibrosis, and ocular scarring.
In vitro TGaseII protein knockdown in cell systems described in the document (e.g., HeLa cells and NRK49-TGaseII cells).
In vivo kidney fibrosis modeling in systems described as having TGaseII expression linked to fibrotic phenotype (including ZDF rats, fa/fa rats, UUO, 5/6 nephrectomy, and TGaseII KO mice).
TGaseII inhibition by neutralizing monoclonal anti-TGaseII antibodies via measured inhibitory crosslinking activity.
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