Two-tailed self-delivery of SIRNA and related methods
阅读说明：本技术 双尾自递送sirna及相关的方法 (Two-tailed self-delivery of SIRNA and related methods ) 是由 阿纳斯塔西娅·科威罗娃 茱莉亚·奥尔特曼 马修·哈斯勒 于 2018-06-22 设计创作，主要内容包括：提供了包含展现出前所未有的细胞摄取和沉默的双尾siRNA(tt-siRNA)的组合物和方法。还提供了用本发明的双尾siRNA治疗神经性疾病和其他疾病的方法。(Compositions and methods comprising two-tailed siRNA (tt-siRNA) that exhibit unprecedented cellular uptake and silencing are provided. Methods of treating neurological and other diseases with the double-tailed siRNA of the invention are also provided.)
1. A double-stranded nucleic acid compound comprising:
a) a sense strand having a 5 'end, a 3' end, and a region complementary to the antisense strand;
b) an antisense strand having a 5 'end, a 3' end, a region complementary to a target RNA;
c) a first overhang at the 3' end of the sense strand, said first overhang having at least 3 consecutive phosphorothioated nucleotides; and
d) a second overhang region at the 3' end of the antisense strand, the second overhang region having at least 3 consecutive phosphorothioate nucleotides.
2. The compound of claim 1, wherein the antisense strand comprises a 5' phosphate moiety.
3. The compound of claim 1, wherein the antisense strand comprises a moiety R at the 5' end, wherein R is selected from the group consisting of:
4. the compound of claim 1, wherein the sense strand and the antisense strand each independently comprise at least 15 contiguous nucleotides.
5. The compound of claim 1, wherein the sense strand and the antisense strand each independently comprise one or more chemically modified nucleotides.
6. The compound of claim 1, wherein the sense strand and the antisense strand each consist of chemically modified nucleotides.
7. The compound of claim 6, wherein the sense strand and the antisense strand each comprise alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides.
8. The compound of claim 6, wherein the nucleotides in the complementary region of the sense strand are alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides, and wherein the nucleotides in the complementary region of the antisense strand are alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides.
9. The compound of claim 8, wherein each complementary base pair consists of a 2 '-methoxy nucleotide and a 2' -fluoro nucleotide.
10. The compound of claim 8, wherein the overhang regions of the sense strand and the antisense strand independently comprise a 2 '-methoxy nucleotide and a 2' -fluoro nucleotide.
11. The compound of claim 10, wherein the overhang of the sense strand and the antisense strand independently consist of at least four consecutive 2' -methoxy nucleotides.
12. The compound of claim 10, wherein the overhang of the sense strand and the antisense strand consist of 2' -methoxy nucleotides.
13. The compound of claim 11, wherein the nucleotides at positions 1,2, 3, and 4 from the 3 'end of the sense strand and the antisense strand consist of 2' -methoxy nucleotides.
14. The compound of any one of claims 1-13, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense strand and the antisense strand are connected to adjacent nucleotides via phosphorothioate linkages.
15. The compound of claim 1, wherein the overhang of the sense strand and the antisense strand independently consist of 4,5, 6, 7, or 8 phosphorothioate nucleotides.
16. The compound of claim 15, wherein the nucleotides at positions 1-7 or 1-8 from the 3 'end of the sense strand or from the 3' end of the antisense strand are independently linked to an adjacent nucleotide via a phosphorothioate linkage.
17. The compound of claim 1 or 15, wherein the sense strand and the overhang region of the antisense strand have the same number of phosphorothioate nucleotides.
18. The compound of claim 1 or 15, wherein the sense strand and the overhang region of the antisense strand have different numbers of phosphorothioate nucleotides.
19. The compound of any one of claims 1-15, wherein the overhang comprises an abasic nucleotide.
20. The compound of claim 1, having a structure selected from formulas (I-VIII):
wherein:
for each occurrence, X is independently selected from: adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
for each occurrence, Y is independently selected from: adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
-represents a phosphodiester internucleoside linkage;
represents a phosphorothioate internucleoside linkage;
for each occurrence-individually represents a base pairing interaction or mismatch; and is
For each occurrence, R is a nucleotide comprising a 5' phosphate, or R1, R2, R3, R4, R5, R6, R7, or R8 as defined above.
21. The compound of claim 20, wherein the sense strand and the antisense strand each comprise one or more chemically modified nucleotides.
22. The compound of claim 20, wherein the sense strand and the antisense strand each consist of chemically modified nucleotides.
23. The compound of claim 22, wherein the sense strand and the antisense strand independently comprise alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides.
24. The compound of any one of claims 1-23, wherein the antisense strand has perfect complementarity to the target RNA.
25. The compound of any one of claims 1-23, wherein the antisense strand has 80% to 99% complementarity to the target RNA.
26. A pharmaceutical composition comprising one or more double stranded nucleic acid compounds of any one of claims 1-23, and a pharmaceutically acceptable carrier.
27. A method of treating a disease or disorder comprising administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical composition of claim 26.
28. The method of claim 27, wherein the disease or disorder is neurological.
29. The method of claim 28, wherein the neurological disease is huntington's disease.
30. The method of claim 27, wherein the subject in need of such treatment is a human.
31. A method of selectively delivering a compound of any one of claims 1-23 in vivo to a target organ, tissue or cell, comprising administering the compound to a subject.
32. The method of claim 31, wherein the target organ is the brain.
33. The method of claim 31, wherein the target cell is a primary cortical neuron.
34. The method of any one of claims 31-33, wherein delivery of the compound is not mediated by a lipid formulation.
35. The method of claim 31, wherein the compound is administered by intravenous injection, intraperitoneal injection, intracranial injection, intrathecal injection, intrastriatal injection, or intracerebroventricular injection.
36. A method of treating a neurological disease or disorder comprising administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical composition of claim 26.
37. The method of claim 36, wherein the double stranded nucleic acid compound has a structure of formula (I) or formula (VI).
38. The method of claim 36, wherein the double stranded nucleic acid compound has a structure of formula (IV) or formula (VII).
39. The method of claim 37 or 38, wherein the pharmaceutical composition is administered by intravenous injection, intraperitoneal injection, intracranial injection, intrathecal injection, intrastriatal injection, or intracerebroventricular injection.
40. The method of claim 37 or 38, wherein the pharmaceutical composition is administered by intracerebroventricular injection.
41. The method of claim 37 or 38, wherein the neurological disease or disorder is huntington's disease.
42. A double-stranded nucleic acid compound comprising:
a) a sense strand having a 5 'end, a 3' end, and a region complementary to the antisense strand;
b) an antisense strand having a 5 'end, a 3' end, a region complementary to a target RNA;
c) a first overhang at the 3' end of the sense strand, said first overhang having 7 consecutive phosphorothioate nucleotides; and
d) a second overhang at the 3' end of the antisense strand, the second overhang having 7 consecutive phosphorothioate nucleotides.
43. A double-stranded nucleic acid compound comprising:
a) a sense strand having a 5 'end, a 3' end, and a region complementary to the antisense strand;
b) an antisense strand having a 5 'end, a 3' end, a region complementary to a target RNA;
c) a first overhang region at the 3' end of the sense strand, the first overhang region comprising a phosphorothioate nucleotide; and
d) a second overhang region at the 3' end of the antisense strand, the second overhang region comprising a phosphorothioate nucleotide,
wherein the nucleotides in the complementary region of the sense strand are alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides, wherein the nucleotides in the complementary region of the antisense strand are alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides, and wherein the region complementary to the antisense strand is at least 15 nucleotides in length.
Technical Field
The present disclosure relates to novel double-tailed siRNA compounds for RNA interference (RNAi) consisting of chemically modified ribonucleotides and two overhanging single-stranded tails. The chemically modified nucleotides are patterned to achieve unexpectedly high potency, uptake and tissue distribution.
Background
Therapeutic RNA oligonucleotides (e.g., sirnas) comprising chemically modified ribonucleotides (e.g., 2 '-fluoro and 2' -methoxy modifications) and/or chemically modified linkers (e.g., phosphorothioate modifications) are known to exhibit increased nuclease resistance while maintaining the ability to promote RNAi relative to corresponding unmodified oligonucleotides. See, e.g., Fosnaugh et al (U.S. publication No. 2003/0143732).
However, there remains a need for potent and non-toxic delivery to specific cell types in vivo, particularly to central nervous system tissues, for effective delivery of RNAi to treat neurological and other diseases.
SUMMARY
The present invention is based on the discovery of novel double-tailed, chemically modified oligonucleotides that are useful as a new class of siRNA therapeutics. Surprisingly, it was found that the two-tailed, chemically modified siRNA (tt-siRNA) demonstrated broad distribution and retention both in striatal and after intraventricular injection, over that observed with single-tailed siRNA.
Accordingly, in one aspect of the invention, provided herein is a double stranded nucleic acid compound comprising: a sense strand having a 5 'end, a 3' end, and a region complementary to the antisense strand; an antisense strand having a 5 'end, a 3' end, and a region complementary to the sense strand, and a region complementary to an mRNA target; an overhang at the 3 'end of the sense strand, said overhang at the 3' end of the sense strand having at least 3 consecutive phosphorothioate nucleotides; and a overhang at the 3 'end of the antisense strand, the overhang at the 3' end of the antisense strand having at least 3 consecutive phosphorothioate nucleotides.
In another embodiment, the antisense strand comprises a 5' phosphate moiety. In another embodiment, the antisense strand comprises a moiety R at the 5' end. In embodiments, R is selected from:
in another embodiment, the sense strand and the antisense strand each independently comprise at least 15 contiguous nucleotides.
In another embodiment, the sense strand and the antisense strand each independently comprise one or more chemically modified nucleotides. In another embodiment, the sense strand and the antisense strand each independently consist of chemically modified nucleotides.
In another embodiment, the sense strand and the antisense strand each comprise alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides. In another embodiment, the nucleotides in the complementary region of the sense strand are alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides, and wherein the nucleotides in the complementary region of the antisense strand are alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides. In another embodiment, each complementary base pair consists of a 2 '-methoxy nucleotide and a 2' -fluoro nucleotide. In another embodiment, the overhang regions of the sense strand and the antisense strand independently comprise a 2 '-methoxy nucleotide and a 2' -fluoro nucleotide. In another embodiment, the overhang of the sense strand and the antisense strand independently consist of at least 4 contiguous 2' -methoxy nucleotides. In another embodiment, the overhang of the sense strand and the antisense strand consists of 2' -methoxy nucleotides.
In another embodiment, the nucleotides at
In another embodiment, the sense strand and the overhang of the antisense strand each independently consist of 4,5, 6, 7, or 8 phosphorothioate nucleotides. In another embodiment, the nucleotides at positions 1-7 or 1-8 from the 3 'end of the sense strand or from the 3' end of the antisense strand are each linked to an adjacent nucleotide via a phosphorothioate linkage.
In another embodiment, the overhang regions of the sense strand and the antisense strand have the same number of phosphorothioate nucleotides. In another embodiment, the overhang regions of the sense strand and the antisense strand have different numbers of phosphorothioate nucleotides.
In another embodiment, the overhang region comprises an abasic nucleotide.
In another embodiment, the structure is selected from formulas (I-VIII):
wherein, for each occurrence, X is independently selected from: adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
for each occurrence, Y is independently selected from: adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof;
-represents a phosphodiester internucleoside linkage;
represents a phosphorothioate internucleoside linkage;
for each occurrence-individually represents a base pairing interaction or mismatch; and is
For each occurrence, R is a nucleotide comprising a 5' phosphate, or R1, R2, R3, R4, R5, R6, R7, or R8 as defined above.
In embodiments of formulas I-VIII, the sense strand and the antisense strand each comprise one or more chemically modified nucleotides. In another embodiment of formulas I-VIII, the sense strand and the antisense strand each consist of chemically modified nucleotides. In another embodiment of formulas I-VIII, the sense strand and the antisense strand independently comprise alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides.
In another embodiment, wherein the antisense strand has perfect complementarity with the target. In another embodiment, the antisense strand has 80% to 99% complementarity with the target.
In another aspect, provided herein are pharmaceutical compositions comprising one or more double stranded nucleic acid compounds as described herein and a pharmaceutically acceptable carrier.
In another aspect, provided herein is a method of treating a disease or disorder comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound or pharmaceutical composition as described herein.
In embodiments, the disease or disorder is neurological. In embodiments, the disease or disorder is huntington's disease. In embodiments, the subject in need of such treatment is a human.
In another aspect, provided herein is a method of selectively delivering a compound as described herein in vivo to a target organ, tissue or cell, comprising administering the compound to a subject. In embodiments, the target organ is the brain. In embodiments, the target cell is a primary cortical neuron. In embodiments, the delivery of the compound is not mediated by a lipid formulation.
In embodiments, the compound is administered by intravenous injection, intraperitoneal injection, intracranial injection, intrathecal injection, intrastriatal injection, or intracerebroventricular injection.
In another aspect, provided herein is a method of treating a neurological disease or disorder comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound or pharmaceutical composition as described herein.
In embodiments, the double stranded nucleic acid compound has a structure of formula (I) or formula (VI). In embodiments, the double stranded nucleic acid compound has a structure of formula (IV) or formula (VII).
In another aspect, provided herein is a method of inhibiting HTT gene expression in a subject in need thereof, comprising introducing into the subject a nucleic acid compound as described herein.
In another aspect, provided herein is a method of treating or managing huntington's disease, comprising administering to a patient in need of such treatment or management a therapeutically effective amount of a nucleic acid compound as described herein.
In embodiments, the nucleic acid compound is administered to the brain of the patient. In another embodiment, the nucleic acid compound is administered by intrastriatal injection. In another embodiment, the nucleic acid compound is administered by intracerebroventricular injection.
In another embodiment, administration of a nucleic acid compound as described herein to the brain results in a reduction of HTT mRNA in the striatum. In another embodiment, administration of a nucleic acid compound as described herein to the brain results in a decrease in HTT mRNA in the cortex.
In another aspect, provided herein are double-stranded nucleic acid compounds comprising: a sense strand having a 5 'end, a 3' end, and a region complementary to the antisense strand; an antisense strand having a 5 'end, a 3' end, a region complementary to a target RNA; a first overhang at the 3' end of the sense strand, said first overhang having 7 consecutive phosphorothioate nucleotides; and a second overhang at the 3' end of the antisense strand, the second overhang having 7 consecutive phosphorothioate nucleotides.
In another aspect, provided herein are double-stranded nucleic acid compounds comprising: a sense strand having a 5 'end, a 3' end, and a region complementary to the antisense strand; an antisense strand having a 5 'end, a 3' end, a region complementary to a target RNA; a first overhang region at the 3' end of the sense strand, the first overhang region comprising a phosphorothioate nucleotide; and a second overhang region at the 3 ' end of the antisense strand, the second overhang region comprising phosphorothioate nucleotides, wherein the nucleotides in the complementary region of the sense strand are alternating 2 ' -methoxy nucleotides and 2 ' -fluoro nucleotides, wherein the nucleotides in the complementary region of the antisense strand are alternating 2 ' -methoxy nucleotides and 2 ' -fluoro nucleotides, and wherein the region complementary to the antisense strand is at least 15 nucleotides in length.
The foregoing summary is not limiting, and other features and advantages of the disclosed compounds and methods will become apparent from the following detailed description and from the claims.
Brief Description of Drawings
The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments taken together with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee. FIG. 1 depicts representative structures of siRNA, single stranded RNA (ssRNA), and single-tailed siRNA (hsiRNA), and line graphs depicting mRNA expression in HeLa cells after treatment with siRNA, ssRNA, or hsiRNA.
FIG. 2 depicts four examples of two-tailed siRNA (tt-siRNA) with phosphorothioate tails of different lengths while maintaining the same number of total phosphorothioates. In all cases, the antisense strand has a chemically linked 5' phosphate.
FIG. 3 depicts a line graph demonstrating that tt-siRNA exhibits effective mRNA silencing in HeLa cells. Primary cortical mouse neurons were treated with the indicated concentrations of two-tailed siRNA for 1 week. mRNA was measured using Affymetrix QuantiGene 2.0. Data were normalized to housekeeping gene (PPIB) and plotted as% of untreated control.
FIGS. 4A-B depict the efficiency of mRNA silencing by tt-siRNA in primary cortical neurons. Fig. 4A shows a line graph depicting huntingtin mRNA expression in primary cortical neuronal cells after treatment with four different tt-sirnas at increasing concentrations. FIG. 4B is a line graph showing the expression of Huntington protein mRNA in primary cortical neurons after treatment with single-tailed siRNA or tt-siRNA (7-13-7).
FIG. 5 depicts four examples of two-tailed siRNA with phosphorothioate tails of varying lengths and increasing numbers of total phosphorothioates.
Fig. 6 depicts primary neuron immunofluorescence images at 6 and 24 hours after treatment with tt-siRNA (red ═ tt-siRNA, blue ═ DAPI).
Fig. 7 depicts immunofluorescence images of 300 μm brain sections 48 hours after intrastriatal injection of negative control, single-tailed siRNA or tt-siRNA (red ═ tt-siRNA, blue ═ DAPI).
FIG. 8 depicts a line graph representing Huntington protein mRNA expression in HeLa cells after treatment with a single-tailed siRNA or one of four different tt-siRNAs.
FIGS. 9A-B depict silencing of Huntington protein mRNA in primary cortical neurons. FIG. 9A is a line graph showing Huntington protein mRNA expression in primary cortical neurons after 1 week of treatment with increasing concentrations of single-tailed siRNA or one of three different tt-siRNAs. Fig. 9B depicts a line graph showing huntingtin mRNA expression in primary cortical neurons 1 week after treatment with increasing concentrations of single-tailed siRNA or four different tt-sirnas.
Figure 10 depicts a two-tailed siRNA with stereoselective phosphorothioate content.
Fig. 11 depicts backbone ligation of double-tailed siRNA according to certain exemplary embodiments.
Fig. 12 depicts sugar modifications of double-tailed siRNA according to certain exemplary embodiments.
Figure 13 depicts an asymmetric compound comprising two double stranded sirnas.
Detailed description of the invention
Provided herein are novel double-tailed, chemically modified double-stranded nucleic acids effective for in vivo gene silencing. In one aspect, a double stranded nucleic acid compound is provided, comprising: (a) a sense strand having a 5 'end, a 3' end, and a region complementary to the antisense strand; (b) an antisense strand having a 5 'end, a 3' end, and a region complementary to the sense strand; (c) an overhang at the 3 'end of the sense strand, said overhang at the 3' end of the sense strand having at least 3 consecutive phosphorothioate nucleotides; and (d) a overhang region at the 3 'end of the antisense strand, the overhang region at the 3' end of the antisense strand having at least 3 consecutive phosphorothioate nucleotides.
In an embodiment, the double stranded nucleic acid compound is a tt-siRNA that does not comprise a second tt-siRNA, wherein the two tt-sirnas are linked to each other at a 3' position.
In embodiments, the double stranded nucleic acid compound does not consist of two tt-sirnas linked to each other at the 3' position by a linker having the structure:
in embodiments, the double-stranded nucleic acid compound does not have the structure of the compound depicted in fig. 13.
In embodiments, the sense strand and the antisense strand each independently comprise at least 15 contiguous nucleotides. In embodiments, the sense strand and the antisense strand each independently consist of 18-22 contiguous nucleotides. In embodiments, the sense strand and the antisense strand each independently consist of at least 20 contiguous nucleotides. In embodiments, the sense strand and antisense strand each independently consist of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
In embodiments, the overhang of the sense strand has the same number of nucleotides as the overhang of the antisense strand (i.e., the double-stranded nucleic acid is symmetric). In embodiments, the overhang of the sense strand has a different number of nucleotides than the overhang of the antisense strand (i.e., the double stranded nucleic acid is asymmetric).
In embodiments, the overhang of the sense strand and the antisense strand independently consist of 3, 4,5, 6, 7, 8,9, or 10 nucleotides. In embodiments, the overhang of the sense strand and the antisense strand independently consist of 5, 6 or 7 nucleotides. In embodiments, the overhang of the sense strand and the antisense strand each consist of 5, 6, or 7 nucleotides.
The complementary regions of the sense and antisense strands of the double stranded nucleic acid compound together constitute the "double stranded region" of the double stranded nucleic acid compound. In embodiments, the double-stranded region is 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length (i.e., the complementary regions of the sense and antisense strands are 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length). In specific embodiments, the double-stranded region is 13, 14, or 15 nucleotides in length.
In embodiments, the sense strand and the antisense strand each independently comprise one or more chemically modified nucleotides. In embodiments, the sense strand or antisense strand consists of chemically modified nucleotides. In embodiments, the sense strand and antisense strand each consist of chemically modified nucleotides.
In embodiments, both the sense strand and the antisense strand comprise alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides. In embodiments, the nucleotides in the double-stranded region of the sense strand are alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides, and/or the nucleotides in the double-stranded region of the antisense strand are alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides.
In embodiments, each complementary base pair of the double-stranded region consists of a 2 '-methoxy nucleotide and a 2' -fluoro nucleotide.
In embodiments, the overhang regions of the sense strand and the antisense strand each independently comprise a 2 '-methoxy nucleotide and a 2' -fluoro nucleotide. In embodiments, the overhang of the sense strand and the antisense strand consists of 2' -methoxy nucleotides. In embodiments, the overhang regions of the sense strand and the antisense strand each independently consist of at least four (e.g., 4,5, 6, 7, or 8) consecutive 2' -methoxy nucleotides.
In embodiments, one or more of the nucleotides at positions 1-4 (i.e., 1,2, 3, and 4), positions 1-5, positions 1-6, or positions 1-7 of the 3 'end of the sense and antisense strands consists of 2' -methoxy nucleotides. In embodiments, the nucleotides at positions 1-4 of the 3 'end of the sense and antisense strands consist of 2' -methoxy nucleotides.
In embodiments, the nucleotide at one or both of
In embodiments, the overhang of the sense strand and the antisense strand independently consist of 3, 4,5, 6, 7, 8,9, or 10 phosphorothioate nucleotides.
In embodiments, one or more nucleotides at positions 1-4 (i.e., 1,2, 3, and 4), positions 1-5, positions 1-6, positions 1-7, positions 1-8 of the 3 'end of the sense strand or at the 3' end of the antisense strand are linked to adjacent nucleotides via phosphorothioate linkages.
In embodiments, the overhang regions of the sense strand and the antisense strand have the same number of phosphorothioate nucleotides. In embodiments, the overhang regions of the sense strand and the antisense strand have different numbers of phosphorothioate nucleotides relative to each other.
In embodiments, the overhang region of the sense strand and the antisense strand comprise one or more abasic nucleotides. In another embodiment, each nucleotide of the overhang region is abasic.
In embodiments, the sense strand of the double-stranded nucleic acid has homology to the target. In particular embodiments, the sense strand has complete homology to the target.
In embodiments, the antisense strand of the double-stranded nucleic acid has complementarity to the target. In particular embodiments, the antisense strand has complete complementarity with the target. In another embodiment, the antisense strand has partial complementarity to the target. In another embodiment, the antisense strand has 95%, 90%, 85%, 80%, 75%, 70%, or 65% complementarity to the target. In embodiments, the antisense strand has 80% to 99% complementarity with the target. In a specific embodiment, the target is an HTT gene.
In particular embodiments, the target mRNA is a mammalian or viral mRNA. In another specific embodiment, the target is an intron region of the target mRNA. In particular embodiments, the target mRNA is produced by a gene associated with a neurological disorder (e.g., HTT).
In embodiments, the double stranded nucleic acid compound has a structure selected from the group consisting of formulas (I-VIII):
wherein: for each occurrence, X is independently selected from: adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; for each occurrence, Y is independently selected from: adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; -represents a phosphodiester internucleoside linkage; represents a phosphorothioate internucleoside linkage; for each occurrence-individually represents a base pairing interaction or mismatch; and for each occurrence R is a nucleotide comprising a 5' phosphate, or R1, R2, R3, R4, R5, R6, R7, or R8 as defined above.
In embodiments, the sense strand and the antisense strand each comprise one or more chemically modified nucleotides. In embodiments, the sense strand and antisense strand each consist of chemically modified nucleotides. In embodiments, the sense strand and the antisense strand independently comprise alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides. In exemplary embodiments, the double stranded nucleic acid compound has a structure of formula (I) or formula (VI). In another exemplary embodiment, the double stranded nucleic acid compound has a structure of formula (IV) or formula (VII).
In another aspect, provided herein are pharmaceutical compositions comprising one or more double stranded nucleic acid compounds as described herein and a pharmaceutically acceptable carrier.
In another aspect, provided herein is a method of treating a disease or disorder comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound or pharmaceutical composition as described herein.
In an embodiment of the method, the disease or disorder is neurological. In an embodiment, the neurological disease is huntington's disease. In another embodiment, the disease is selected from: alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal dementia (corticobasal dementia), Parkinson's disease with dementia with Lewy bodies, post-traumatic neurodegeneration and chronic traumatic encephalopathy.
In embodiments, the subject in need of such treatment is a human. In another embodiment, the subject in need of such treatment is a mouse. In another embodiment, the subject in need of such treatment is a rat. In another embodiment, the subject in need of such treatment is a monkey. In another embodiment, the subject in need of such treatment is sheep. In another embodiment, the subject in need of such treatment is a dog.
In another aspect, provided herein is a method of treating a neurological disease or disorder comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound or pharmaceutical composition as described herein.
Definition of
Unless defined otherwise herein, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. If there is any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of protein molecules.
Generally, the terms used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genomics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques provided herein are generally performed according to conventional methods well known in the art and described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The terms used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.
In order that the disclosure may be more readily understood, selected terms are defined as follows.
The term "complementary" refers to a relationship between nucleotides that exhibit Watson-Crick base pairing, or to an oligonucleotide that forms a double-stranded nucleic acid via Watson-Crick base-pairing hybridization. The term "complementarity" refers to the state of an oligonucleotide (e.g., sense strand or antisense strand) that is partially or fully complementary to other oligonucleotides. An oligonucleotide described herein as having complementarity to a second oligonucleotide can be 100%, > 95%, > 90%, > 85%, > 80%, > 75%, > 70%, > 65%, > 60%, > 55%, or > 50% complementary to the second oligonucleotide.
As used herein in the context of an oligonucleotide sequence, "a" represents a nucleoside comprising the base adenine (e.g., adenosine or a chemically modified derivative thereof), "G" represents a nucleoside comprising the base guanine (e.g., guanosine or a chemically modified derivative thereof), "U" represents a nucleoside comprising the base uracil (e.g., uridine or a chemically modified derivative thereof), and "C" represents a nucleoside comprising the base cytosine (e.g., cytidine or a chemically modified derivative thereof).
As used herein, the term "3 'end" refers to the end of a nucleic acid that contains an unmodified hydroxyl group at the 3' carbon of its ribose ring.
As used herein, the term "5 'end" refers to the end of a nucleic acid that contains a phosphate group attached to the 5' carbon of its ribose ring.
As used herein, the term "nucleoside" refers to a molecule consisting of a heterocyclic base and its sugar.
As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group on its 3 'or 5' sugar hydroxyl group.
An RNAi agent having a strand with a "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" (e.g., tt-siRNA) means that the strand has a sequence sufficient to initiate destruction of the target mRNA by RNAi.
As used herein, the term "isolated RNA" (e.g., "isolated tt-siRNA", "isolated siRNA" or "isolated siRNA precursor") refers to an RNA molecule that is substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
The term "discriminatory RNA silencing" refers to, for example, the ability of an RNA molecule to substantially inhibit the expression of a "first" or "target" polynucleotide sequence, while not substantially inhibiting the expression of a "second" or "non-target" polynucleotide sequence, when the two polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene and the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele and the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is a DNA sequence encoding a regulatory region (e.g., a promoter or enhancer element) of a target gene. In other embodiments, the target polynucleotide sequence is a target mRNA encoded by a target gene.
As used herein, the term "siRNA" refers to small interfering RNAs that induce the RNA interference (RNAi) pathway. siRNA molecules can vary in length (typically 18-30 base pairs) and have varying degrees of complementarity to their target mRNA. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form a hairpin structure comprising a duplex region.
As used herein, the term "antisense strand" refers to a strand of an siRNA duplex that has a degree of complementarity to a target gene or mRNA, and has complementarity to the sense strand of the siRNA duplex.
As used herein, the term "sense strand" refers to a strand of an siRNA duplex that has complementarity to an antisense strand of the siRNA duplex.
As used herein, the term "overhang" or "tail" refers to 3, 4,5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous nucleotides at the 3' end of one or both of the sense and antisense strands that are single-stranded, i.e., do not base pair with the other strand of the siRNA duplex (i.e., do not form a duplex with the other strand of the siRNA duplex).
As used herein, the term "double-tail oligonucleotide" or "tt-siRNA" refers to a double-stranded siRNA comprising a sense strand and an antisense strand, a duplex region in which the sense and antisense strands are base-paired, and one overhanging single-stranded tail located at each of the 3 'end of the sense strand and the 3' end of the antisense strand. Each single-stranded tail independently comprises three, four, five, six, seven, eight or more overhanging nucleotides that do not form a duplex with nucleotides from another strand. Each overhanging single-stranded tail of the tt-siRNA comprises or consists of a phosphorothioate nucleotide.
In certain exemplary embodiments, the tt-siRNA of the invention comprises a duplex region that is about 8-20 nucleotides or nucleotide analogs in length, about 10-18 nucleotides or nucleotide analogs in length, about 12-16 nucleotides or nucleotide analogs in length, or about 13-15 nucleotides or nucleotide analogs in length (e.g., a duplex region of about 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs).
In certain exemplary embodiments, each overhang of the tt-siRNA of the invention comprises at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 consecutive nucleotides. In certain embodiments, each overhang of the tt-siRNA of the invention is about 4, about 5, about 6, or about 7 nucleotides in length. In certain embodiments, the sense strand overhang has the same number of nucleotide lengths as the antisense strand overhang. In other embodiments, the sense strand overhang has fewer nucleotides than the antisense strand overhang. In other embodiments, the antisense strand overhang has fewer nucleotides than the sense strand overhang.
In certain exemplary embodiments, the tt-siRNA of the present invention comprises a sense strand and/or an antisense strand, each of which is about 10, about 15, about 20, about 25, or about 30 nucleotides in length. In a specific embodiment, the tt-siRNA of the invention comprises a sense strand and/or an antisense strand, each of which is about 15 to about 25 nucleotides in length. In a specific embodiment, the tt-siRNA of the invention comprises a sense strand and an antisense strand, each of which is about 20 nucleotides in length. In certain embodiments, the sense strand and the antisense strand of the tt-siRNA are the same length. In other embodiments, the sense and antisense strands of the tt-siRNA are different lengths.
In certain exemplary embodiments, the total length of the tt-siRNA of the invention (from the 3 'end of the antisense strand to the 3' end of the sense strand) is about 20, about 25, about 30, about 35, about 40, about 45, about 50, or about 75 nucleotides. In certain exemplary embodiments, the tt-siRNA of the invention has an overall length of about 15 to about 35 nucleotides. In other exemplary embodiments, the total length of the tt-siRNA of the invention is from about 20 to about 30 nucleotides. In other exemplary embodiments, the total length of the tt-siRNA of the invention is from about 22 to about 28 nucleotides. In particular embodiments, the tt-siRNA of the invention has an overall length of about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides.
As used herein, the term "chemically modified nucleotide" or "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a nonstandard nucleotide that includes a non-naturally occurring ribonucleotide or deoxyribonucleotide. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide, while still retaining the ability of the nucleotide analog to perform its intended function. Examples of the position of the nucleotide which can be derivatized include the 5 position, for example, 5- (2-amino) propyluridine, 5-bromouridine, 5-propynyluridine, 5-propenyl uridine and the like; 6 position, e.g., 6- (2-amino) propyluridine; the 8-position of adenosine and/or guanosine, for example, 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine and the like. Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza adenosine; o-modified and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or otherwise as known in the art) nucleotides; and other heterocyclic modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev.,2000Aug.10(4): 297-.
Nucleotide analogs may also include modifications to the sugar portion of the nucleotide. For example, the 2' OH group may be replaced by a group selected from: H. OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2COOR OR OR, wherein R is substituted OR unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, and the like. Other possible modifications include those described in U.S. patent nos. 5,858,988 and 6,291,438.
As used herein, the term "metabolically stable" refers to an RNA molecule containing ribonucleotides that have been chemically modified from a 2 '-hydroxy group to a 2' -O-methyl group. In particular embodiments, the duplex region of the tt-siRNA comprises one or more 2 '-fluoro modifications and/or one or more 2' -methoxy modifications. In certain exemplary embodiments, the duplex region comprises alternating 2 '-fluoro modifications and alternating 2' -methoxy modifications in one or both of the sense and antisense strands.
As used herein, the term "phosphorothioate" refers to a phosphate group of a nucleotide that is modified by the substitution of one or more oxygens of the phosphate group with sulfur. Thiophosphoric acid also contains a cationic counter ion (e.g., sodium, potassium, calcium, magnesium, etc.). The term "phosphorothioate nucleotide" refers to a nucleotide having one or two phosphorothioate linkages to other nucleotides. In certain embodiments, the single stranded tail of the tt-siRNA of the invention comprises or consists of phosphorothioate nucleotides.
In some embodiments, the compounds, oligonucleotides, and nucleic acids described herein can be modified to include one or more of the internucleotide linkages provided in figure 12. In particular embodiments, the compounds, oligonucleotides, and nucleic acids described herein comprise one or more internucleotide linkages selected from phosphodiesters and phosphorothioates.
It is understood that certain internucleotide linkages provided herein, including, for example, phosphodiesters and phosphorothioates, contain a formal charge of-1 at physiological pH and that the formal charge will be balanced by a cationic moiety (e.g., an alkali metal such as sodium or potassium, an alkaline earth metal such as calcium or magnesium, or ammonium or guanidinium ions).
In some embodiments, the compounds, oligonucleotides, and nucleic acids described herein can be modified to comprise one or more of the internucleotide backbone linkages provided in fig. 11.
As used herein, the term "lipid preparation" may refer to a liposomal preparation, for example, wherein liposomes are used to form aggregates with nucleic acids to facilitate penetration of the nucleic acids into cells. Without being bound by theory, liposomes are used to penetrate into cells because the phospholipid bilayer readily fuses with the phospholipid bilayer of the cell membrane, allowing nucleic acids to penetrate the cell.
In some embodiments, the compounds, oligonucleotides, and nucleic acids described herein can be modified to include one or more of the internucleotide linkages provided in figure 12. In particular embodiments, the compounds, oligonucleotides, and nucleic acids described herein comprise one or more internucleotide linkages selected from phosphodiesters and phosphorothioates.
It is understood that certain internucleotide linkages provided herein (including, for example, phosphodiesters and phosphorothioates) contain a formal charge of-1 at physiological pH, and that formal charge will be balanced by a cationic moiety (e.g., an alkali metal such as sodium or potassium, an alkaline earth metal such as calcium or magnesium, or ammonium or guanidinium ion).
SiRNA design
In some embodiments, the tt-siRNA molecules of the invention are duplexes consisting of a sense strand and a complementary antisense strand having sufficient complementarity to htt mRNA to mediate RNAi. In certain exemplary embodiments, the tt-siRNA molecule is about 10-50 or more nucleotides in length, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs, or a combination of nucleotides and nucleotide analogs). In other exemplary embodiments, the siRNA molecule is about 16-30 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length in each strand, wherein one strand is sufficiently complementary to the target region to mediate RNAi. In certain exemplary embodiments, the strands are aligned such that at least 4,5, 6, 7, 8,9, 10 or more bases at the ends of the strands are not aligned (i.e., they have no complementary bases in the opposing strands), such that there is a overhang of 4,5, 6, 7, 8,9, 10 or more residues at each or both ends of the duplex when the strands anneal. In certain exemplary embodiments, the siRNA molecule is about 10-50 or more nucleotides in length, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs, or a combination of nucleotides and nucleotide analogs). In specific exemplary embodiments, the siRNA molecule is about 16 to 30 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length in each strand, wherein one strand is substantially complementary to the target sequence and the other strand is identical or substantially identical to the first strand.
In general, the tt-siRNA can be designed by using any method known in the art, for example, by using the following protocol:
tt-siRNA should be specific for the target sequence. The first strand should be complementary to the target sequence and the other strand is substantially complementary to the first strand. In one embodiment, the target sequence is outside the CAG repeat of the amplified mutant htt allele. In another embodiment, the target sequence is outside the coding region of the htt allele. Exemplary target sequences are selected from the 5 'untranslated region (5' -UTR) or intron regions of the target gene. Cleavage of the mRNA at these sites should eliminate translation of the corresponding mutant protein. Target sequences from other regions of the htt gene are also suitable for targeting. The sense strand is designed based on the target sequence. In addition, siRNAs with lower G/C content (35-55%) may be more effective than those with G/C content higher than 55%. Thus, in one embodiment, the invention includes nucleic acid molecules having a G/C content of 35-55%.
2. The sense strand of the tt-siRNA was designed based on the sequence of the selected target site. In certain exemplary embodiments, the sense strand comprises about 19-25 nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides. In specific exemplary embodiments, the sense strand comprises 19, 20, or 21 nucleotides. However, the skilled artisan will appreciate that sirnas less than 19 nucleotides in length or greater than 25 nucleotides in length may also be used to mediate RNAi. Therefore, sirnas of such length are also within the scope of the invention, as long as they retain the ability to mediate RNAi. Longer RNA silencing agents have been shown to elicit interferon or Protein Kinase R (PKR) responses in certain mammalian cells, which may be undesirable. In certain exemplary embodiments, the RNA silencing agents of the invention do not elicit a PKR response (i.e., are of sufficiently short length). However, longer RNA silencing agents may be useful, for example, in cell types that are unable to produce a PRK response, or where a PKR response has been down-regulated or inhibited by alternative means.
The tt-siRNA molecules of the invention have sufficient complementarity with a target sequence such that the tt-siRNA can mediate RNAi. Generally, particularly suitable are tt-sirnas that contain a nucleotide sequence sufficiently identical to a target sequence portion of a target gene to effect RISC-mediated cleavage of the target gene. Thus, in an exemplary embodiment, the sense strand of the tt-siRNA is designed to have a sequence that is substantially identical to the target moiety. For example, the sense strand may be 100% identical to the target site. However, 100% identity is not required. Greater than 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity) between the sense strand and the target RNA sequence is particularly suitable. The present invention has the advantage of being able to tolerate certain sequence variations to enhance the efficiency and specificity of RNAi. In one embodiment, the sense strand has 4, 3,2, 1, or 0 mismatched nucleotides to the target region, such as a target region that differs by at least 1 base pair between the wild-type and mutant alleles, e.g., a target region that comprises gain-of-function mutations, and the other strand is identical or substantially identical to the first strand. In addition, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides may also be effective in mediating RNAi. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
Sequence identity can be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps (gaps) can be introduced in the first or second sequences that are optimally aligned). The nucleotides (or amino acid residues) at the corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e. percent (%) homology-number of identical positions/total number of positions x 100), optionally with penalties for the number of gaps introduced and/or the length of gaps introduced.
Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment is generated on certain portions of the aligned sequences that have sufficient identity, rather than on portions that have a low degree of identity (i.e., local alignment). An illustrative, non-limiting example of a local alignment algorithm for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77. Such algorithms are incorporated into the BLAST program (version 2.0) of Altschul et al (1990) J.mol.biol.215: 403-10.
In another embodiment, the alignment is optimized by introducing appropriate gaps, and the percent identity over the length of the aligned sequences (i.e., the gapped alignment) is determined. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic acids Res.25(17): 3389-. In another embodiment, the alignment is optimized by introducing appropriate gaps and determining the percent identity over the entire length of the aligned sequences (i.e., the overall alignment). An illustrative, non-limiting example of a mathematical algorithm for the overall comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such algorithms are integrated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The antisense or guide strand of the tt-siRNA is generally the same length as the sense strand and includes complementary nucleotides. In one embodiment, the strands of the siRNA are paired in such a way as to have a 3' overhang of 4 to 15 (e.g., 4,5, 6, or 7) nucleotides.
4. Using any method known in the art, potential targets are compared to appropriate genomic databases (human, mouse, rat, etc.) and any target sequences with significant homology to other coding sequences are disregarded. One such method for such sequence homology searches is known as BLAST, which is available on the national center for Biotechnology Information (national center for Biotechnology Information) website.
5. One or more sequences are selected that meet your evaluation criteria.
Other general information on The design and use of tt-siRNA can be found in The "siRNA User Guide (The siRNA User Guide)" available on The Max-Plank-institut fur Biophysikalise Chemie website.
Alternatively, the tt-siRNA can be functionally limited to a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing to a target sequence (e.g., 400mM NaCl, 40mM PIPES, pH 6.4, 1mM EDTA, hybridization at 50 ℃ or 70 ℃ for 12-16 hours, followed by washing). Additional exemplary hybridization conditions include hybridization at 70 ℃ in 1 XSSC or at 50 ℃ in 1 XSSC, 50% formamide followed by a wash at 70 ℃ in 0.3 XSSC, or hybridization at 70 ℃ in 4 XSSC, or at 50 ℃ in 4 XSSC, 50% formamide followed by a wash at 67 ℃ in 1 XSSC. It is expected that hybridization temperatures for hybridizations less than 50 base pairs in length should be 5-10 ℃ lower than the melting temperature (Tm) of the hybridization, where Tm is determined according to the following equation. For a hybridization less than 18 base pairs in length, Tm (° C) is 2 (# of a + T bases) +4 (# of G + C bases). For hybridizations 18 to 49 base pairs in length, Tm (° C) 81.5+16.6(log 10[ Na + ]) +0.41 (% G + C) - (600/N), where N is the number of bases in the hybridization and [ Na + ] is the sodium ion concentration of the hybridization buffer (1 × SSC [ Na + ]0.165M). Additional examples of stringent conditions for polynucleotide hybridization are provided in Sambrook, j., e.f. fritsch and t.manitis, 1989, molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual), Cold spring harbor Laboratory Press, Cold spring harbor, N.Y.,
The negative control tt-siRNA should have the same nucleotide composition as the selected tt-siRNA, but no significant sequence complementarity to the appropriate genome. Such negative controls can be designed by randomly scrambling (scrambling) the nucleotide sequence of selected sirnas. Homology searches can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, a negative control tt-siRNA can be designed by introducing one or more base mismatches in the sequence.
6. To verify the effectiveness of the siRNA to destroy a target mRNA (e.g., wild-type or mutant Huntington protein mRNA)Alternatively, sirnas can be incubated with target cDNA (e.g., huntingtin cDNA) in a Drosophila (Drosophila) -based in vitro mRNA expression system. Automated radiographic detection on agarose gels (autoradiography)32P radiolabeled newly synthesized target mRNA (e.g., huntingtin mRNA). The presence of cleaved target mRNA is indicative of mRNA nuclease activity. Suitable controls include omitting the siRNA and using non-target cDNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA, but no significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected tt-siRNA. Homology searches can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, a negative control tt-siRNA can be designed by introducing one or more base mismatches in the sequence.
Modified nucleotides
In embodiments, the tt-siRNA comprises one or more chemically modified nucleotides. In embodiments, the double-tail oligonucleotide consists of chemically modified nucleotides. In certain embodiments of a double-tail oligonucleotide, > 95%, > 90%, > 85%, > 80%, > 75%, > 70%, > 65%, > 60%, > 55% or > 50% of the nucleic acid comprises a chemically modified nucleotide.
In embodiments, the sense strand and the antisense strand of the tt-siRNA each comprise one or more chemically modified nucleotides. In embodiments, each nucleotide of the sense strand and the antisense strand is chemically modified. In embodiments, both the sense strand and the antisense strand comprise alternating 2 '-methoxy nucleotides and 2' -fluoro nucleotides. In embodiments, the nucleotides at
Delivery and distribution
In another aspect, provided herein is a method of selectively delivering a nucleic acid as described herein to a specific organ of a patient, comprising administering to the patient a double-tail siRNA as described herein, such that the double-tail siRNA is selectively delivered. In one embodiment, the organ is a liver. In another embodiment, the organ is a kidney. In another embodiment, the organ is the spleen. In another embodiment, the organ is a heart. In another embodiment, the organ is the brain.
The compositions described herein facilitate simple, effective, non-toxic delivery of metabolically stable double-tailed siRNA and promote effective silencing of therapeutic targets in a range of tissues in vivo.
In another aspect, provided herein is a method of selectively delivering a compound as described herein to a target organ, tissue or cell in vivo, comprising administering the compound to a subject. In embodiments, the target organ is the brain. In embodiments, the target cell is a primary cortical neuron. In embodiments, the delivery of the compound is not mediated by a lipid formulation.
In embodiments, the method has a selectivity for the target organ of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, i.e., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the tt-siRNA administered to the subject is localized to the target organ.
In certain exemplary embodiments, the compound or pharmaceutical composition is administered by intravenous injection, intraperitoneal injection, intracranial injection, intrathecal injection, intrastriatal injection, or intracerebroventricular injection. In particular embodiments, the compound or pharmaceutical composition is administered by intracerebroventricular injection.
The synthetic tt-siRNA can be delivered into cells by methods known in the art, including cationic lipofection and electroporation. To obtain longer term suppression of the target gene (i.e., the htt gene) and to facilitate delivery in some cases, one or more tt-sirnas from the recombinant DNA construct may be expressed intracellularly. Such methods for expressing siRNA duplexes from recombinant DNA constructs in cells to allow longer term target gene suppression in cells are known in the art and include mammalian Pol III promoter systems capable of expressing functional double stranded siRNA (e.g., H1 or U6/snRNA promoter system (Tuschl, T.,2002, supra); Bagella et al, 1998; Lee et al, 2002, supra; Miyagishi et al, 2002, supra; Paul et al, 2002, supra; Yu et al, 2002, supra; Sui et al, 2002, supra). Termination of transcription by RNAPol III occurs at the run of four consecutive T residues in the DNA template, providing a mechanism for terminating siRNA transcription at a specific sequence. The siRNA is complementary to the sequence of the target gene in the 5 '-3' and 3 '-5' directions, and both strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs driven by H1 or U6 snRNA promoters and expressed in cells can suppress the expression of a target gene (Bagella et al, 1998; Lee et al, 2002, supra; Miyagishi et al, 2002, supra; Paul et al, 2002, supra; Yu et al, 2002, supra); sui et al, 2002, supra). Constructs containing siRNA sequences under the control of the T7 promoter also constitute functional siRNAs when co-transfected into cells with a vector expressing T7 RNA polymerase (Jacque et al, 2002, supra). A single construct may contain multiple sequences encoding sirnas targeting the same gene or multiple genes (e.g., multiple regions of the gene encoding htt) and may be driven, for example, by separate PolIII promoter sites.
Viral-mediated delivery mechanisms can also be used to induce specific silencing of target genes by expression of siRNAs (e.g., by generating recombinant adenoviruses carrying siRNAs under the transcriptional control of an RNA Pol II promoter) (Xia et al, 2002, supra). Infection of HeLa cells by these recombinant adenoviruses allows for the reduction of the expression of endogenous target genes. Injection of the recombinant adenoviral vector into a transgenic mouse expressing a target gene of the siRNA results in a reduction in target gene expression in vivo. As above, whole embryo electroporation can efficiently deliver synthetic sirnas into post-implantation mouse embryos in animal models (Calegari et al, 2002). In adult mice, effective siRNA delivery can be achieved by "high pressure" delivery techniques (techniques in which a solution containing a large amount of siRNA is rapidly injected (within 5 seconds) into the animal via the tail vein) (Liu et al, 1999, supra; McCaffrey et al, 2002, supra; Lewis et al, 2002. nanoparticles and liposomes can also be used to deliver siRNA into animals.
Modified tt-siRNA
In certain aspects of the invention, an RNA silencing agent of the invention (or any portion thereof) as described herein (e.g., tt-siRNA) can be modified such that the activity of the RNA silencing agent is further increased. For example, the RNA silencing agents described above can be modified with any of the modifications described herein. Modifications may be used, in part, to further enhance target recognition, to enhance stability of the agent (e.g., to prevent degradation), to facilitate cellular uptake, to enhance targeting efficiency, to enhance efficacy of binding (e.g., to a target), to enhance tolerance of a patient to the agent, and/or to reduce toxicity.
1) Modification to enhance target recognition
In certain embodiments, the tt-sirnas of the present invention can be substituted with destabilizing nucleotides to enhance single nucleotide target recognition (see U.S. application serial No. 11/698,689, filed 1-25, 2007, and U.S. provisional application No. 60/762,225, filed 1-25, 2006, both of which are incorporated herein by reference). Such modifications may be sufficient to abolish the specificity of the tt-siRNA for a non-target mRNA (e.g., a wild-type mRNA) without significantly affecting the specificity of the tt-siRNA for a target mRNA (e.g., a gain-of-function mutated mRNA).
In certain exemplary embodiments, the tt-siRNA of the present invention is modified by introducing at least one universal nucleotide in its antisense strand. Universal nucleotides comprise a base portion that can optionally base pair with any of the four conventional nucleotide bases (e.g., A, G, C, U). Universal nucleotides are particularly suitable because they have relatively little effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base moiety or an inosine analog base moiety selected from: deoxyinosine (e.g., 2 '-deoxyinosine), 7-deaza-2' -deoxyinosine, 2 '-aza-2' -deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramide-inosine, 2 '-O-methoxyethyl-inosine, and 2' -OMe-inosine. In specific exemplary embodiments, the universal nucleotide is an inosine residue or a naturally occurring analog thereof.
In certain embodiments, the tt-siRNA of the invention is modified by introducing at least one destabilizing nucleotide among the 5 nucleotides from the nucleotide determining specificity (i.e., the nucleotide recognizing the disease-associated polymorphism). For example, destabilizing nucleotides can be introduced at positions located within 5, 4, 3,2, or 1 nucleotides from the nucleotide determining specificity. In an exemplary embodiment, a destabilizing nucleotide is introduced at 3 nucleotide positions from the specificity-determining nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destabilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g., tt-siRNA and shRNA), destabilizing nucleotides can be introduced into the strand or strand portion that does not contain the specificity-determining nucleotides. In certain exemplary embodiments, destabilizing nucleotides are introduced into the same strand or strand portion that contains the specificity-determining nucleotide.
2) Modifications to enhance potency and specificity
In certain embodiments, the tt-siRNA of the present invention can be modified to promote enhanced efficiency and specificity in mediating RNAi according to asymmetric design rules (see U.S. patent nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705). Such alterations facilitate entry of the antisense strand of an siRNA (e.g., an siRNA designed using the methods of the invention or an siRNA generated from an shRNA) into RISC in favor of the sense strand, such that the antisense strand preferentially directs cleavage or translational inhibition of the target mRNA, and thus increases or enhances the efficiency of target cleavage and silencing. In particular embodiments, the asymmetry of an RNA silencing agent is enhanced by decreasing the base pair strength between the 5 'end of the antisense strand (AS 5') and the 3 'end of the sense strand (S3') of the RNA silencing agent relative to the bond strength or base pair strength between the 3 'end of the antisense strand (AS 3') and the 5 'end of the sense strand (S5') of the RNA silencing agent.
In one embodiment, the asymmetry of the tt-siRNA of the invention can be enhanced such that fewer G: C base pairs are present between the 5 'end of the first or antisense strand and the 3' end of the sense strand portion than between the 3 'end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the invention may be enhanced such that there is at least one mismatched base pair between the 5 'end of the first or antisense strand and the 3' end of the sense strand portion. In certain exemplary embodiments, the mismatched base pairs are selected from the group consisting of: g: A, C: A, C: U, G: G, A: A, C: C and U: U. In other embodiments, the asymmetry of the tt-siRNA of the invention can be enhanced such that there is at least one unstable base pair, e.g., G: U, between the 5 'end of the first or antisense strand and the 3' end of the sense strand portion. In other embodiments, the asymmetry of the RNA silencing agents of the invention may be enhanced such that there is at least one base pair comprising a rare nucleotide (e.g., inosine (I)). In certain exemplary embodiments, the base pairs are selected from: a, I: U and I: C. In another embodiment, the asymmetry of the tt-siRNA of the invention can be enhanced such that there is at least one base pair comprising a modified nucleotide. In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of: 2-amino-G, 2-amino-A, 2, 6-diamino-G and 2, 6-diamino-A.
3) tt-siRNA with enhanced stability
The tt-siRNA of the present invention can be further modified to improve stability in serum or growth media used for cell culture. To enhance stability, the 3' -residues may be stabilized against degradation, for example, they may be selected such that they consist of purine nucleotides, in particular adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogs (e.g., substitution of uridine by 2' -deoxythymidine) is acceptable and does not affect the efficiency of RNA interference.
In an exemplary aspect, the invention features a tt-siRNA comprising a first and a second strand, wherein the second strand and/or the first strand is modified by replacing internal nucleotides with modified nucleotides such that in vivo stability is enhanced as compared to a corresponding unmodified RNA silencing agent. As defined herein, an "internal" nucleotide is a nucleotide present at any position other than the 5 'or 3' end of a nucleic acid molecule, polynucleotide or oligonucleotide. The internal nucleotides may be located within a single-stranded molecule or within the strands of a duplex or double-stranded molecule. In one embodiment, the sense strand and/or antisense strand is modified by substitution of at least one internal nucleotide. In another embodiment, the sense strand and/or antisense strand are modified by substitution of at least 2, 3, 4,5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense strand and/or antisense strand are modified by substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In another embodiment, the sense strand and/or antisense strand are modified by substituting all internal nucleotides.
In exemplary embodiments of the invention, the tt-siRNA can contain at least one modified nucleotide analog. The nucleotide analogs can be located at a location where target-specific silencing activity (e.g., RNAi-mediating activity or translational inhibitory activity) is substantially unaffected, e.g., in the region of the 5 'end and/or the 3' end of the siRNA molecule. In particular, the ends may be stabilized by incorporating modified nucleotide analogs.
Exemplary nucleotide analogs include sugar and/or backbone modified ribonucleotides (i.e., including modifications to the phosphate-sugar backbone). For example,the phosphodiester bond of native RNA can be modified to include at least one N or S heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate group attached to the adjacent ribonucleotide is replaced by a modified group (e.g., a phosphorothioate group). In exemplary sugar-modified ribonucleotides, the 2' OH group is replaced by a group selected from: H. OR, R, halogen, SH, SR, NH2、NHR、NR2Or ON, wherein R is C1-C6Alkyl, alkenyl or alkynyl, and halogen is F, Cl, Br or I.
In particular embodiments, the modification is a 2 ' -fluoro, 2 ' -amino and/or 2 ' -thio modification. Specific exemplary modifications include 2 '-fluorocytidine, 2' -fluorouridine, 2 '-fluoroadenosine, 2' -fluoroguanosine, 2 '-aminocytidine, 2' -aminouridine, 2 '-aminoadenosine, 2' -aminoguanosine, 2, 6-diaminopurine, 4-thiouridine and/or 5-amino-allyluridine. In a specific embodiment, the 2' -fluororibonucleotide is each of uridine and cytidine. Additional exemplary modifications include 5-bromouridine, 5-iodouridine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2' -amino-butyryl-pyreneuridine, 5-fluorocytidine and 5-fluorouridine. 2 '-deoxynucleotides and 2' -Ome nucleotides can also be used in the modified RNA silencing reagent portion of the invention. Additional modified residues include deoxyabasic inosine, N3-methyluridine, N6, N6-dimethyladenosine, pseudouridine, purine ribonucleotides, and triazole nucleosides. In certain exemplary embodiments, the 2 'moiety is methyl, such that the linking moiety is a 2' -O-methyl oligonucleotide.
In exemplary embodiments, the RNA silencing agent of the invention comprises a Locked Nucleic Acid (LNA). LNA comprises sugar-modified nucleotides which are resistant to nuclease activity (are highly stable) and have single nucleotide recognition for mRNA (Elmen et al, Nucleic Acids Res. (2005),33(1):439 447; Braasch et al, (2003) Biochemistry 42: 7967-. These molecules have 2 ' -O,4 ' -C-ethylene bridged nucleic acids with possible modifications such as 2 ' -deoxy-2 "-fluorouridine. In addition, LNA increases the specificity of an oligonucleotide by confining the sugar moiety within the 3' -inner conformation, thereby pre-organizing the nucleotides for base pairing and increasing the melting temperature of the oligonucleotide by as much as 10 ℃/base.
In another exemplary embodiment, the RNA silencing agent of the invention comprises a Peptide Nucleic Acid (PNA). PNAs comprise modified nucleotides in which the sugar-phosphate moiety of the nucleotide is replaced by a neutral 2-aminoethylglycine moiety capable of forming a polyamide backbone that is highly resistant to nuclease digestion and confers increased binding specificity to the molecule (Nielsen et al, Science, (2001),254: 1497-1500).
Nucleobase-modified ribonucleotides, i.e., ribonucleotides, that contain at least one non-naturally occurring nucleobase in place of a naturally occurring nucleobase are also exemplified. The base may be modified to block adenosine deaminase activity. Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5- (2-amino) propyl uridine, 5-bromouridine; adenosine and/or guanosine modified at the 8-position, for example, 8-bromoguanosine; deaza nucleotides, for example, 7-deaza adenosine; o-alkylated and N-alkylated nucleotides, for example, N6-methyladenosine, are suitable. It should be noted that the above modifications may be combined.
In other embodiments, cross-linking can be used to alter the pharmacokinetics of the RNA silencing agent, e.g., to increase half-life in vivo. Accordingly, the invention includes an RNA silencing agent having two complementary nucleic acid strands, wherein the two strands are cross-linked. The invention also includes RNA silencing agents conjugated or unconjugated (e.g., at their 3' ends) to other moieties (e.g., non-nucleic acid moieties such as peptides), organic compounds (e.g., dyes), etc.). Modifying an siRNA derivative in this manner can increase cellular uptake or improve cellular targeting activity of the resulting siRNA derivative compared to the corresponding siRNA, for tracking the siRNA derivative in a cell, or improve stability of the siRNA derivative compared to the corresponding siRNA.
Other exemplary modifications include: (a)2 'modifications, e.g., providing a 2' OMe moiety on U in the sense or antisense strand (but particularly on the sense strand), or providing a 2 'OMe moiety in a 3' overhang, e.g., at the 3 'terminus (as context indicates, the 3' terminus means at the 3 'atom or at the 3' most portion of the molecule, e.g., the 3 'P or 2' most position); (b) modification of the backbone, e.g., replacement of O with S in the phosphate backbone, e.g., providing phosphorothioate modifications at U or a or both (particularly on the antisense strand); for example, P is replaced with S; (c) replacement of U with C5 amino linker; (d) replacement of a with G (in particular embodiments, the sequence change is on the sense strand rather than the antisense strand); and (d) a modification at position 2 ', 6', 7 'or 8'. Exemplary embodiments are those in which one or more of these modifications are present on the sense strand rather than the antisense strand, or embodiments in which the antisense strand has fewer such modifications. Other exemplary modifications include: methylated P is used in the 3 'overhang, e.g., at the 3' terminus; in a 3 'overhang, e.g., at the 3' end, 2 'modifications are combined, e.g., to provide a 2' O Me moiety and a backbone modification, e.g., to replace P with S, e.g., to provide a phosphorothioate modification, or to use methylated P; modified with a 3' alkyl group; in the 3 'overhang, e.g., at the 3' end, modified with abasic pyrrolidones; modified with naproxen, ibuprofen, or other moieties that inhibit degradation of the 3' end.
4) Modifications to enhance cellular uptake
In other embodiments, the compounds of the invention can be modified with chemical moieties, for example, to enhance cellular uptake by target cells (e.g., neuronal cells). Thus, the invention encompasses tt-siRNA conjugated or unconjugated (e.g., at its 3' end) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), and the like. Conjugation can be accomplished by methods known in the art, for example using methods in the following references: lambert et al, Drug Deliv. Rev.:47(1),99-112(2001) (describing nucleic acids loaded into Polyalkylcyanoacrylate (PACA) nanoparticles); fattal et al, J.control Release 53(1-3):137-43(1998) (describes nucleic acids bound to nanoparticles); schwab et al, Ann. Oncol.5suppl.4:55-8(1994) (describes nucleic acids attached to intercalators, hydrophobic groups, polymeric cations or PACA nanoparticles); and Godard et al, Eur.J.biochem.232(2):404-10(1995) (nucleic acids attached to nanoparticles are described).
In a specific embodiment, the tt-siRNA is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand comprising a cationic group. In another embodiment, the lipophilic moiety is linked to one or both strands of the tt-siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the tt-siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from: cholesterol, vitamin D, DHA, DHAg2, EPA, vitamin E, vitamin K, vitamin a, folic acid or a cationic dye (e.g., Cy 3).
Pharmaceutical compositions and methods of administration
In one aspect, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of one or more double-tailed siRNA compounds as described herein and a pharmaceutically acceptable carrier. In another specific embodiment, a pharmaceutical composition comprises a compound of formulae I-VIII as described herein and a pharmaceutically acceptable carrier.
The present invention relates to the use of the above agents for prophylactic and/or therapeutic treatment as described herein. Thus, a modulator of the present invention (e.g., a tt-siRNA agent) can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise a nucleic acid molecule, protein, antibody or modulatory compound and a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.
The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., Intravenous (IV), intradermal, subcutaneous (SC or SQ), intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following ingredients: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide). The parenteral formulations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor ELTM (BASF, pasiboni, n.j.) or Phosphate Buffered Saline (PBS). In all cases, the compositions must be sterile and should flow to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures of the foregoing. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by: if desired, the active compounds in the required amounts are admixed with one or a combination of the ingredients listed above in a suitable solvent and then sterile filtered. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/
Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for use in humans. The dosage of such compounds generally lies within a range of circulating concentrations that include ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, a therapeutically effective dose can first be estimated from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range that includes EC50 (i.e., the concentration of the test compound at which half maximal response is achieved) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.
Method of treatment
In one aspect, the invention provides prophylactic and therapeutic methods of treating a subject at risk of a (pre-disposed) disease or disorder. In one embodiment, the disease or disorder is a neurological disease or disorder. In a specific embodiment, the disease or disorder is huntington's disease.
In another aspect, the invention provides prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease or condition caused, in whole or in part, by a functionally acquired mutein. In one embodiment, the disease or disorder is a trinucleotide repeat disease or disorder. In another embodiment, the disease or disorder is a polyglutamine disorder. In certain exemplary embodiments, the disease or disorder is a disorder that is associated with the expression of huntingtin protein, and wherein an alteration of huntingtin protein, particularly an amplification of the copy number of CAG repeats, causes a defect in the huntingtin gene (structure or function) or huntingtin protein (structure or function or expression) such that clinical manifestations include those seen in huntington's patients.
As used herein, "Treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or vector or transgene encoding the same) to a patient, or to a tissue or cell line isolated from a patient, who has a disease or disorder, has symptoms of a disease or disorder, or has a predisposition to a disease or disorder, with the purpose of curing, healing, alleviating, relieving, altering, remediating, improving, ameliorating, or affecting the disease or disorder, the symptoms of the disease or disorder, or the predisposition to the disease.
In one aspect, the invention provides a method of preventing a disease or disorder as described above in a subject by administering to the subject a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding the same). A subject at risk for a disease can be identified by any one or combination of diagnostic or predictive assays, e.g., as described herein. Administration of a prophylactic agent can occur prior to manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented, or alternatively, its progression is delayed.
Another aspect of the invention relates to methods of therapeutically treating a subject (i.e., altering the onset of symptoms of a disease or disorder). In exemplary embodiments, the modulation methods of the invention involve contacting a cell expressing a gain-of-function mutation with a therapeutic agent (e.g., a tt-siRNA or vector or transgene encoding the same) specific for one or more target sequences in a gene such that gene sequence-specific interference is achieved. These methods can be performed in vitro (e.g., by culturing cells with an agent), or alternatively, in vivo (e.g., by administering an agent to a subject).
The modified tt-siRNA for enhanced uptake into neural cells can be administered at the following unit doses: less than about 1.4mg/kg body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001mg/kg body weight, and less than 200 nanomolar RNA agents (e.g., about 4.4 x 1016Copies/kg body weight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nanomole of RNA silencing agent/kg body weight. For example, a unit dose may be administered by injection (e.g., intravenous or intramuscular, intrathecal or directly into the brain), inhalation dose, or topical administration. Particularly suitable doses are less than 2, 1 or 0.1mg/kg body weight.
tt-siRNA can be delivered directly to an organ (e.g., directly into the brain, spine, etc.) at approximately the following dose: about 0.00001mg to about 3mg per organ, or about 0.0001-0.001mg per organ, about 0.03-3.0mg per organ, about 0.1-3.0mg per eye, or about 0.3-3.0mg per organ. The dose can be an amount effective to treat or prevent a neurological disease or disorder (e.g., huntington's disease). In one embodiment, the unit dose is administered less frequently than once daily, e.g., less frequently than once every 2 days, 4 days, 8 days, or 30 days. In another embodiment, unit doses are not administered at a certain frequency (e.g., a non-periodic frequency). For example, a unit dose may be administered in a single dose. In one embodiment, an effective dose is administered in an otherwise conventional therapeutic form.
In one embodiment, an initial dose and one or more maintenance doses of tt-siRNA are administered to a subject. The maintenance dose is typically lower than the initial dose, e.g., half less than the initial dose. The maintenance regimen may comprise treating the subject with a dose ranging from 0.01 μ g to 1.4mg/kg body weight per day (e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001mg/kg body weight per day). Maintenance doses are generally administered no more than once every 5 days, every 10 days, or every 30 days. In addition, the treatment regimen may last for a period of time, which will vary depending on the nature of the particular disease, its severity, and the overall condition of the patient. In certain exemplary embodiments, the dose may be delivered no more than once a day, e.g., no more than once every 24 hours, every 36 hours, every 48 hours or more, e.g., no more than once every 5 days or every 8 days. After treatment, the patient can be monitored for changes in condition and reduction in symptoms of the disease state. The dose of the compound may be increased if the patient does not respond significantly to the current dose level, or the dose may be decreased if a reduction in the symptoms of the disease state is observed, if the disease state has resolved, or if undesirable side effects are observed.
An effective dose may be administered in a single dose or in two or more doses as appropriate to the particular circumstances and considerations. If it is desired to facilitate repeated or frequent infusions, it may be desirable to implant a delivery device, such as a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal, or intrathecal), or reservoir. In one embodiment, the pharmaceutical composition comprises a plurality of RNA silencing agent species. In another embodiment, the species of RNA silencing agent has a non-overlapping and non-adjacent sequence with respect to the naturally occurring target sequence with another species. In another embodiment, the plurality of RNA silencing agent species are specific for different naturally occurring target genes. In another embodiment, the species of RNA silencing agent is allele-specific. In another embodiment, multiple RNA silencing agent species target two or more target sequences (e.g., two, three, four, five, six or more target sequences).
After successful treatment, it is desirable to have the patient undergo maintenance therapy to prevent recurrence of the disease state, wherein the compounds of the present invention are administered in maintenance doses (ranging from 0.01 μ g to 100g/kg body weight) (see U.S. Pat. No. 6,107,094).
In another aspect, provided herein is a method of treating or managing huntington's disease, comprising administering to a patient in need of such treatment or management a therapeutically effective amount of a compound, siRNA or nucleic acid as described herein, or a pharmaceutical composition comprising the compound, siRNA or nucleic acid.
In certain exemplary embodiments, compositions comprising the RNA silencing agents of the invention can be delivered to the nervous system of a subject by a variety of routes. Exemplary routes include intrathecal, parenchymal (e.g., in the brain), nasal, and ocular delivery. The compositions can also be delivered systemically, e.g., by intravenous, subcutaneous, or intramuscular injection, which is particularly useful for delivering RNA silencing agents to peripheral neurons. Exemplary delivery routes are directed to the brain, e.g., into the ventricles or hypothalamus of the brain, or into the lateral or dorsal regions of the brain. RNA silencing agents for nerve cell delivery can be incorporated into pharmaceutical compositions suitable for administration.
For example, a composition can include one or more RNA silencing agents and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may be administered in a variety of ways depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including ocular, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular (e.g., intracerebroventricular) administration. In certain exemplary embodiments, various suitable compositions and methods described herein are sought to deliver the RNA silencing agents of the present invention across the Blood Brain Barrier (BBB).
The route of delivery may depend on the condition of the patient. For example, a tt-siRNA of the invention (e.g., to the globus pallidus or within the striatum of the basal ganglia and proximal to the striatum, medium spiny neurons) can be administered directly into the brain of a subject diagnosed with huntington's disease. In addition to the tt-siRNA of the invention, a second therapy, e.g., a palliative therapy and/or a disease-specific therapy, can be administered to the patient. The second therapy can be, for example, symptomatic (e.g., for alleviating symptoms), neuroprotective (e.g., for slowing or arresting the progression of the disease), or restorative (e.g., for reversing the course of the disease). For the treatment of huntington's disease, for example, the symptomatic therapy may include the drugs haloperidol, carbamazepine, or sodium 2-propylvalerate. Other therapies may include psychotherapy, physiotherapy, speech therapy, communication and memory assistance, social support services, and dietary recommendations.
The RNA silencing agent can be delivered to neural cells of the brain. Delivery methods that do not require the composition to cross the blood-brain barrier can be utilized. For example, a pharmaceutical composition containing an RNA silencing agent can be delivered to a patient by direct injection into a region containing cells affected by a disease. For example, the pharmaceutical composition may be delivered by direct injection into the brain. Injection into a specific region of the brain (e.g., substantia nigra, cortex, hippocampus, striatum, or globus pallidus) can be by stereotactic injection. The RNA silencing agent can be delivered to multiple regions of the central nervous system (e.g., to multiple regions of the brain, and/or to the spinal cord). The RNA silencing agent can be delivered to a diffuse region of the brain (e.g., diffuse delivery to the cortex of the brain).
In one embodiment, the RNA silencing agent may be delivered by way of a cannula or other delivery device having one end implanted into a tissue (e.g., the brain, e.g., substantia nigra, cortex, hippocampus, striatum, or globus pallidus of the brain). The cannula may be connected to a reservoir of RNA silencing agent. Flow or delivery may be mediated by a pump (e.g., osmotic pump or a micropump, such as an Alzet pump (Durect, cupertino), CA). In one embodiment, the pump and reservoir are implanted in a region remote from the tissue (e.g., in the abdomen) and delivery is achieved by a catheter leading from the pump or reservoir to the release site. Devices for delivery to the brain are described, for example, in U.S. patent nos. 6,093,180 and 5,814,014.
The tt-siRNA of the present invention may be further modified such that it is capable of crossing the Blood Brain Barrier (BBB). For example, the RNA silencing agent may be conjugated to a molecule that enables the agent to cross the barrier. Such modified RNA silencing agents may be administered by any desired method, e.g., such as by intraventricular or intramuscular injection, or by pulmonary delivery.
In certain embodiments, exosomes are used to deliver the RNA silencing agents of the invention. Exosomes can cross the BBB upon systemic injection and specifically deliver siRNA, antisense oligonucleotides, chemotherapeutic agents and proteins to neurons (see Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. (2011). Delivery of siRNA to the mouse braine by system injection of targeted exosomes. nature biotechnol.2011apr; 29(4):341-5.doi: 10.1038/nbt.1807; El-andalogusis S, Lee Y, Lakhal-Littleton, Li J, serpy Y, Gardiner C, Alvarez-Erviti L, sargeusil, woodjj. (MJ.) expression-mediated approach of protein 20135. seq.
in certain embodiments, one or more lipophilic molecules are used to allow delivery of the RNA silencing agents of the invention across the BBB (Alvarez-Ervit (2011)). The RNA silencing agent will then be activated into its active form, for example, by enzymatically degrading the lipophilic camouflage to release the drug.
In certain embodiments, one or more receptor-mediated permeable compounds can be used to increase the permeability of the BBB to allow delivery of the RNA silencing agents of the invention. These drugs temporarily increase the permeability of the BBB by increasing the osmotic pressure in the blood (tight junctions between these endothelial cells become loosened) (El-Andaloussi (2012)) by loosening the tight junctions, normal intravenous injection of RNA silencing agents can be performed.
In certain embodiments, nanoparticle-based delivery systems are used to deliver the RNA silencing agents of the present invention across the BBB. As used herein, "Nanoparticle" refers to a polymeric Nanoparticle, which is generally a solid biodegradable colloidal system, which has been extensively studied as a drug or gene carrier (s.p. egusquiaguirre, m.igartua, r.m.hernandez, and j.l.pedraz, "nanoparticel delivery systems for cancer therapy: advanced in Clinical and Clinical research," Clinical and Clinical information on-purity, vol.14, No.2, pp.83-93,2012). Polymeric nanoparticles are divided into two main categories, namely natural polymers and synthetic polymers. Natural polymers for siRNA delivery include, but are not limited to: cyclodextrin, chitosan and atelocollagen (y.wang, z.li, y.han, l.h.liang, and a.ji, "Nanoparticle-based delivery system for application of siRNA in vivo," Current drug metabolism, vol.11, No.2, pp.182-196,2010). Synthetic polymers include, but are not limited to: polyethyleneimine (PEI), poly (dl-lactide-co-glycolide) (PLGA), and dendrimers, which have been extensively studied (x.yuan, s.naguib, and z.wu, "Recent advances of siRNA Delivery by nanoparticles," expert opinion on Drug Delivery, vol.8, No.4, pp.521-536,2011). For a review of nanoparticles and other suitable Delivery systems see Jong-Min Lee, Tae-Jong Yoon, and Young-Seok Cho, "centralized developments in Nanoparticle-Based siRNA Delivery for Cancer Therapy," BioMedResearch International, vol.2013, article ID 782041,
The RNA silencing agents of the invention can be administered ocularly, e.g., to treat retinal disorders, e.g., retinopathy. For example, the pharmaceutical composition may be applied to the surface of the eye or near tissue (e.g., within the eyelid). They may be applied topically, for example by spraying, drops, as eye washes or ointments. The ointment or drippable liquid may be delivered by an ophthalmic delivery system known in the art, such as an applicator or eye dropper. Such compositions may include: mucus mimetics (mucomimetics), such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose, or poly (vinyl alcohol); preservatives, such as sorbic acid, EDTA or benzylchromium chloride, and customary amounts of diluents and/or carriers. The pharmaceutical composition may also be administered to the interior of the eye and may be introduced through a needle or other delivery device capable of introducing it into a selected area or structure. Compositions containing RNA silencing agents can also be applied via an ophthalmic patch.
In general, the RNA silencing agents of the invention can be administered by any suitable method. As used herein, local delivery may refer to the direct application of the RNA silencing agent to any surface of the body, including the surface of the eye, mucosa, body cavity, or any internal surface. Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, sprays and liquids. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Topical administration may also be used as a means of selectively delivering the RNA silencing agent to the epidermis or dermis of a subject, or a specific layer or underlying tissue thereof.
Compositions for intrathecal or intraventricular (e.g., intracerebroventricular) administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Compositions for intrathecal or intraventricular administration typically do not include a transfection reagent or an additional lipophilic moiety other than, for example, a lipophilic moiety linked to an RNA silencing reagent.
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. The injection in the chamber may be facilitated by, for example, a chamber conduit connected to the reservoir. For intravenous use, the total concentration of solutes should be controlled to render the formulation isotonic.
The tt-siRNA of the present invention can be administered to a subject via pulmonary delivery. Pulmonary delivery of the composition can be achieved by inhalation of the dispersion so that the composition in the dispersion can reach the lungs where it can be readily absorbed directly into the blood circulation through the alveolar region. Pulmonary delivery can be effective for systemic delivery and for local delivery to treat pulmonary diseases. In one embodiment, the RNA silencing agent administered by pulmonary delivery has been modified such that it is capable of crossing the blood brain barrier.
Pulmonary delivery can be achieved by different methods, including the use of nebulized, aerosolized, microporous (micellar), and dry powder based formulations. Delivery can be achieved using liquid nebulizers, aerosol-based inhalers, and dry powder dispensing devices. A metered dose device is particularly suitable. One of the benefits of using a nebulizer or inhaler is that the possibility of contamination is minimized, since the device is self-contained. For example, dry powder dispensing devices deliver drugs that can be easily made into dry powders. The RNA silencing reagent composition may be stably stored as a lyophilized or spray-dried powder alone, or in combination with a suitable powder carrier. Delivery of the composition for inhalation may be mediated by a dose timing element, which may include a timer, dose counter, time measuring device provision, or time indicator, which when integrated into the device enables dose tracking, compliance monitoring, and/or dose triggering to the patient during aerosol drug administration.
Types of pharmaceutical excipients that can be used as carriers include: stabilizers, such as Human Serum Albumin (HSA); bulking agents, such as carbohydrates, amino acids, and polypeptides; a pH adjusting or buffering agent; salts such as sodium chloride and the like. These carriers may be in crystalline form or amorphous form, or may be a mixture of both.
Particularly valuable bulking agents include compatible carbohydrates, polypeptides, amino acids, or combinations thereof suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like, disaccharides such as lactose, trehalose, and the like, cyclodextrins such as 2-hydroxypropyl- β -cyclodextrin, and polysaccharides such as raffinose, maltodextrin, dextran, and the like, sugar alcohols such as mannitol, xylitol, and the like.
pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
The RNA silencing agent of the invention can be administered by oral and nasal delivery. For example, drugs administered through these membranes act rapidly, provide therapeutic plasma levels, avoid the first pass effects of liver metabolism, and avoid exposure of the drug to adverse Gastrointestinal (GI) environments. Additional advantages include easy access to the membrane site, allowing for easy application, location, and removal of the drug. In one embodiment, the RNA silencing agent administered by oral or nasal delivery has been modified to be able to cross the blood brain barrier. It is to be understood that the methods described in this disclosure are not limited to the particular methods and experimental conditions disclosed herein; as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Furthermore, unless otherwise indicated, the experiments described herein use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled person and are explained fully in the literature. See, e.g., authored by Ausubel et al, guidelines for molecular biology (Current Protocols in molecular biology), John Wiley & Sons, inc., NY, n.y. (1987-: a laboratory Manual (Molecular Cloning: A laboratory Manual) (fourth edition), antibody: a Laboratory Manual (Antibodies: A Laboratory Manual),
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations to the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, they will be more clearly understood by reference to the following examples, which are included merely for purposes of illustration and are not intended to be limiting.
Examples
- 上一篇：一种医用注射器针头装配设备
- 下一篇：用于检测肺癌的试剂盒、装置和方法