Phagocytic cell delivery of RNAI
The present invention provides a particulate delivery system for delivering an RNAi construct to phagocytic cells such as macrophages, comprising various configurations of a complex comprising a phagocytic cell-targeting moiety and an RNAi construct. The invention further provides methods of making the delivery system, and their uses, such as treating phagocytic cell-associated disease conditions.
1. A delivery system for delivering an RNAi construct to a phagocytic cell and inhibiting expression of a target gene in the phagocytic cell, comprising an RNAi construct and a phagocytic cell-targeting moiety,
wherein the RNAi construct is a double-stranded RNA (dsRNA) construct of 25-30 nucleotides in length, comprising:
(1) a sense strand having a 5′-end and a 3′-end, wherein the 5′-end 12 nucleotides and the 3′-end 10 nucleotides of the sense strand comprise 2′-modified ribose sugars and,
(2) an antisense strand having a 5′-end and a 3′-end, which hybridizes to the sense strand,
wherein the phagocytic cell-targeting moiety comprises beta-glucan, and wherein the RNAi construct inhibits expression of the target gene in the phagocytic cell.
2. The delivery system of claim 1 , wherein the dsRNA construct is blunt-ended.
3. The delivery system of claim 1 , wherein each end of the sense strand comprises non-hydrolyzable internucleotide linkages.
4. The delivery system of claim 1 , wherein the 2′-modified ribose sugars are 2′-O-alkyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, 2′-H (deoxyribonucleotides), or combination thereof.
5. The delivery system of claim 4 , wherein the 2′-O-alkyl nucleotides are 2′-O-methyl nucleotides or 2′-O-allyl nucleotides.
6. The delivery system of claim 1 , wherein the antisense strand comprises a 2′-O-methyl modified nucleotide at the 2nd nucleotide on the 5′-end of the antisense strand and no other modified nucleotides or the antisense strand comprises at least four consecutive 2′-O-methyl modified 3′-end nucleotides with phosphothioate linkages.
7. The delivery system of claim 6 , wherein the dsRNA has enhanced target specificity or reduced off-target silencing compared to a similar construct without the 2′-modification at said position(s).
8. The delivery system of claim 1 , wherein the sense strand of the dsRNA comprises a mismatch nucleotide at the 2nd nucleotide from the 3′-end of the sense strand and/or the last 2nd-8th nucleotides at the 3′-end of the sense strand mis-match their corresponding antisense strand nucleotides.
9. The delivery system of claim 1 , wherein the dsRNA has improved stability in serum and/or cerebral spinal fluid compared to an unmodified dsRNA having the same sequence and/or the dsRNA does not induce interferon response in primary cells.
10. The delivery complex of claim 1 , wherein the phagocytic cell-targeting moiety further comprises one or more ligands for phagocytic cell receptor(s).
11. The delivery system of claim 1 , wherein the antisense strand is unmodified.
12. A pharmaceutical composition comprising the delivery system of claim 1 , and a pharmaceutically acceptable excipient.
13. A method of treating an individual for a disease condition associated with a phagocytic cell defect, comprising administering to the individual a delivery system of claim 1 , wherein the RNAi construct antagonizes the function of a target gene causing the disease condition.
14. The method of claim 13 , wherein the disease condition is: macular degeneration, osteoporosis, non-immune inflammation, chronic obstructive pulmonary disease (COPD), Alzheimer's disease, sepsis and septic shock, neutrophilic alveolitis, hepatitis, inflammatory bowel disease, ischemia/reperfusion, glomerulonephritis, or rheumatoid arthritis.
15. A method for delivering an RNAi construct to a phagocytic cell in a subject, comprising:
administering a delivery system complex to the subject, wherein the delivery system complex comprises an RNAi construct and a phagocytic cell-targeting moiety,
wherein the RNAi construct is a double-stranded RNA (dsRNA) construct of 19-49 nucleotid in length, comprising:
(1) a sense strand having a 5′-end and a 3′-end,
wherein the 5′-end 12 nucleotides and the 3′-end 10 nucleotides of the sense strand are 2′-modified sugars, and,
(2) an antisense strand having a 5′-end and a 3′-end, which hybridizes to the sense strand, and
wherein the phagocytic cell-targeting moiety comprises beta-glucan.
16. The method of claim 15 , wherein the phagocytic cell-targeting moiety further comprises one or more ligands for phagocytic cell receptor(s).
17. The method of claim 15 , wherein the dsRNA construct is blunt-ended.