IP Library › Granted Patent US 12,258,566

Granted Patent B2

US 12,258,566 · App. 17/187,129 · Granted Mar 25, 2025

Oligonucleotides for PRNP modulation

Inventors: Anastasia Khvorova (Westborough, MA); Zachary Kennedy (Worcester, MA)

Assignee: UNIVERSITY OF MASSACHUSETTS

C12N15/1138 C12N2310/14 C12N2310/315 C12N2310/3515 C12N2320/30

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Quick Facts

Patent No.: US 12,258,566
App. No.: 17/187,129
Granted: Mar 25, 2025
Kind: B2

External Links

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Abstract

This disclosure relates to novel PRNP targeting sequences. Novel PRNP targeting oligonucleotides for the treatment of neurodegenerative diseases are also provided.

Claims (156)

1. An RNA molecule comprising:

about 8 nucleotides to 80 nucleotides in length; and

a double stranded RNA (dsRNA) comprising a sense strand and an antisense strand, each strand with a 5′ end and a 3′ end, wherein a portion of the antisense strand is complementary to a portion of the sense strand,

wherein the antisense strand comprises a sequence substantially complementary to a prion protein (PRNP) nucleic acid sequence of any one of SEQ ID Nos: 90, 159, 176, 179, and 181-183 to direct target-specific silencing, and

wherein the antisense strand comprises at least 70% 2′-O-methyl modifications.

2. The RNA molecule of claim 1 , wherein the dsRNA comprises complementarity to at least 10, 11, 12, or 13 contiguous nucleotides of the PRNP nucleic acid sequence of any one of SEQ ID Nos: 90, 159, 176, 179, and 181-183.

3. The RNA molecule of claim 1 , wherein the dsRNA comprises at least 80% chemically modified nucleotides.

4. The RNA molecule of claim 1 , wherein:

A:

(1) the nucleotide at position 14 from the 5′ end of the antisense strand is not a 2′-methoxy-ribonucleotide;

(2) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(3) the sense strand comprises at least 70% 2′-O-methyl modifications; and

(4) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages; or

B:

(1) the antisense strand comprises at least 85% 2′-O-methyl modifications;

(2) the nucleotides at positions 2 and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(3) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(4) the sense strand comprises 100% 2′-O-methyl modifications; and

(5) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages; or

C:

(1) the antisense strand comprises at least 75% 2′-O-methyl modifications;

(2) the nucleotides at positions 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(3) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(4) the sense strand comprises 100% 2′-O-methyl modifications; and

(5) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages; or

D:

(1) the antisense strand comprises at least 75% 2′-O-methyl modifications;

(2) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(3) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(4) the sense strand comprises 100% 2′-O-methyl modifications; and

(5) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages; or

E:

(1) the antisense strand comprises at least 75% 2′-O-methyl modifications;

(2) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(3) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(4) the sense strand comprises at least 65% 2′-O-methyl modifications;

(5) the nucleotides at positions 7, 9, 10, and 11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and

(6) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages; or

F:

(1) the antisense strand comprises at least 75% 2′-O-methyl modifications;

(2) the nucleotides at positions 2 and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(3) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(4) the sense strand comprises at least 75% 2′-O-methyl modifications;

(5) the nucleotides at positions 7, 10, and 11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and

(6) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

5. The RNA molecule of claim 1 , wherein a functional moiety is linked to the 5′ end and/or the 3′ end of the antisense strand.

6. The RNA molecule of claim 5 , wherein the functional moiety is linked to the antisense strand and/or the sense strand by a linker.

7. A pharmaceutical composition for inhibiting the expression of a prion protein (PRNP) gene in an organism, comprising the RNA molecule of claim 1 and a pharmaceutically acceptable carrier.

8. A branched RNA compound comprising two or more RNA molecules comprising 15 to 35 nucleotides in length,

wherein each RNA molecule comprises a double stranded (ds) RNA comprising a sense strand and an antisense strand,

wherein each antisense strand independently comprises a sequence substantially complementary to a prion protein (PRNP) nucleic acid sequence of any one of SEQ ID NOs: 90, 159, 176, 179, and 181-183 to direct target-specific silencing,

wherein the antisense strand comprises at least 70% 2′-O-methyl modifications, and

wherein the two or more RNA molecules are connected to one another by one or more moieties independently selected from a linker, a spacer, and a branching point.

9. A compound of formula (I):

L-(N) n (I),

wherein:

L comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or combinations thereof, wherein formula (I) optionally further comprises one or more branch point B, and one or more spacer S, wherein:

the one or more branched point B is independently for each occurrence a polyvalent organic species or derivative thereof;

the one or more spacer S comprises independently for each occurrence an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or combinations thereof; and

N is a double stranded RNA comprising 15 to 35 bases in length comprising a sense strand and an antisense strand; wherein:

the antisense strand comprises at least 70% 2′-O-methyl modifications;

the antisense strand comprises a sequence substantially complementary to a PRNP nucleic acid sequence of any one of SEQ ID NOs: 90, 159, 176, 179, and 181-183 to direct target-specific silencing; and

the sense strand and antisense strand each independently comprise one or more chemical modifications; and

n is 2, 3, 4, 5, 6, 7, or 8.

10. The compound of claim 9 , wherein:

the compound has a structure selected from formulas (I-1)-(I-9):

11. The compound of claim 9 , wherein:

L is structure L1:

or

L is structure L2:

12. A pharmaceutical composition for inhibiting the expression of a PRNP gene in an organism, comprising the branched RNA compound of claim 8 , and a pharmaceutically acceptable carrier.

13. The RNA molecule of claim 1 , wherein the RNA molecule comprises about 15 nucleotides to 25 nucleotides in length.

14. The RNA molecule of claim 1 , wherein the dsRNA comprises no more than 3 mismatches with the PRNP nucleic acid sequence of any one of SEQ ID NOs: 90, 159, 176, 179, and 181-183.

15. The RNA molecule of claim 1 , wherein:

the antisense strand comprises no more than 3 mismatches with the PRNP nucleic acid sequence of any one of SEQ ID NOs: 90, 159, 176, 179, and 181-183;

the antisense strand comprises full complementarity to the PRNP nucleic acid sequence of any one of SEQ ID NOs: 90, 159, 176, 179, 181-183;

the antisense strand comprises about 15 nucleotides to 25 nucleotides in length, optionally 20, 21, or 22 nucleotides in length;

the sense strand comprises about 15 nucleotides to 25 nucleotides in length, optionally 15, 16, 18, or 20 nucleotides in length;

the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs, optionally 15, 16, 18, or 20 base pairs;

the dsRNA comprises a blunt-end;

the dsRNA comprises at least one single stranded nucleotide overhang, optionally about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang;

the sense strand comprises at least one of a 2′-O-methyl modified nucleotide, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, or a mixture thereof;

the dsRNA comprises at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage comprises a phosphorothioate internucleotide linkage;

the dsRNA comprises 4-16 phosphorothioate internucleotide linkages or 8-13 phosphorothioate internucleotide linkages;

the nucleotides at positions 1 and 2 from the 3′ end of the sense strand, and the nucleotides at positions 1 and 2 from the 5′ end of the antisense strand are connected to adjacent ribonucleotides via phosphorothioate linkages; and/or

the dsRNA comprises at least one modified internucleotide linkage of Formula I:

wherein:

B is a base pairing moiety;

W is selected from the group consisting of O, OCH 2 , OCH, CH 2 , and CH;

X is selected from the group consisting of halo, hydroxy, and C 1-6 alkoxy;

Y is selected from the group consisting of O − , OH, OR, NH − , NH 2 , S − , and SH;

Z is selected from the group consisting of O and CH 2 ;

R is a protecting group; and

is an optional double bond.

16. The RNA molecule of claim 1 , wherein the dsRNA is fully chemically modified.

17. The RNA molecule of claim 1 , wherein the dsRNA comprises at least 70% 2′-O-methyl nucleotide modifications.

18. The RNA molecule of claim 1 , wherein the antisense strand comprises 70% to 90% 2′-O-methyl nucleotide modifications.

19. The RNA molecule of claim 1 , wherein the sense strand comprises at least 65% 2′-O-methyl nucleotide modifications or 100% 2′-O-methyl nucleotide modifications.

20. The RNA molecule of claim 1 , wherein the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

21. The RNA molecule of claim 20 , wherein the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of the sense strand.

22. The RNA molecule of claim 1 , wherein the antisense strand comprises a 5′ phosphate, a 5′-alkyl phosphonate, a 5′ alkylene phosphonate, or a 5′ alkenyl phosphonate.

23. The RNA molecule of claim 22 , wherein the antisense strand comprises a 5′ vinyl phosphonate.

24. The RNA molecule of claim 5 , wherein the functional moiety comprises a hydrophobic moiety.

25. The RNA molecule of claim 24 , wherein the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

26. The RNA molecule of claim 25 , wherein the steroid selected from the group consisting of cholesterol and lithocholic acid (LCA).

27. The RNA molecule of claim 25 , wherein the fatty acid selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosanoic acid (DCA).

28. The RNA molecule of claim 25 , wherein the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, retinoic acid, alpha-tocopheryl succinate, and derivatives or metabolites thereof.

29. The RNA molecule of claim 6 , wherein the linker comprises a divalent linker or a trivalent linker.

30. The RNA molecule of claim 29 , wherein the divalent linker or the trivalent linker is selected from the group consisting of:

wherein n is 1, 2, 3, 4, or 5.

31. The RNA molecule of claim 6 , wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

32. The RNA molecule of claim 6 , wherein when the linker is a trivalent linker, the linker further links a phosphodiester or a phosphodiester derivative.

33. The RNA molecule of claim 32 , wherein the phosphodiester or the phosphodiester derivative is selected from the group consisting of:

wherein X is O, S, or BH 3 .

34. The pharmaceutical composition of claim 7 , wherein the RNA molecule inhibits the expression of the PRNP gene by at least 50% or by at least 80%.

35. The branched RNA compound of claim 8 , wherein each RNA molecule comprises 15 to 25 nucleotides in length.

36. The compound of claim 9 , wherein the antisense strand comprises a 5′ terminal group R selected from the group consisting of:

37. The compound of claim 9 , wherein the compound has the structure of formula (II):

wherein:

X, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof;

Y, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof;

- represents a phosphodiester internucleoside linkage;

= represents a phosphorothioate internucleoside linkage; and

--- represents, individually for each occurrence, a base-pairing interaction or a mismatch.

38. The compound of claim 9 , wherein the compound has the structure of formula (IV):

wherein:

X, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof;

Y, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof;

- represents a phosphodiester internucleoside linkage;

= represents a phosphorothioate internucleoside linkage; and

--- represents, individually for each occurrence, a base-pairing interaction or a mismatch.

39. The compound of claim 36 , wherein R is R 3 and n is 2.

40. The compound of claim 36 , wherein L is structure L2:

41. The compound of claim 40 , wherein R is R 3 and n is 2.

42. The pharmaceutical composition of claim 12 , wherein the branched RNA compound inhibits the expression of the PRNP gene by at least 50% or at least 80%.

43. The RNA molecule of claim 1 , wherein the antisense strand comprises no more than 3 mismatches with the PRNP nucleic acid sequence of any one of SEQ ID NOs: 90, 159, 176, 179, and 181-183.

44. The RNA molecule of claim 1 , wherein the antisense strand comprises full complementarity to the PRNP nucleic acid sequence of any one of SEQ ID NOs: 90, 159, 176, 179, and 181-183.

45. The RNA molecule of claim 1 , wherein the antisense strand comprises about 15 nucleotides to 25 nucleotides in length, optionally 20, 21, or 22 nucleotides in length.

46. The RNA molecule of claim 1 , wherein the sense strand comprises about 15 nucleotides to 25 nucleotides in length, optionally 15, 16, 18, or 20 nucleotides in length.

47. The RNA molecule of claim 1 , wherein the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs, optionally 15, 16, 18, or 20 base pairs.

48. The RNA molecule of claim 1 , wherein the dsRNA comprises a blunt-end.

49. The RNA molecule of claim 1 , wherein the dsRNA comprises at least one single stranded nucleotide overhang, optionally about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang.

50. The RNA molecule of claim 1 , wherein:

the sense strand comprises at least one modified nucleotide, wherein the sense strand comprises a 2′-O-methyl modified nucleotide, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, or a mixture thereof;

the dsRNA comprises at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage comprises a phosphorothioate internucleotide linkage;

the dsRNA comprises 4-16 phosphorothioate internucleotide linkages or 8-13 phosphorothioate internucleotide linkages;

the nucleotides at positions 1 and 2 from the 3′ end of sense strand, and the nucleotides at positions 1 and 2 from the 5′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate linkages; and/or

the dsRNA comprises at least one modified internucleotide linkage of Formula I:

wherein:

B is a base pairing moiety;

W is selected from the group consisting of O, OCH 2 , OCH, CH 2 , and CH;

X is selected from the group consisting of halo, hydroxy, and C 1-6 alkoxy;

Y is selected from the group consisting of O − , OH, OR, NH − , NH 2 , S − , and SH;

Z is selected from the group consisting of O and CH 2 ;

R is a protecting group; and

is an optional double bond.

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 19, 2025

From: KHVOROVA, ANASTASIA; KENNEDY, ZACHARY

To: UNIVERSITY OF MASSACHUSETTS

Reel/Frame 070266/0050 →

Continuity (2)

Provisional Application 62982941 · Feb 28, 2020

Related Publication 20210317460A1 · Oct 14, 2021

References Cited (111)

US 4522811A · Eppstein et al. · 1985 [cited by applicant]

US 5328470A · Nabel et al. · 1994 [cited by applicant]

US 5684143A · Grayaznov et al. · 1997 [cited by applicant]

US 5814014A · Elsberry et al. · 1998 [cited by applicant]

US 5858988A · Wang · 1999 [cited by applicant]

US 6093180A · Elsberry et al. · 2000 [cited by applicant]

US 6107094A · Crooke · 2000 [cited by applicant]

US 6168587B1 · Bellhouse et al. · 2001 [cited by applicant]

US 6194389B1 · Johnston et al. · 2001 [cited by applicant]

US 6291438B1 · Wang · 2001 [cited by applicant]

US 6471996B1 · Sokoll et al. · 2002 [cited by applicant]

US 6472375B1 · Hoon et al. · 2002 [cited by applicant]

US 7750144B2 · Zamore et al. · 2010 [cited by applicant]

US 8304530B2 · Zamore et al. · 2012 [cited by applicant]

US 8309704B2 · Zamore et al. · 2012 [cited by applicant]

US 8309705B2 · Zamore et al. · 2012 [cited by applicant]

US 8329892B2 · Zamore et al. · 2012 [cited by applicant]

US 20050053583A1 · Sakaguchi et al. · 2005 [cited by applicant]

US 20050220766A1 · Amalfitano et al. · 2005 [cited by applicant]

US 20060078542A1 · Mah et al. · 2006 [cited by applicant]

US 20070031844A1 · Khvorova · 2007 [cited by examiner]

US 20070259827A1 · Aronin et al. · 2007 [cited by applicant]

US 20080269149A1 · Bowles et al. · 2008 [cited by applicant]

US 20100186103A1 · Gao et al. · 2010 [cited by applicant]

US 20110269818A1 · Bennett · 2011 [cited by examiner]

US 20140296486A1 · Gao et al. · 2014 [cited by applicant]

US 20150166984A1 · Liu et al. · 2015 [cited by applicant]

US 20170096683A1 · Scaria · 2017 [cited by examiner]

US 20170312367A1 · Khvorova · 2017 [cited by examiner]

US 20180023094A1 · Gao · 2018 [cited by examiner]

US 20180298380A1 · Gao · 2018 [cited by examiner]

US 20200087663A1 · Aronin · 2020 [cited by applicant]

US 20220025366A1 · Freier · 2022 [cited by examiner]

EP 4110913A2 · 2023 [cited by applicant]

WO WO2003029459A2 · 2003 [cited by applicant]

WO WO2010019270A1 · 2010 [cited by applicant]

WO WO2011038575A1 · 2011 [cited by applicant]

WO WO2016161388A1 · 2016 [cited by applicant]

WO WO2017030973A1 · 2017 [cited by applicant]

WO WO2017132669A1 · 2017 [cited by applicant]

WO WO2018031933A2 · 2018 [cited by applicant]

WO WO2020041769A1 · 2020 [cited by applicant]

WO WO2021173984A2 · 2021 [cited by applicant]

Vickers et al. The Journal of Biological Chemistry vol. 278(9):7108-7118, 2003. [cited by examiner]

Alisky, et al., “Gene Therapy for Amyotrophic Lateral Sclerosis and Other Motor Neuron Diseases”, Human Gene Therapy, vol. 11, Issue 17, pp. 2315-2329, Nov. 20, 2000. [cited by applicant]

Alvarez-Erviti, et al., “Delivery of siRNA to the Mouse Brain By Systemic Injection of Targeted Exosomes”, Nature Biotechnology, vol. 29, No. 4, pp. 341-345, Apr. 2011. [cited by applicant]

Ambros, et al., MicroRNAs and Other Tiny Endogenous RNAs in C. elegans, Current Biology, vol. 13, Issue 10, pp. 807-818, May 13, 2003. [cited by applicant]

Atwell, et al., Stable Heterodimers From Remodeling the Domain Interface of a Homodimer Using a Phage Display Library, Journal of Molecular Biology, vol. 270, Issue 1, pp. 26-35, Jul. 4, 1997. [cited by applicant]

Braasch, et al., “RNA Interference in Mammalian Cells by Chemically-Modified RNA”, Biochemistry, vol. 42, No. 26, pp. 7967-7975, Jun. 11, 2003. [cited by applicant]

Brummelkamp, et al., A System for Stable Expression of Short Interfering RNAs in Mammalian Cells, Science, vol. 296, Issue 5567, pp. 550-553, Apr. 19, 2002. [cited by applicant]

Carter, “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155-168, 1990. [cited by applicant]

Chen, et al., “Gene Therapy for Brain Tumors: Regression of Experimental Gliomas by Adenovirus-Mediated Gene Transfer In Vivo”, Proceedings of the National Academy of Sciences, vol. 91, No. 8, pp. 3054-3057, 1994. [cited by applicant]

Davidson, et al., A Model System for In Vivo Gene Transfer Into the Central Nervous System Using an Adenoviral Vector, Nature Genetics, vol. 3, No. 3, pp. 219-223, Mar. 1, 1993. [cited by applicant]

Davidson, et al., Recombinant Adeno-Associated Virus Type 2, 4, and 5 Vectors: Transduction of Variant Cell Types and Regions in the Mammalian Central Nervous System, Proceedings of the National Academy of Sciences, vol… [cited by applicant]

Doench, et al., siRNAs can Function as miRNAs, Genes & Development, vol. 17, pp. 438-442, 2003. [cited by applicant]

Ducruix, et al., Crystallization of Nucleic Acids and Proteins: A Practical Approach, Second Edition, 1999, pp. 201-216. [cited by applicant]

Eckstein, Phosphorothioate Oligodeoxynucleotides: What is Their Origin and What is Unique About Them?, Antisense and Nucleic Acid Drug Development, vol. 10, Issue 2, pp. 117-121, Jan. 30, 2009. [cited by applicant]

Egusquiaguirre, et al., “Nanoparticle Delivery Systems for Cancer Therapy: Advances in Clinical and Preclinical Research”, Clinical and Translational Oncology, vol. 14, pp. 83-93, 2012. [cited by applicant]

El Andaloussi, et al., “Exosome-Mediated Delivery of siRNA In Vitro and In Vivo”, Nature Protocols, vol. 7, No. 12, pp. 2112-2126, Nov. 15, 2012. [cited by applicant]

El Andaloussi, et al., “Exosomes for Targeted siRNA Delivery Across Biological Barriers”, Advanced Drug Delivery Reviews, vol. 65, pp. 391-397, 2013. [cited by applicant]

El Andaloussi, et al., “Extracellular Vesicles: Biology and Emerging Therapeutic Opportunities”, Nature Reviews Drug Discovery, vol. 12, pp. 347-357, May 2013. [cited by applicant]

Elmen, et al., Locked Nucleic Acid (LNA) Mediated Improvements in siRNA Stability And Functionality, Nucleic Acids Research, vol. 33, Issue 1, pp. 439-447, Jan. 14, 2005. [cited by applicant]

Fattal, et al., Biodegradable Polyalkylcyanoacrylate Nanoparticles for the Delivery of Oligonucleotides, Journal of Controlled Release, vol. 53, pp. 137-143, May 1998. [cited by applicant]

Fisher, et al., Transduction With Recombinant Adeno-Associated Virus For Gene Therapy is Limited by Leading-Strand Synthesis, Journal of virology, vol. 70, No. 1, pp. 520-532, Jan. 1996. [cited by applicant]

Godard, et al., “Antisense Effects of Cholesterol-Oligodeoxynucleotide Conjugates Associated with Poly(alkylcyanoacrylate) Nanoparticles”, European Journal of Biochemistry banner, vol. 232, pp. 404-410, 1995. [cited by applicant]

Grad, et al., Computational and Experimental Identification of C. elegans microRNAs, Molecular Cell, vol. 11, Issue 5, pp. 1253-1263, May 2003. [cited by applicant]

Griffiths-Jones, San, The microRNA Registry, Nucleic Acids Research, vol. 32, Issue Supplement 1, pp. D109-D111, Jan. 1, 2004. [cited by applicant]

Hamajima, et al., Intranasal Administration of HIV-DNA Vaccine Formulated with a Polymer, Carboxymethylcellulose, Augments Mucosal Antibody Production and Cell-Mediated Immune Response, Clinical Immunology and Immunopat… [cited by applicant]

Herdewijn, Piet, Heterocyclic Modifications of Oligonucleotides and Antisense Technology, Antisense and Nucleic Acid Drug Development, vol. 10, Issue 4, pp. 297-310, Jul. 8, 2004. [cited by applicant]

International Search Report and Written Opinion for PCT International Patent Application No. PCT/US2021/019888, mailed Sep. 1, 2021. [cited by applicant]

Karlin, et al., Applications and Statistics for Multiple High-Scoring Segments in Molecular Sequences, Proceedings of the National Academy of Sciences of the USA, vol. 90, pp. 5873-5877, Jun. 1993. [cited by applicant]

Karlin, et al., Methods for Assessing the Statistical Significance of Molecular Sequence Features by Using General Scoring Schemes, Proceedings of the National Academy of science of the USA, vol. 87, No. 6, pp. 2264-226… [cited by applicant]

Lagos-Quintana, et al., Identification of Novel Genes Coding for Small Expressed RNAs, Science, vol. 294, Issue 5543, pp. 853-858, Oct. 26, 2001. [cited by applicant]

Lagos-Quintana, et al., Identification of Tissue-Specific MicroRNAs from Mouse, Current Biology, vol. 12, Issue 9, pp. 735-739, Apr. 30, 2002. [cited by applicant]

Lagos-Quintana, et al., New microRNAs From Mouse and Human, RNA, vol. 9, No. 2, pp. 175-179, 2003. [cited by applicant]

Lai, et al., Computational Identification of Drosophila microRNA Genes, Genome Biology, vol. 4, No. 7, pp. 1-20, Jun. 30, 2003. [cited by applicant]

Lam, et al., “A New Type of Synthetic Peptide Library for Identifying Ligand-Binding Activity”, Nature, vol. 354, pp. 82-84, Nov. 7, 1991. [cited by applicant]

Lambert, et al., “Nanoparticulate Systems for the Delivery of Antisense Oligonucleotides”, Advanced Drug Delivery Reviews, vol. 47, pp. 99-112, 2001. [cited by applicant]

Lau, et al., An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans, Science, vol. 294, Issue 5543, pp. 858-862, Oct. 26, 2001. [cited by applicant]

Lee, et al., “Recent Developments in Nanoparticle-Based siRNA Delivery for Cancer Therapy”, BioMed Research International, vol. 2013, Article ID 782041, 10 Pages, Jun. 2013. [cited by applicant]

Lee, et al., An Extensive Class of Small RNAs in Caenorhabditis elegans, Science, vol. 294, Issue 5543, pp. 862-864, Oct. 26, 2001. [cited by applicant]

Lim, et al., The microRNAs of Caenorhabditis elegans, Genes & Development, vol. 17, No. 8, pp. 991-1008, 2003. [cited by applicant]

Lim, et al., Vertebrate MicroRNA Genes, Science, vol. 299, Issue 5612, p. 1540, Mar. 7, 2003. [cited by applicant]

McCaffrey, et al., Gene Expression: RNA Interference in Adult Mice, Nature, vol. 418, No. 6893, pp. 38-39, Jul. 4, 2002. [cited by applicant]

Miyagishi, et al., U6 promoter-driven siRNAs With Four Uridine 3′ Overhangs Efficiently Suppress Targeted Gene Expression in Mammalian Cells, Nature Biotechnology, vol. 20, No. 5, pp. 497-500, May 1, 2002. [cited by applicant]

Mourelatos, et al., miRNPs: A Novel Class of Ribonucleoproteins Containing Numerous microRNAs, Genes & Development, vol. 16, No. 6, pp. 720-728, 2002. [cited by applicant]

Nielsen, et al., Sequence-Selective Recognition of DNA by Strand Displacement With a Thymine-Substituted Polyamide, Science, vol. 254, Issue 5037, pp. 1497-1500, Dec. 6, 1991. [cited by applicant]

Petersen, et al., LNA: A Versatile Tool for Therapeutics and Genomics, Trends in Biotechnology, vol. 21, Issue 2, pp. 74-81, Feb. 2003. [cited by applicant]

Putnam, David A., Antisense Strategies and Therapeutic Applications, American Journal of Health System Pharmacy, vol. 53, No. 2, pp. 151-160, Jan. 15, 1996. [cited by applicant]

Reinhart, et al., Small RNAs Correspond to Centromere Heterochromatic Repeats, Science, vol. 297, No. 5588, 1 Page, Sep. 13, 2002. [cited by applicant]

Rusckowski, et al., Biodistribution and Metabolism of a Mixed Backbone Oligonucleotide (GEM 231) Following Single and Multiple Dose Administration in Mice, Antisense and Nucleic Acid Drug Development, vol. 10, Issue 5, … [cited by applicant]

Schwab, et al., An Approach for New Anticancer Drugs:Oncogene-Targeted Antisense DNA, Annals of Oncology, vol. 5, Issue 4, pp. 55-58, 1994. [cited by applicant]

Stein, et al., Inhibition of Vesivirus Infections in Mammalian Tissue Culture with Antisense Morpholino Oligomers, Antisense and Nucleic Acid Drug Development, vol. 11, Issue 5, pp. 317-325, Oct. 2001. [cited by applicant]

Stein, et al., Systemic and Central Nervous System Correction of Lysosomal Storage in Mucopolysaccharidosis Type VII Mice, Journal of Virology, vol. 73, No. 4, pp. 3424-3429, Apr. 1999. [cited by applicant]

Vorobjev, et al., Nuclease Resistance and RNase H Sensitivity of Oligonucleotides Bridged by Oligomethylenediol and Oligoethylene Glycol Linkers, Antisense and Nucleic Acid Drug Development, vol. 11, No. 2, pp. 77-85, A… [cited by applicant]

Wang, et al., Nanoparticle-Based Delivery System for Application of siRNA In Vivo, Current Drug Metabolism, vol. 11, No. 2, pp. 182-196, 2010. [cited by applicant]

Xia, et al., siRNA-Mediated Gene Silencing in Vitroand In Vivo, Nature Biotechnology, vol. 20, No. 10, pp. 1006-1010, Sep. 16, 2002. [cited by applicant]

Yuan, et al., Recent Advances of siRNA Delivery by Nanoparticles, Expert Opinion on Drug Delivery vol. 8, Issue 4, pp. 521-536, 2011. [cited by applicant]

Zamore, et al., Ancient Pathways Programmed by Small RNAs, Science, May 17, 2002, 296(5571): 1265-1269. [cited by applicant]

Zeng, et al., Both Natural and Designed Micro RNAs Can Inhibit the Expression of Cognate mRNAs When Expressed in Human Cells, Molecular Cell, vol. 9, pp. 1327-1333, Jun. 2002. [cited by applicant]

Zeng, et al., Sequence Requirements for Micro RNA Processing and Function in Human Cells, RNA, vol. 9, pp. 112-123, 2003. [cited by applicant]

Zhang, et al., “Several rAAV Vectors Efficiently Cross the Blood-brain Barrier and Transduce Neurons and Astrocytes in the Neonatal Mouse Central Nervous System”, Molecular Therapy, vol. 19, Issue 8, pp. 1440-1448, Aug.… [cited by applicant]

Avino, et al., “Branched RNA: A New Architecture for RNA Interference”, Journal of Nucleic Acids, Mar. 6, 2011, 2011(586935): 1-7. [cited by applicant]

Hu, et al., “Pharmacological Prion Protein Silencing Accelerates Central Nervous System Autoimmune Disease via T Cell Receptor Signalling”, Brain, Feb. 2010, 133(2): 375-388. [cited by applicant]

Kang, et al., “Dual MicroRNA to Cellular Prion Protein Inhibits Propagation of Pathogenic Prion Protein in Cultured Cells”, Molecular Neurobiology, Mar. 2018, 55(3): 2384-2396. [cited by applicant]

Kang, et al., “Establishment and Characterization of Prnp Knockdown Neuroblastoma Cells Using Dual microRNA-mediated RNA Interference”, Prion, Apr. 2011, 5(2): 93-102. [cited by applicant]

Kim, “Utility of RNAi-mediated prnp Gene Silencing in Neuroblastoma Cells Permanently Infected by Prions: Potentials and Limitations”, Antiviral Research, Nov. 2009, 84(2): 185-193. [cited by applicant]

Partial Supplementary European Search Report received in European Patent Application No. 21761310.8, dated Apr. 2, 2024. [cited by applicant]

Shi, et al., “Prion Protein Participates in the Regulation of Classical and Alternative Activation of BV2 Microglia”, Journal of Neurochemistry, Jan. 2013, 124(2): 168-174. [cited by applicant]

Supplementary European Search Report received in European Patent Application No. 21761310.8, dated Jun. 24, 2024. [cited by applicant]

Sutou, et al., “Knockdown of the Bovine Prion Gene PRNP by RNA Interference (RNAi) Technology”, BMC Biotechnology, Jul. 26, 2007, 7(44): 1-10. [cited by applicant]