AU2023203042A1 - Compositions and methods for delivery of therapeutic agents - Google Patents
Compositions and methods for delivery of therapeutic agents Download PDFInfo
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Abstract
This disclosure provides improved lipid-based compositions, including lipid nanoparticle compositions, and
methods of use thereof for delivering agents in vivo including nucleic acids and proteins. These
compositions are not subject to accelerated blood clearance and they have an improved toxicity profile in
vivo.
Description
Cd COMPOSITIONS AND METHODS FOR DELIVERY OF AGENTS
',C 5 RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § II9(e) of U.S. provisional application number 62/265,973, filed December 10, 2015, U.S. provisional application number 62/266,581, filed December 12, 2015, U.S. provisional application number 62/311,388, filed March 21, 2016, U.S. provisional application number 62/350,172, filed o June 14, 2016, U.S. provisional application number 62/413,027, filed October 26, 2016, U.S. provisional application number 62/311,386, filed March 21, 2016, U.S. provisional N application number 62/350,165, filed June 14, 2016, U.S. provisional application number 62/311,380, filed March 26, 2016, and U.S. provisional application number 62/413,050, iled October 26, 2016, each ofwhich is incorporated by reference herein in its entirety. 15 BACKGROUND Effective in vivo delivery of active agents such as small molecule drugs, proteins, peptides, and nucleic acids represents a continuing medical challenge. Some active agents are recognized by the immune system, resulting in decreased efficacy. To address this issue, !o certain active agent formulations have incorporated polymers such as polyethylene glycol which was thought to cloak or mask the agent, thereby reducing its antigenicity and immunogenicity. However, even these "stealth" formulations have their limitations, including an inability to be repeatedly and frequently dosed, for example, over a period of days without loss of activity. 25 In addition, some agents or formulations when administered in vivo may interact with one or more cells or factors, potentially interfering with their functions, and ultimately resulting in adverse effects. Such adverse effects may limit the administration frequency and/or administered dose of the agent, or may preclude in vivo use altogether.
30 SUMMARY The present disclosure is based, at least in part, on the discoveries that components of lipid nanoparticles (LNPs) may induce an innate immune response. In some embodiments components of the LNPs, such as phosphatidylcholine, may induce the production of natural IgM and/or IgG molecules, which may be mediated by activation of Bl cells, such as Bl a and/or BI b cells. These biological mechanisms may contribute to drug responses caused by LNPs, including accelerated blood clearance (ABC) and dose-limiting toxicity such as acute phase response (APR) and complement activation-related pseudoallergy (CARPA). Both Bla cells and platelets express CD36, which can bind phosphatidylcholine. 5 The activation of Bl cells and platelets by LNPs may be mediated by activation of the CD36 receptor by a component in the LNPs such as phosphatidylcholine. Additionally, the PEG-lipid on the LNPs may contribute to the production of natural IgM and/or anti-PEG IgG and IgM. Accordingly, provided herein are methods and compositions for delivering LNPs to N a subject without promoting the same degree of LNP-related drug responses as noted herein N i10 by using LNPs that do not trigger an innate immune response characterized by natural IgM N production, natural IgG production, anti-PEG IgM, anti-PEG IgG, Bla cell activation, Blb cell activation, pDC cell activation and/or platelet aggregation and/or activation, and/or using secondary agents, in particular, pharmacological agents, that inhibit the production of this innate immune response, as well as suppress the downstream signaling pathways leading to 15 the LNP-related drug responses. This disclosure provides, in part, novel lipid nanoparticles (LNP) and LNP formulations that are less susceptible to recognition and thus clearance, by the immune system. The LNP provided herein have surprisingly improved clearance and in some instances, toxicity profiles. While not intending to be bound by any particular mechanism or 20 theory, the improved clearance profiles are believed to have reduced recognition by and/or binding to certain immune cells and less overall effect on those and other immune cells and factors. More specifically, certain of the LNPs provided herein have no or low binding to Bla and/or B I b cells, Bla and/or Bl b activating activity, pDC activating activity, platelet aggregating activity, and/or platelet activating activity. This activity may be due at least in 25 part to the components of the LNP, or an agent that inhibits immune responses induced by the LNP components. Such an agent may be incorporated within the LNP administered or or formulated separately. Also provided in this disclosure are compounds and compositions, including formulations, that modulate immune responses to administered nanoparticles such as LNP. 30 Also provided herein are methods ofuse of such compounds and compositions, particularly relating to immune modulation in vivo. Also provided are methods of use of LNPs together with certain classes of secondary agents, including for example use of LNPs in subjects that have been co-medicated, e.g., pre-medicated with certain secondary agents. Also provided are pre-administration and/or pre-treatment screening methods that identify patients that respond to LNP administration and optionally classify such patients according the degree of their response. Identifying such subjects may lead, in some instances, to a modified treatment regimen.
5 Certain of the LNPs provided herein comprise a cationic lipid, a helper lipid, a structural lipid, and a stabilizer which may or may not be provided conjugated to another
lipid. The cationic lipid may be but is not limited to DLin-DMA, DLin-D-DMA, DLin N MC3-DMA, DLin-KC2-DMA and DODMA. The cationic lipid may be an ionizable lipid. N 0 The structural lipid may be but is not limited to a sterol such as for example N cholesterol. The helper lipid is an amphiphilic surface active lipid, or surfactant. In some embodiments it is a non-cationic lipid The helper lipid may comprise at least one non-polar chain and at least one polar headgroup moiety. A helper lipid may also be referred to as a 5 complementary lipid i.e. the lipid functions to "complement" the amino lipid and increase the fusogenicity of the bilayer to help endosomal escape. In some embodiments the non-polar chain is a lipid. In other embodiments it is a fatty acid of at least 8C. In exemplary embodiments, the helper lipid is non-naturally occurring (e.g., not naturally occurring in human subjects) or is exogenous. !o Certain ofthe LNPs lack any phosphatidyl choline (PC) lipids (i.e., are free of phosphatidyl choline (PC)). Certain of the LNPs provided herein lack specific phosphatidyl choline lipids such as but not limiting to DSPC. Certain of the LNPs comprise a phosphatidyl choline analog, such analogs comprising modified head groups (e.g., a modified quaternary amine head group), modified core group, and/or modified lipid tail group. Such 25 analogs may comprise a zwitterionic group that is a non-PC zwitterionic group. The helper lipid may be a lipid of any one or any combination of Formulae I, I-a, I-b, I-b-1, 1-b-2, 1-b-3, I-b-4, I-c, I-c-1, 1-c-2, I-c-3, or 11 as provided herein. Certain LNPs comprise other helper non-cationic lipids including for example oleic acid or oleic acid analogs. The helper lipid may be a lipid ofFormula IV as provided herein. 30 The stabilizer may be polyethylene glycol (PEG). PEG may be conjugated to a lipid and thus may be provided as PEG-c-DOMG or PEG-DMG, for example. The stabilizer, whether provided in a conjugated or an unconjugated form, may comprise 1.5 mol % ofthe LNP, or it may comprise less than 0.5 mol % of the LNP. For example, it may comprise less than 0.4 mol %, less than 0.3 mol %, less than 0.2 mol %, or less than 0.1 mol %. Each possibility represents a separate embodiment of the present invention. The LNP may comprise a PEGylated lipid of FormulaIII, including Formulae III-OH, 111-a-1, 111-a-2, I-b-1, III1-b-2, 1II-b-I-OH, 111-b-2-OH, V, V-OH. Each possibility 5 represents a separate embodiment of the present invention. Certain of the LNPs provided herein comprise no or low levels of PEGylated lipids, including no or low levels of alkyl-PEGylated lipids, and may be referred to herein as being free of PEG or PEGylated lipid. Thus, some LNPs comprise less than 0.5 mol % PEGylated N lipid. In some instances, PEG may be an alkyl-PEG such as methoxy-PEG. Still other LNPs N lo comprise non-alkyl-PEG such as hydroxy-PEG, and/or non-alkyl-PEGylated lipids such as N hydroxy-PEGylated lipids. Each possibility represents a separate embodiment of the present invention. The PEGylated lipid may be a Cmpd420, a Cmpd396, a Cmpd394, Cmpd397, Cmpd395, Cmpd4l7, Cmpd48, or Cmpd4I9. Each possibility represents a separate 15 embodiment of the present invention. In some instances, the LNP may comprise about 50 mol %,10 mol % helper lipid, 1.5 mol % PEGylated lipid, and 38.5 mol % structural lipid. In some instances, the LNP may comprise about 50 mol %,10 mol % helper lipid, less than 0.5 mol % PEGylated lipid, and 39.5 mol % structural lipid. Each possibility 20 represents a separate embodiment of the present invention. In some embodiments, the stabilizer is a non-PEG moiety such as an XTEN peptide that may or may not be conjugated to a lipid. The XTEN peptide is capable of forming a hydrated shell around the LNP due to its hydrophilic nature. It further serves to increase the half-life of the LNP, compared to an LNP lacking (or free of) any stabilizer. Unlike PEG, 25 however, it is biodegradable and has been reported to be non-immunogenic. The X FEN peptide may have an amino acid sequence of MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS (SEQ ID NO: I) or MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS (SEQ ID NO:2). Other XTEN amino acid sequences are known in the art, including for example those 30 reported in U.S. Patent No. 9,062,299. Examples of XTEN conjugated lipids include but are not limited to Cmpd431, and Cmpd432 and Cmpd433. Click chemistry may be used to conjugate the XTEN peptide to the lipid.
In some embodiments, the stabilizer is a non-PEG moiety such as a PAS peptide. A PAS peptide is a peptide comprising primarily if not exclusively proline, alanine and serine. Like PEG and XTEN peptides, the PAS peptide is capable of forming a hydrated shell around the LNP. It too serves to increase the half-life of an LNP, compared to an LNP lacking (or 5 free of) a stabilizer. Unlike XTEN peptides, however, PAS peptides tend to be neutral in charge, and thus at least in this respect more similar to PEG. The PAS peptide may have an amino acid sequence of
SAPSSPSPSAPSSPSPASPSSAPSSPSPSAPSSPSPASPSSAPSSPSPSAPSSPSPASPS(SEQ N ID NO:3 or N i0 AASPAAPSAPPAAASPAAPSAPPAAASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO:4). N Other PAS amino acid sequences are known in the art, including for example, those reported
in WO 2008155134. The disclosure contemplates LNPs having any combination of the foregoing characteristics. Such LNPs may be further characterized as having reduced binding to BI a 15 cells and/or reduced Bla cell activation activity. Additionally or alternatively, they maybe further characterized as having reduced platelet aggregation activity, which may be indicated
as reduced platelet activation activity.
This disclosure further contemplates that such LNPs may be used in vivo to deliver an agent, such as a protein or a nucleic acid, without triggering accelerated blood clearance 20 (ABC). Thus, such LNPF can beadministered to a subject repeatedly and within short time periods without risk of enhanced clearance by the immune system, as has been previously
reported for a variety of administered agents including lipid formulated agents. Thus, the LNPs and more importantly their cargo can be administered more frequently, and effectively at higher doses over these short time periods, than was previously possible. 25 Even more surprisingly, certain of these LNPs also demonstrate reduced toxicity upon administration. Again, while not intending to be bound by any particular mechanism or
theory, this is believed to result from the reduced platelet aggregation activity of these LNPs. This inability or reduced ability to aggregate platelets reduces the likelihood and severity of coagulopathy-related toxicity that has been observed following LNP administration in vivo. 30 It was wholly unexpected that certain LNPs would have the dual benefit of reduced susceptibility to ABC and reduced toxicity in vivo. As a result, these LNPs allow for higher doses of encapsulated agent to be administered to a subject, due in part to the reduced toxicity
profile of their encapsulating LNP. The LNPs also lead to a longer half-life for the LNPs and thus their cargo, due in part to their reduced susceptibility to ABC. This results in higher and more stable levels of cargo between administrations. Moreover, in the case of cargo requires repeated frequent administration, the LNPs provided herein facilitate such administration. This serves to increase the efficacy of certain agents by allowing more frequent dosing than 5 may currently be possible. This also serves to render useful other agents that may have not been used previously in vivo due to these restrictions.
This disclosure further provides other novel formulations and methods of use of LNPs, including LNP formulations. Specifically, provided herein are methods of use of N LNPs and LNP formulations together with anti-platelet agents including but not limited to N i10 platelet aggregation inhibitors. This disclosure contemplates that such agents may be N administered to a subject prior to and/or substantially simultaneously with, and/or even after
administration of the LNP. Thus, such agents may be formulated together with the LNP or they may be separatly formulated but administered together, via the same route, and/or at the
same or substantially the same time. Significantly, pre-medication or co-medication ofa
15 subject with these anti-platelet agents results in reduced toxicity, including coagulopathy
related toxicity, and in a lower and less severe incidence of ABC. According to certain
embodiments, subjects may be pre-medicated or co-medicated with a combination of secondary agents including platelet aggregation inhibitors, anti-histamines, and NSAIDS or COX enzyme inhibitors. Certain secondary agents that may he used have dual functionality 20 (i.e., they are ahle to inhibit platelet aggregation, in whole or in part, and also have a general
anti-inflammatory effect). One such example is aspirin.
A lipid nanoparticle (LNP) encapsulating an mRNA encoding a protein is provided in some aspects of the invention. The LNP has a cationic lipid, a non-cationic helper lipid comprising at least one fatty acid chain of at least 8C and at least one polar head group 2S moiety, and wherein the helper lipid is not a phosphatidyl choline (PC), a PEG lipid, and a sterol. In some aspects the LNP further comprises an agent that inhibits immune responses by
the LNP. In other aspects the non-cationic helper lipid is a zwitterionic non-cationic helper lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1,2-distearoyl-su-glycero-3-phosphocholine (DSPC) substitute. 30 In certain embodiments, non-cationic lipids useful in the present invention are DSPC
analogs wherein the phosphocholine moiety is replaced by a different zwitterionic group. In certain embodiments, the different zwitterionic group is not a phosphocholine group. In certain embodiments, a non-cationic lipid useful in the present invention is a compound of
Formula (II). Provided herein are compounds of Formula (II): Z A
(II), 5 or a salts thereof, wherein: N Z is a zwitterionic moiety, 8 (D 0 O O wherein the zwitterionic moiety is not of the formula: 0 m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
B (R 2)p A is of the formula: \ L2-R2 or 2 10 each instance of L is independently a bond or optionally substituted C-6 alkylene, wherein one methylene unit of the optionally substituted Ci-6 alkylene is optionally replaced with -0-, -N(R')-, -S-, -C(O)-, -C(O)N(RN), -NRNC(O)-, -C()O-, -OC(O)-, OC(0)0-, -OC(O)N(RN)-, -NRNC(0)0, or -NRNC(O)N(RN)-; each instance of R2 is independently optionally substituted C 30 alkyl, optionally 15 substituted C 3 0 alkenyl, or optionally substituted C 3 0 alkynyl; optionally wherein one or more methylene units of R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -0-, -S-, -C(O)-, -C(O)N(RN)-, NRNC(O)-, -NReC(O)N(RN)-, -C(0)0-, -OC(O)-, -OC(0)0-, -OC(O)N(R)-, 20 NRNC(0)0-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=NRN), NRNC(=NR)N(R)-,-C(S)-,-C(S)N(R)-, -NRNC(S)-, -NR NC(S)N(RN)-, -S(O)-,
OS(O)-, -S(0)0-, -OS(0)0-, -OS() 2-, -S(0) 2 0-, -OS(0)20-, -N(R N)S(0)_, S(O)N(R N)-, -N(R N)S(O)N(R N)-, -OS(O)N(RN)-, -N(R N)S(o)O)--, -S(0)2-, -N(R N)S (0)2-,
-S(0) 2N(RN), -N(RN)S(0) 2 N(RN)-, -OS(0) 2N(RN)-, or -N(RN)S(0) 20-; 25 each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted hetcroaryl; and
p is I or 2.
In certain embodiments, Z is an amino acid or a derivative thereof. In certain embodiments, Z is of one of the following formulae:
(RN) 3N RN 0 0®R N)/ 0y/ (y
(RN 0 N O N(RN) 3 0 O N Of'" Cl 0 ~0 0 eRO0-P.. 00 00
(RN) 3N N' O's/ 5 (N3(RN) 3 NON R H N(R ) 3 0N e 0 0 OS0 3 (RN) 3 X~'P 7.O (RN) NfS 0,7 0 0 db
protecting group. In certain wherein ROis hydrogen, optionally substituted alkyl oran oxygen//I0) \NN) embodiments, acompound of Formula (II) is ofone of the following formulae: 0 0 (RN) 3 N0 RN 0 0R ) 0-3N R N 00 N O A N O 10AN 0 A O 0 0 0 OOP
(RN N 3 RNN 0D 0(N) N0 N O, A0A H, 0 0 RN emor iens a saltun thereof.1 sofoeofteolwigfrm le 0 ®N~O(RN) D3 N 0RN(N A(N ½AA (RNN)N
10 0 H
orasaltthereof.
In certain embodiments, acompound of Formula (11)is ofone of the following formulae: 0 R2 0 R2
__(RN )3 N( RN 0 0 OE 0 'I I 0 N 2 0CO R 0O'1 0R GO 00 E)N(RN )3
0 R2 00 R2 00 (DN(RN )3 0 0-/ 0 00 eo 0 AR 2 0 R-l' 2 2N) 2 0 R 0 0R 0~
G0 0 0S0 3 0
(RN) N, s"O' O' R2 R2. 5 00 NR)3
oy R 2 0 yR 2
0 0 e 00 (RN )3N( RN 00 (RN) 30 0P 0) R 2 0KjR 2 II 0 0 0
0y R2 0)O R2
0 00 (RN )3N RN 0 00 00 N ")N0 00 RN
(R )3N H 0 R2
or asalt thereof. 10 ~For example, in certain embodiments, acompound of Formula (11)is one of the following:
0'
0 NMe 3
00 u(Kto, 0
NMe 3 (D
E) 0 cdC0 2 00 H 3N p 11 0
0K 00 0 Me 3 N 0
00 0 ClH 3N 0 O,
0 0 0
0
0 0 NH3 H 0
0 N
0 0
0G0 00 Cd O H 3N N H
(D0I 0
00K 0 0N3H
N 0
0
or salts thereof. Non-cationic lipids useful in the present invention also include analogs of oleic acid. As described herein, an oleic acid analog can comprise a modified oleic acid tail, a modified 5 carboxylic acid moiety, or both. In certain embodiments, an analog of oleic acid is a compound of Formula (IV). Provided herein are compounds of Formula (IV): O HO R4 (IV), or a salt thereof, wherein: 10 R4 is optionally substituted, C1 0 -4 0 alkyl; optionally substituted, Cio-40 alkenyl; 4 optionally substituted, CIO. 40 alkynyl; wherein at least one methylene group of R is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN) , -0-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, NReC(O)N(RN)-, -C(O)O-, -OC(O)-, 15 OC(O)O-, -OC(O)N(RN)-, -NRNC()O-, -C(O)S-, -SC(O)-, -C(=NRN),
C(=NRN)N(RN)-, -NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, NRNC(S)-, -NRNC(S)N(RN) , -S(O)-, -OS(O)-, -S(0)0-, -OS(0)0-, -OS(0)2-, S(0) 2 0-, -OS(0) 20-, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(R)N) N)()_SN) pN N(RN)S(O)O, -S(0)2-, -N(R)S(O) 2 -, -S(O) 2N(RN), -N(RN)S() 2 N(R
20 OS(0) 2N(RN)-, ur -N(RN)S(0) 20-; and each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In certain embodiments, the compound of Formula (IV) is one of the following:
0
0 0 (Cmpd148) HO
0 0 (Cmpd149) 0
0
HO 0
(Cmpd159), or salts thereof. In certain embodiments, an oleic acid analog is a compound wherein the carboxylic acid moiety of oleic acid replaced by a different group. In certain embodiments, an oleic acid analog useful in the present invention is one of the following: H 0S, 0 0 (Cmpd157) H O NA
(Cmpd158) H
6O160
N lo 1 e H N0N
H CF 3 CON ,-, 2
Cl N N00
H N SN 0
EtO0 P11 0
H O P11 0
HO3S
HN--N or salts thereof.
Examples of non-cationic lipids include, but are not limited to the following: 0
10 0 (Cmpd]8) 0
(Cmpd393)
H 0 0 N O
(Cmpd125)
HO N 0
0O 0 S N H
0 S Ell
0 HO N
0O 0 N H
0 HO
I0 HO 0 ~SN H HO N
WO 2017/099823 PCTUS2O16OOO129 16
00
0
__________ H lI
0
0l 0
c-I NOH,
N~OH
H 3 CIOH
0
10 00HO n
HOH Ho-OH
0 OH0 OHO
CH 2OH
HO O HO10 "OH _0H OH OH ,and
A lipid nanoparticle (LNP) encapsulating an mRNA encoding aprotein is provided in some aspects ofthe invention. The LNP has acationic lipid,anon-cationic helper lipid comprising at least one fatty acid chain of atleast8C and at least one polar head group moiety, aPEG lipid,and asterol. In some aspects the LNP further comprises an agent that inhibits immune responses by the LNP. In some embodiments the PEG lipid isan alkyl PEGylated lipids, non-alkyl-PEG such as hydroxy-PEG, anon-alkyl-PEGylated lipid such as hydroxy-PEGylated lipid, aCmpd420, aCmpd396, aCmpd394, Cmpd397, Cmpd395, Cmpd4l7, Cmpd4l8, orCmpd4l9, Cmpd42l, Cmpd422, or wherein the PEG lipid contains less than 0.5% molar ratio of PEG lipid to the other components. In certain embodiments, aPEG lipid useful in the present invention is acompound of Formula (III). Provided herein are compounds of Formula (III):
(III). or salts thereof, wherein: R3 is -OR 0 .
RO is hydrogen, optionally substituted alkyl, oran oxygen protecting group; r isaannintegerbeteenand100,inclusive; L' isoptionally substitutedC.o alkylene, whereinat leastone methylene ofvthe optionallysubstituted Ciioalkylene isindependentlyreplaced withoptionally substituted carbocyclylene, optionally substitutedheterocyclylene,optionallysubstituted arylene, optionallysubstituted heteroarylene, -N(RN)-, -S-, -C(O)-, -C(O)N(RN)-,-
NRNC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, or NRNC(O)N(RN
D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is0,1,2,3,4,5,6,7,8,9,or10;
L2-RB (R 2)p IIL2-R2 A is of the formula: or ; 2 each instance of L is independently a bond or optionally substituted C. 6 alkylene, wherein one methylene unit of the optionally substituted C. 6 alkylene is optionally replaced with -0-, -N(RN)-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C(O)O-, -OC(O)-, OC(O)O-, -OC(O)N(RN)-, -NRNC(O)O-, or -NRC(O)N(RN each instance of R2 is independently optionally substituted C 30 alkyl, optionally substituted Cs3 0 alkenyl, or optionally substituted C. 30 alkynyl; optionally wherein one or more methylene units of R 2 are independently replaced with optionally substituted
carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene,
optionally substituted heteroarylene, -N(RN)-, -0-, --,-C(O)-, -C(O)N(R)-, NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)_,_ NRNC(O)O-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN), -NRNC(=NRN)_, NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-,-S()-, OS(O)-, -S(O)O-, -OS(O)O-, -OS(O) 2 -, -S(0)20-,-5(0)20-, -N(RN)S(O)_,_ S(O)N(RN)-, -N(R N)S(O)N(RN-, -OS(O)N(RN-, -N(RN)S(O)O-, -S(O)2-, -N(RN)S C)2
-S(O) 2 N(RN)-, -N(RN)S(O) 2N(RN)-, -OS(O) 2 N(RN)-, or -N(RN)S(O) 2 -; each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;
Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and
p is I or 2. 3 In certain embodiments, the compound of Fomula (III) is a PEG-OH lipid (i.e., R is -ORO, and Ro is hydrogen). In certain embodiments, the compound of Formula (III) is of Formula (111-011):
.lO ()L-DmA
(III-OH), or a salt thereof. In certain embodiments, D is a moiety obtained by click chemistry (e.g., triazole). In certain embodiments, the compound of Formila (III) is of Formula (III-a-1) or (III-a-2): N.::.N, A ,NzN 'I R3 O L Nt)m R3 O or rA 5 (III-a-i) (III-a-2), or a salt thereof. In certain embodiments, the compound of Formula (III) is of one of the following formulae: R2 R2 O 0 N=N L2 R2 O NsrN L R2 R3 N L2'Rm R3 O< 4 sN ~~lmL 2
' O2 N=N L 2 I2 O N :N L I2 2 2 HO N L HO R r sO"
. 10
or a salt thereof, wherein s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the compound of Formula (III) is of one of the following formulae: O R2 O R2
O N=N 00 0 NN 0
R3 O R2 R3 OO R2 15 2 CO K2 U
O N=N 0 O N:N 0 HO O)S 4A N 9ER2 HO O s O R2
or a salt thereof. In certain embodiments, a compound of Formula (III) is of one of the following formulae:
R2 > OyR2 O-.Z Cd 00 N=N O N -N O R2 0 1 O R2 0 N
R3 0 R3 0
R2 2 O R
0 -N -N O N=N 0 O O N R
O R200 2 R O HO HO
or a salt thereof. In certain embodiments, a compound of Formula (III) is of one ofthe following formulae: O
0 N==N NN 00 O
(Cmpd394),
N=N 0 0O
(Cmpd396), 0
N=N 0 0 %
/0 (Cmpd395),
C0 N=N 0
(Cmpd397), or a salt thereof. In certain embodiments, D is a moiety cleavable under physiological conditions (e.g., 5 ester, amide, carbonate, carbamate, urea). In certain embodiments, a compound of Formula (III) is of Formula (III-b-1) or (1II-b-2):
R3 L'O m 3 1 m ROy-r M R3 .L>
(III-b-1) (III-b-2), or a salt thereof. to In certain embodiments, a compound of Formula (III) is of Formula (III-b-i-OH)or (III-b 2-OH):
HO OLiO>m HO L0 0 A 0r
(111-b-I-OH) (III-b-2-OH), or a salt thereof 15 In certain embodiments, the compound of Formula (III) is of one of the following formulae: R R22
R3 L O L2'R2 R3 O 2R2 r ' L1' oj1>«L2
0 0 2 2 R 2R2
SHO OLO mL2 0
or a salt thereof. 20 In certain embodiments, a compound of Formula (III) is of one of the following formulae:
O R2 O R2 00 R3 OL O O R2 R3 O
OyR2Oy<R2 2 0 R 0 R2 Nl O
HO OL O OR2 HO L O O R2 0 or a salt thereof. In certain embodiments, a compound of Formula (III) is of one of the following 5 formulae: O R2 O R2
0 00 R3 O0 O 0 R2 R3 OOr O R
O R2 O R2
0O 00 0 00 HO Or s 0 R2 HO OR2 0
or a salt thereof. In certain embodiments, a compound of Formula (III) is of one of the following 10 formulae:
0OO0 00 O 0
0 0 O 00
0t 0 0 00
or salts thereof.
In certain embodiments, a PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (V). Provided herein are compounds of Formula (V): 0 R3<O R5
5 (V), Cl or a salts thereof, wherein: R' is-ORO; Ro is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between I and 100, inclusive; 10 R5 is optionally substituted C1 0 .40 alkyl, optionally substituted C0o40 alkenyl, or optionally substituted C 1 040 alkynyl; and optionally one or more methylene groups of Rs are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN), -S-, C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NR C(O)N(R N)-, -C(O)O-, -OC(O)-, -OC(O)O-, 15 OC(O)N(RN)-, -NRNC(O)O, -C(O)S-, -SC(O)-, -C(=N R N)-, -C(=N R N N
NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(Sy NRNC(S)N(RN)-,-S(O)-,-OS(O)-,-S(0)0-,-OS(O)O-,-OS(O)r,-S(0)20-,
OS(0)20-, -N(R)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(R)-, N(RN)S(0)O-, -S(0)2-, -N(RN)S(0) 2 -, -S(0) 2N(RN)-, -N(RN)S(0) 2N(RN)_ 20 OS(O) 2N(RN)-, or -N(RN)S(O) 2 0-; and each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In certain embodiments, the compound of Formula (V) is of Formula (V-Oil): 0 HO O) R5
25 (V-OH), or a salt thereof. In certain embodiments, a compound of Formula (V) is of one of the following formulae: 0 30 ( p4O
30 (Cmpd400),
Cd
(Cmpd401), 0
N (Cm pd401), O
5 0 (Crnpd402), 0
0 HOO
H HO N r 0 HOO 10 H
or a salt thereof. In other aspects the non-cationic helper lipid is a zwitterionic non-cationic helper lipid, a lipid that is not a phosphatidyl choline (PC), a DSPC analog, oleic acid, an oleic acid analog, or a 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC) substitute. 15 In some embodiments the agent that inhibits immune responses by the LNP comprises a miR binding site. In other embodiments the miR binding site is selected from miR 126, miR 155, and miR 142 3p. The miR binding site is incorporated into a mRNA in some embodiments. In other embodiments the miR binding site is separate from the mRNA. In some embodiments the agent that inhibits immune responses by the LNP comprises 20 an mRNA comprising a miR binding site. In various embodiments, the mRNA comprises 1-4, one, two, three or four miR binding sites, wherein at least one of the miR binding sites is a miR-126 binding site. In one embodiment, the mRNA, comprises at least two microRNA binding sites, wherein at least one of the microRNA binding sites is a miR-126 binding site. In one embodiment, the mRNA, e.g., mmRNA, comprises a miR-126 binding site and a 25 second microRNA binding site for a miR selected from the group consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24 and miR-27. In another embodiment, the mRNA, comprises amiR-126 (e.g., miR-126-3p) binding site and a miR-142 (e.g., miR-142-3p) binding site. A miR referred to by number herein can refer to either of the two mature microRNAs originating from opposite arms of the 5 same pre-miRNA (e.g., either the 3p or 5p microRNA). All miRs referred to by number herein are intended to include both the 3p and 5p arms/sequences. It has now been discovered that incorporation of at least one microRNA binding site for a microRNA expressed in immune cells (e.g., miR-126, miR-142, miR-155 and combinations thereof) into N an mRNA construct can reduce or inhibit ABC when the lipid-comprising compound or N i0 composition comprising the mRNA is administered to a subject. In one embodiment, the N mechanism of action of the miRNA binding site(s) is a microRNA "sponge", wherein the miRNA binding site(s) in the construct or LNP "soaks up" microRNAs that bind to the binding site(s). The DSPC analog may have a modified head group that is a modified quaternary 15 amine head group, a modified core group, or a modified lipid tail group. In some embodiments. The PEG lipid in other embodiments contains at least 0.0001%, at least 0.0005%, at least 0.001%, at least 0.005%, at least 0.01%, at least 0.05%, at least 0.1%, at least 0.15%, at least 0.2%, at least 0.25%, at least 0.3%, at least 0.35%, at least 0.4%, at least 0.45%, and less than 0.5% molar ratio of PEG lipid to the other components. ?0 In some embodiments the LNP may have a molar ratio of about 45-65% cationic lipid, about 0.15-15% PEG lipid, about 15-45% cholesterol and about 5-25% non-cationic helper lipid or a molar ratio of about 55% cationic lipid, about 2.5% PEG lipid, about 32.5% cholesterol and about 10% non-cationic lipid. In other embodiments the cationic lipid is selected from DLin-DMA, DLin-D-DMA, 25 DLin-MC3-DMA, DLin-KC2-DMA or DODMA. In other embodiments the cationic lipid is selected from the lipid Cmpd numbers provided herein. The invention in some aspects is a method for delivering lipid nanoparticles (LNPs) to a subject without producing an immune response that promotes accelerated blood clearance (ABC) in response to subsequent doses of the LNP. The method involves administering a 30 first dose of LNPs to the subject, wherein the first dose of'LNPs does not induce an immune response that promotes ABC upon administration of a second dose of LNP, and administering a second dose of LNPs to the subject, wherein the subject does not have an ABC response to the second dose of LNPs. In some embodiments the LNPs encapsulate a therapeutic agent and wherein the subject receives an effective amount of the therapeutic agent for treating a disease. In other aspects the invention is a method for delivering lipid nanoparticles (LNPs) to a subject without producing an immune response that promotes accelerated blood clearance
5 (ABC) in response to subsequent doses of the LNP, by administering a first dose of LNPs to the subject, wherein the LNPs are capable of inducing an immune response that promotes
ABC upon administration of a second dose of LNP, administering an agent that inhibits immune responses induced by the LNPs and administering a second dose of LNPs to the N subject, wherein the subject does not have an ABC response to the second dose of LNPs. N 10 In yet other aspects the invention is a method for reducing dose-limiting toxicity N (DLT) in a subject being treated with a therapeutic regimen involving lipid nanoparticle (LNP)-mediated drug delivery, by administering LNPs to the subject, wherein the LNPs do not induce an immune response associated with B Icell activation or platelet activation, and optionally administering an agent that inhibits immune responses induced by the LNPs, such 15 that DLT is reduced in the subject being treated with the therapeutic regimen. In yet other aspects the invention is a method in a subject of increasing the therapeutic
index of a therapeutic regimen involving lipid nanoparticle (LNP)-mediated drug delivery, by administering LNPs to the subject, wherein the LNPs do not induce an immune response associated with B Icell activation or platelet activation, and optionally administering an agent 20 that inhibits immune responses induced by the LNPs, such that DLT is reduced in the subject being treated with the therapeutic regimen.
A method for reducing dose-limiting toxicity (DLT) in a subject being treated with therapeutic regimen involving lipid nanoparticle (LNP)-mediated drug delivery is provided in other aspects of the invention. The method involves administering to the subject LNPs and an
25 agent that inhibits platelet activation, such that DLT is reduced in the subject being treated with therapeutic regimen.
A method for delivering a therapeutic level of a protein of interest to a subject is provided in other aspects of the invention. The method involves administering to the subject a first dose of lipid nanoparticles (LNPs), which encapsulate an mRNA coding for the protein 30 of interest, wherein the first dose of LNPs does not induce an immune response that promotes
accelerated blood clearance (ABC) upon administration of a second dose of LNP. According to other aspects the invention is a method for reducing dose-limiting toxicity (DLT) and/or accelerated blood clearance (ABC) in a subject being treated with a therapeutic regimen involving lipid nanoparticle (LNP)-mediated drug delivery, by administering to the subject a first dose of lipid nanoparticles (LNPs), which encapsulates an mRNA coding for the protein of interest, wherein the first dose of LNPs do not activate a CD36-dependent signaling pathway in an immune cell upon administration of a second dose 5 of LNP. An accelerated blood clearance (ABC) insensitive lipid nanoparticle (LNP) having a cationic lipid, a PEG-lipid, a sterol, and a helper lipid, wherein the helper lipid does not comprise a phosphatidyl choline (PC) is provided in other aspects of the invention. N According to other aspects the invention is a lipid nanoparticle (LNP) having a N 10 cationic lipid, a non-cationic, non-PC lipid, less than 0.5% (w/w) of a PEGylated lipid, and a N sterol. The LNP is insensitive to accelerated blood clearance upon repeated administration in vivo within 2 days - 3 weeks. In some embodiments the LNP is insensitive to accelerated blood clearance upon repeated administration in vivo within 4-12 days.
In some aspects, the invention is a method for reducing accelerated blood clearance 15 (ABC) in a subject being treated with a therapeutic regimen involving repeat dosing of lipid nanoparticles (LNPs), the method comprising administering LNPs to the subject, wherein the LNPs do not activate Bla cells and/or do not induce production of natural IgM molecules capable of binding to the LNPs, such that ABC is reduced upon repeat administration of the LNPs to the subjot. 20 In some aspects, the invention is a method for reducing accelerated blood clearance (ABC) in a subject being treated with a therapeutic regimen involving multiple dosing of lipid nanoparticles (LNPs), the method comprising administering a dose of LNPs to the subject, wherein the LNPs do not activate B1a cells and/or do not induce production of natural IgM molecules capable of binding to the LNPs, such that ABC is reduced upon 25 administration of one or more subsequent doses of the LNPs to the subject. In some aspects, the invention is a method for reducing accelerated blood clearance (ABC) of lipid nanoparticles (LNPs) in a subject being treated with a multi-dose or repeat dosing therapeutic regimen, the method comprising administering LNPs to the subject, wherein the LNPs do not activate Bla cells and/or do not induce production of natural IgM 30 molecules capable ofbinding to the LNPs. such that ABC is reduced upon subsequent or repeat dosing of LNPs in the subject. In some aspects, the invention is a method for decelerating blood clearance ofLNPs, the method comprising administering LNPs to a subject, wherein the LNPs do not activate
Bla cells and/or do not induce production of natural IgM molecules capable of binding to the LNPs, such that upon administration of a subsequent dose of the LNPs to the subject blood clearance of the LNPs is decelerated. As used herein, "decelerating" or "decelerated" refers to slow, delay or repress blood clearance. 5 In some aspects, the invention is a method for delivering lipid nanoparticles (LNPs) to a subject without promoting accelerated blood clearance (ABC), the method comprising administering LNPs to the subject, wherein the LNPs do not promote ABC. In some embodiments, the LNPs do not induce production of natural IgM molecules capable of binding to the LNPs. N 10 In some embodiments, the LNPs do not activate Bla cells. N In some embodiments, the LNPs do not activate CD36 or BIa cells. In some embodiments, the LNPs are free of an epitope that activates Bla cells. In some embodiments, the LNPs comprise a helper lipid, which comprises at least one fatty acid chain of at least 8C and at least one polar moiety, and wherein the helper lipid does 15 not activate Bla cells. In some embodiments, the LNPs are free of phosphatidyl choline (PC). In other embodiments, the helper lipid is a phosphatidyl choline analog. In some embodiments, the phosphatidyl choline analog comprises a modified PC head group, a modified PC core group, and/or a modified PC lipid tail. In some embodiments, the helper lipid competitively inhibits 20 phosphatidylcholine from binding to CD36. In some embodiments, the helper lipid does not bind or has low binding activity to CD36. In some embodiments, the LNPs comprise oleic acid or an oleic acid analog. In some embodiments, the LNPs are free of PEG or a PEGylated
lipid. In some embodiments, the LNPs further comprise a PEGylated lipid. In other 25 embodiments, the PEGylated lipid is an alkyl-PEGylated lipid. In some embodiments, the PEGylated lipid is a methoxy-PEGylated lipid. In some embodiments, the PEGylated lipid is DMG-PEG. In some embodiments, the PEGylated lipid is a hydroxy-PEGylated lipid. In some embodiments, the PEGlyated lipid is less than 0.5% (w/w). In some embodiments, the PEGylated lipid is less than 0.25% (w/w). 30 In some embodiments, the LNPs further comprise a cationic lipid. In some embodiments, the cationic lipid is MC3 or DLin-MC3-DMA. In some embodiments, the LNPs further comprise a sterol. In some embodiments, the sterol is cholesterol.
In some embodiments, the LNPs encapsulate a therapeutic agent. In some embodiments, the therapeutic agent is a protein or a nucleic acid. In some embodiments, the therapeutic agent is a mRNA coding for a therapeutic protein. In some embodiments, the LNPs are administered to the subject at multiple doses. In some embodiments, wherein the 5 interval of two consecutive doses is less than 2 weeks. In some embodiments, the interval of two consecutive doses is less than I week. In other embodiments the doses are between 2
days - 3 weeks; 3- days -3 weeks, 4 days- 3 weeks, 5 days - 3 weeks, 2 days - 2 weeks; 3 days -2 weeks, 4 days- 2 weeks, 5 days - 2 weeks, 2-15 days; 3-15 days, 3 - 10 days, or 3- 7 day apart. 10 In some aspects, the invention is a method for reducing accelerated blood clearance
N (ABC) of lipid nanoparticles (LNPs) encapsulating an mRNA, the method comprising: administering to a subject in need thereof a first dose of the LNPs, and administering to the
subject a second dose of the LNPs; wherein the first dose, the second dose, or both are equal to or less than about 0.3 mg/kg. 15 In some embodiments, the first dose, the second dose, or both are equal to or less
than about 0.2 mg/kg. In some embodiments, the first dose, the second dose, or both are equal
to or less than about 0.1 mg/kg. In some embodiments, the first dose, the second dose, or
both are about 0.1-0.3 mg/kg. In some embodiments, the interval between the first dose and the second dose is less than 2 weeks. In some embodiments, the interval between the first
20 dose and the second dose is less than I week. In some embodiments, the mRNA encapsulated in LNPs is a therapeutic mRNA. In some embodiments, the mRNA encapsulated in LNPs is a rnRNA encoding a vaccine antigen. In some embodiments, the mRNA encapsulated in LNPs encodes multiple proteins. In some embodiments, the LNPs do not induce production of natural IgM molecules
25 capable of binding to the LNPs. In some embodiments, the LNPs do not activate BIa cells. In some embodiments, the LNPs are free of an epitope that activates Bla cells. In some
embodiments, the LNPs comprise a helper lipid, which comprises at least one fatty acid chain of at least 8C and at least one, polar moiety, and wherein the helper lipid does not activate Bla cells. In some embodiments, the LNPs are free of phosphatidyl choline (PC). 30 In some embodiments, the helper lipid is a phosphatidyl choline analog. In some embodiments, the phosphatidyl choline analog comprises a modified PC head group, a modified PC core group, and/or a modified PC lipid tail. In some embodiments, the helper rNq lipid competitively inhibits phosphatidylcholine from binding to CD36. In some embodiments, the helper lipid does not bind or has low binding activity to CD36. In some embodiments, the LNPs comprise oleic acid or an oleic acid analog. In some embodiments, the LNPs are free of PEG or a PEGylated lipid. In some embodiments, the 5 LNPs further comprise a PEGylated lipid. In some embodiments, the PEGylated lipid is an alkyl-PEGylated lipid. In some embodiments, the PEGylated lipid is a methoxy-PEGylated lipid. In some embodiments, the PEGylated lipid is DMG-PEG. In some embodiments, the PEGylated lipid is a hydroxy-PEGylated lipid. In some embodiments, the PEGlyated lipid is less than 0.5% (w/w). In some embodiments, the PEGylated lipid is less than 0.25% (w/w). i 10 Each possibility represents a separate embodiment of the present invention.
N In some embodiments, the LNPs further comprise a cationic lipid. In some embodiments, the cationic lipid is MC3 or DLin-MC3-DMA. In some embodiments, the LNPs further comprise a sterol. In some embodiments, the sterol is cholesterol. In some aspects, the invention is a method for reducing accelerated blood clearance
15 (ABC) in a subject being treated with a therapeutic regimen involving repeat dosing of lipid nanoparticles (LNPs), the method comprising administering to the subject LNPs and an agent that inhibits Bla cell-mediated immune responses induced by the LNPs, such that ABC is reduced upon repeat administration of the LNPs to the subject. In some aspects, the invention is a method for reducing accelerated blood clearance 20 (ABC) in a subject being treated with a therapeutic regimen involving multiple dosing of lipid nanoparticles (LNPs), the method comprising administering to the subject LNPs and an agent that inhibits BIa cell-mediated immune responses induced by the LNPs, such that ABC is reduced upon administration of one or more subsequent doses of the LNPs to the subject. In some aspects, the invention is a method for reducing accelerated blood clearance
25 (ABC) of lipid nanoparticles (LNPs) in a subject being treated with a multi-dose or repeat dosing therapeutic regimen, the method comprising administering to the subject LNPs and an agent that inhibits BI a cell-mediated immune responses induced by the LNPs, such that ABC is reduced upon subsequent or repeat dosing of LNPs in the subject. In some aspects, the invention is a method for decelerating blood clearance ofLNPs,
30 the method comprising administering to a subject LNPs and an agent that inhibits BI a cell
mediated immune responses induced by the LNPs, such that upon administration of a
subsequent dose of the LNPs to the subject blood clearance of the LNPs is decelerated.
In some aspects, the invention is a method for reducing or inhibiting accelerated
blood clearance (ABC) of lipid nanoparticles (LNPs) in a subject, the method comprising administering to the subject LNPs and an agent that inhibits immune responses induced by the LNPs such that ABC of the LNPs is reduced or inhibited. 5 In some aspects, the invention is a method for reducing or inhibiting accelerated blood clearance (ABC) of lipid nanoparticles (LNPs) in a subject, the method comprising administering to the subject LNPs and an agent to inhibit immune responses induced by the
LNPs such that ABC of the LNPs is reduced or inhibited. In some examples, the amount of the agent used in any of the methods described herein is sufficient to inhibit any of the Ni10 immune responses described herein. N In some embodiments, the agent inhibits production of or neutralizes natural IgM capable of binding to the LNPs. In some embodiments, the immune response induced by the LNPs is activation of BIa cells. In some embodiments, the immune response induced by the LNPs is binding of natural IgM to the LNPs. In some embodiments, the agent binds and/or 15 inhibits CD36 on Bl a cells. I sumeeiibudiiiients, the agent is administered to the subject prior to, after, or
concurrently with the administration ofthe LNPs. In some embodiments, the LNPs
encapsulate a therapeutic agent. In some embodiments, the therapeutic agent is a protein or a
nucleic acid. In some embodiments, the therapeutic agent is a mRNA coding for a therapeutic 20 protein. In some embodiments, the subject is administered with the LNPs at multiple doses.
In some embodiments, the interval between two consecutive doses is less than 2 weeks. In some embodiments, the interval between two consecutive doses is less than I week. In some embodiments, the interval between two consecutive doses is less than 21, 20, 19, 18, 17, 16, 25 15, 14, 13, 12, I1, 10, 9, 8, 7, 6, 5, 4, 3, 2 or I days. In some embodiments, the subject is administered a dose once daily, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. Each possibility represents a separate embodiment of the present invention. In some aspects, the invention is a method for reducing dose-limiting toxicity (DLT) in a subject being treated with therapeutic regimen involving lipid nanoparticle (LNP) 30 mediated drug delivery, the method comprising administering LNPs to the subject, wherein the LNPs do not promote platelet activation, such that DLT is reduced in the subject being
treated with therapeutic regimen.
In some aspects, the invention is a method for reducing toxicity associated with delivery of therapeutic doses of lipid nanoparticle (LNP)-encapsulated drug to a subject, the method comprising administering LNPs to the subject, wherein the LNPs do not promote platelet activation, such that the toxicity is reduced. 5 In some aspects, the invention is a method for delivering lipid nanoparticles (LNPs) to a subject without promoting toxicity associated with LNPs, the method comprising
administering LNPs to the subject, wherein the LNPs do not promote LNP-related toxicity. In some embodiments, the LNP-related toxicity comprises coagulopathy, N disseminated intravascular coagulation (DIC), vascular thrombosis, complement activation N 10 related pseudoallergy (CARPA), or a combination thereof. In some embodiments, the LNPs N do not promote the classical pathway (CP). In some embodiments, the LNPs do not promote the alternative pathway (AP). In some embodiments, the LNPs do not promote platelet activation or aggregation. In some embodiments, the LNPs do not activate CD36. In some embodiments, the LNPs are free of an epitope that activates CD36. 15 In some embodiments, the LNPs comprise a helper lipid, which comprises at least one
fatty acid chain of at least 8C and at least one polar moiety, and wherein the helper lipid does not activate Bla cells. In some embodiments, the LNPs are free of phosphatidyl choline (PC). In some embodiments, the helper lipid is a phosphatidyl choline analog. In some embodiments, the phosphatidyl choline analog comprises a modified PC head group, a 20 modified PC core group, and/or a modified PC lipid tail. In some embodiments, the helper lipid competitively inhibits phosphatidycholine from binding to CD36. In some embodiments, the helper lipid does not bind or has low binding activity to CD36. In some embodiments, the LNPs comprise oleic acid or an oleic acid analog. In some embodiments, the LNPs are free of PFG or a PEGylated lipid 25 In some embodiments, the LNPs further comprise a PEGylated lipid. In some embodiments, the PEGylated lipid is an alkyl-PEGylated lipid. In some embodiments, the PEGylated lipid is a methoxy-PEGylated lipid. In some embodiments, the PEGylated lipid is DMG-PEG. In some embodiments, the PEGylated lipid is a hydroxy-PEGylated lipid. In some embodiments, the PEGlyated lipid is less than 0.5% (w/w). In some embodiments, the
30 PEGylated lipid is less than 0.25% (w/w). Each possibility represents a separate embodiment of the present invention.
In some embodiments, the LNPs further comprise a cationic lipid. In some
embodiments, the cationic lipid is MC3 or DLin-MC3-DMA. In some embodiments, the LNPs further comprise a sterol. In some embodiments, the sterol is cholesterol. In some embodiments, the LNPs encapsulate a therapeutic agent. In some 5 embodiments, the therapeutic agent is a protein or a nucleic acid. In some embodiments, the therapeutic agent is a mRNA coding for a therapeutic protein. In some aspects, the invention is a method for delivering lipid nanoparticles (LNPs) encapsulating an mRNA to a subject without promoting LNP-related toxicity, the method comprising administering an amount of the LNPs to a subject during a period, wherein the N 10 serum level of the LNPs in the subject during the administration period is not sufficient to N induce LNP-related toxicity. In some embodiments, the LNP-related toxicity comprises coagulopathy, disseminated intravascular coagulation (DIC), vascular thrombosis, activation-related pseudoallergy (CARPA), acute phase response (APR), or a combination thereof. In some 15 embodiments, the serum level ofthe LNPs in the subject during the administration period is not sufficient to induce CARPA or APR. In some embodiments, the serum level of the LN's in the subject during the administration period is not sufficient to induce the classical pathway (CP). In some embodiments, the serum level of the LNPs in the subject during the administration period is not sufficient to induce the alternative pathway (AP). In some 20 embodiments, the serum level of the LNPs in the subject during the administration period is
not sufficient to induce platelet activation or aggregation.
In some embodiments, the dose of the LNPs are lower than 0.1 mg/kg, 0.05 mg/kg, 0.02 mg.kg or 0.01 mg/kg. In some embodiments, the administration period is at least 96 hours, 72 hours, 48 hours, 24 hours, or 12 hours. 25 In some embodiments, the mRNA encapsulated in LNPs is a therapeutic mRNA. In
some embodiments, the mRNA encapsulated in LNPs is a mRNA encoding a vaccine antigen. In some embodiments, the mRNA encapsulated in LNPs encodes multiple proteins. In some embodiments, the LNPs comprise a helper lipid, which comprises at least one fatty acid chain of at least 8C and at least one polar moiety, and wherein the helper lipid does not 30 activate Bla cells. In some embodiments, the LNPs are free of phosphatidyl choline (PC). In some embodiments, the helper lipid is a phosphatidyl choline analog. In some embodiments, the phosphatidyl choline analog comprises a modified PC head group, a modified PC core group, and/or a modified PC lipid tail. In some embodiments, the helper lipid competitively inhibits phosphatidycholine from binding to CD36. In some embodiments, the helper lipid does not bind or has low binding activity to CD36. In some embodiments, the LNPs comprise oleic acid or an oleic acid analog. In some embodiments, the LNPs are free of PEG or a PEGylated 5 lipid. In some embodiments, the LNPs further comprise a PEGylated lipid. In some embodiments, the PEGylated lipid is an alkyl-PEGylated lipid, a methoxy-PEGylated lipid, a DMG-PEG, or a hydroxy-PEGylated lipid. In some embodiments, the PEGlyated lipid is less than 0.5% (w/w). In some embodiments, the PEGylated lipid is less than 0.25% (w/w). In 10 some embodiments, the LNPs further comprise a cationic lipid. In some embodiments, N cationic lipid is MC3 or DLin-MC3-DMA. In some embodiments, the LNPs further comprise a sterol. In some embodiments, the sterol is cholesterol. In some aspects, the invention is a method for reducing dose-limiting toxicity (DLT) in a subject being treated with therapeutic regimen involving lipid nanoparticle (LNP) 15 mediated drug delivery, the method comprising administering to the subject LNPs and an agent that inhibits platelet activation, such that DLT is reduced in the subject being treated with therapeutic regimen. In some aspects, the invention is a method of increasing the therapeutic index in a subject being treated with lipid nanoparticle (LNP)-mediated drug delivery, the method 20 comprising administering to the subject LNPs and an agent that inhibits platelet activation, such that dose-limiting toxicity (DLT) is reduced in the subject being treated with LNP. In yet other aspects the invention is a method of a therapeutic regimen involving lipid nanoparticle (LNP)-mediated drug delivery, by administering LNPs to the subject, wherein the LNPs do not induce an immune response associated with B Icell activation or platelet 25 activation, and optionally administering an agent that inhibits immune responses induced by the LNPs, such that DLT is reduced in the subject being treated with the therapeutic regimen. In some aspects, the invention is a method for reducing toxicity associated with delivery of therapeutic doses of lipid nanoparticle (LNP)-encapsulated drug to a subject, the method comprising administering to the subject LNPs and an agent that inhibits platelet 30 activation, such that the toxicity is reduced. In some aspects, the invention is a method for lessening lipid nanoparticle (LNP) related toxicity in a subject, the method comprising administering to the subject LNPs and an agent in an amount effective to inhibit the LNP-related toxicity or alleviate at least one symptom thereof. In some embodiments, the LNP-related toxicity comprises coagulopathy, disseminated intravascular coagulation (DIC), vascular thrombosis, activation-related 5 pseudoallergy (CARPA), acute phase response (APR), or a combination thereof. In some embodiments, the agent is administered to the subject prior to, after, or currently with the administration of the LNPs. In some embodiments, the LNPs encapsulate a therapeutic agent. In some embodiments, the therapeutic agent is a protein or a nucleic acid. In some N embodiments, the therapeutic agent is a mRNA coding for a therapeutic protein. In some N 10 embodiments, the agent alleviates at least one symptom associated with the LNP-related N toxicity. In some embodiments, the agent is a nonsteroidal anti-inflammatory drug (NSAID) or an antihistamine agent, wherein the anti-histamine is a histamine receptor blocker, such as an HI antagonist or an HI inverse agonist. In some embodiments, the NSAID is a COX-2 and/or 5-LOX inhibitor. In some embodiments, the antihistamine is a histamine receptor 15 blocker. In some embodiments, the histamine receptor blocker is an H Iantagonist or an HI1 inverse agonist. In some embodiments, the HI antagonist is diphenhydramine (Benadry), fexofenadine (Allegra) or loratadine (Claritin), and the HI inverse agonist is cetirizine. In some embodiments, the agent inhibits CARPA or ARP. In some embodiments, the agent inhibits the classical pathway (CP). In some embodiments, the agent inhibits the alternative 20 pathway. Each possibility represents a separate embodiment of the present invention. In some embodiments, the agent inhibits platelet activation. In some embodiments, the agent is a platelet aggregation inhibitor. In some embodiments, the platelet aggregation inhibitor is an ADP receptor antagonist. In some embodiments, the platelet aggregation inhibitor is aspirin or clopidrogrel (PLAVIX@). In some embodiments, the platelet 25 aggregation inhibitor is selected from aspirin/pravastatin, cilostazol, prasugrel, aspirin/dipyridamole, ticagrelor, cangrelor, elinogrel, dipyridamole, and ticlopidine. In some embodiments, the agent inhibits CD36. In some embodiments, the agent inhibits aTLR receptor, CD62P, properdin, a component of the complement system, C-reactive protein, or other proteins of the acute phase response. Each possibility represents a separate 30 Ciiabudinicit uf the present inventionI. In some aspects, the invention includes a method for reducing dose-limiting toxicity (DLT) and/or accelerated blood clearance (ABC) in a subject being treated with a therapeutic regimen involving lipid nanoparticle (LNP)-mediated drug delivery, the method comprising administering to the subject LNPs encapsulating the therapeutic agent, wherein the LNPs do not activate an immune cell thrombospondin receptor such as CD36, such that ABC is reduced upon repeat administration of the LNPs to the subject. In some embodiments, the immune cells are platelets and/or B cells, I particular, BI a cells. 5 In some aspects, the invention includes a method for delivering a therapeutically effective amount of a therapeutic agent via lipid nanoparticles to a subject, the method comprising administering to the subject LNPs encapsulating the therapeutic agent, wherein the LNPs do not activate CD36. In some embodiments, the LNPs are free of an epitope that N activates CD36. In some embodiments, the LNPs are free of phosphatidyl choline (PC). In N 10 some embodiments, the LNPs comprise a helper lipid that does not bind or has low binding N activity to CD36, or competitively inhibits phosphatidylcholine from binding to CD36. In some embodiments, the helper lipid comprises at least one fatty acid chain of at least 8C and at least one polar moiety. In some embodiments, the helper lipid is a phosphatidyl choline analog. In some embodiments, the phosphatidyl choline analog comprises a modified PC 15 head group, a modified PC core group, and/or a modified PC lipid tail. In some embodiments, the LNPs comprise oleic acid or an oleic acid analog. In some embodiments, the LNPs are free of PEG or a PEGylated lipid. In some embodiments, the LNPs further comprise a PEGylated lipid. In some embodiments, the PEGylated lipid is an alkyl-PEGylated lipid, a methoxy-PEGylated lipid, 20 DMG-PEG or a hydroxy-PEGylated lipid. In some embodiments, the PEGlyated lipid is less than 0.5% (w/w). In some embodiments, the PEGylated lipid is less than 0.25% (w/w). In some embodiments, the LNPs further comprise a cationic lipid. In some embodiments, the cationic lipid is MC3 or DLin-MC3-DMA. In some embodiments, the LNPs further comprise a sterol. In some embodiments, the sterol is cholesterol. 25 In some embodiments, the LNPs encapsulate a therapeutic agent. In some embodiments, the therapeutic agent is a protein or a nucleic acid. In some embodiments, the therapeutic agent is a mRNA coding for a therapeutic protein. In some embodiments, the LNPs are administered to the subject at multiple doses. In some embodiments, the interval of two consecutive doses is less than 2 weeks. In some
30 embodiments, the interval of two consecutive doses is less than I week. In some aspects, the invention is a method for delivering a therapeutic level of a
protein of interest to a subject, the method comprising administering to the subject lipid
nanoparticles (LNPs), which encapsulate an mRNA coding for the protein of interest, wherein the LNPs do not activate B I a cells and/or do not activate platelets, such that a therapeutic level of the protein of interest is delivered to the subject. In some aspects, the invention is a method for delivering a therapeutic level of a protein of interest to a subject, the method comprising administering to the subject lipid 5 nanoparticles (LNPs), which encapsulate an mRNA coding for the protein of interest, wherein the LNPs do not induce drug responses associated with LNPs. In some embodiments, the drug response associated with LNPs is accelerated blood clearance. In some embodiments, the LNPs do not induce production of natural IgM molecules capable of binding to the LNPs. In some embodiments, the LNPs do not activate Bl a cells. In some Ni10 embodiments, the LNPs is free of an epitope that activates BI a cells. N In some embodiments, the LNPs are administered to the subject at multiple doses. In some embodiments, the interval of two consecutive doses is less than 2 weeks. In some embodiments, the interval of two consecutive doses is less than I week. In some embodiments, the drug response associated with LNPs is an adverse reaction induced by the 15 LNPs. In some embodiments, the adverse reaction comprises coaglopathy, disseminated intravascular coagulation (DIC), vascular thrombosis, activation-related pseudoallergy (CARPA), acute phase response (APR), or a combination thereof. In some embodiments, the LNPs do not promote CARPA or APR. In some embodiments, the LNPs do not promote the classical pathway (CP). In some embodiments, the LNPs do not promote the alternative 20 pathway (AP). In some embodiments, the LNPs do not promote platelet activation or aggregation. In some embodiments, the LNPs do not activate CD36. In some embodiments, the LNPs comprise a helper lipid, which comprises at least one fatty acid chain of at least 8C and at least one polar moiety, and wherein the helper lipid does not induce production of natural IgM capable of binding to the LNPs, do not activate 25 Bl a cells, do not activate CD36, and/or do not activate platelet.
In some embodiments, the LNPs are free of phosphatidyl choline (PC). In some embodiments, the helper lipid is a phosphatidyl choline analog. In some embodiments, the phosphatidyl choline analog comprises a modified PC head group, a modified PC core group, and/or a modified PC lipid tail. In some embodiments, the helper lipid competitively inhibits 30 phosphatidylcholine from binding to CD36. In some embodiments, the helper lipid does not bind or has low binding activity to CD36. In some embodiments, the LNPs comprise oleic acid or an oleic acid analog. In some embodiments, the LNPs are free of PEG or a PEGylated lipid.
In some embodiments, the LNPs further comprise a PEGylated lipid. In some embodiments, the PEGylated lipid is an alkyl-PEGylated lipid. In some embodiments, the PEGylated lipid is a methoxy-PEGylated lipid, DMG-PEG or a hydroxy-PEGylated lipid. In some embodiments, the PEGlyated lipid is less than 0.5% (w/w). In some embodiments, the 5 PEGylated lipid is less than 0.25% (w/w). In some embodiments, the LNPs further comprise a cationic lipid. In some embodiments, the cationic lipid is MC3 or DLin-MC3-DMA. In some embodiments, the LNPs further comprise a sterol. In some embodiments, the sterol is cholesterol.
In some embodiments, the mRNA encodes a therapeutic protein. In some 10 embodiments, the mRNA encodes a vaccine antigen. N In some aspects, the invention is a method for delivering a therapeutic level of a protein of interest to a subject, the method comprising administering to the subject lipid
nanoparticles (LNPs), which encapsulate an mRNA coding for the protein of interest, and an agent in amount effective to inhibit platelet activation and/or B cell activation, in particular, 15 activation of Bl a cells, induced by the LNPs, such that a therapeutic level of the protein of initees is delivered tu tie subject.
In some aspects, the invention is a method for delivering a therapeutic level ofa
protein of interest to a subject, the method comprising administering to the subject lipid nanoparticles (LNPs), which encapsulate an mRNA coding for the protein of interest, and an 20 agent in amount effective to inhibit a drug response induced by the LNPs or alleviate at least one symptom thereof. In some embodiments, the drug response is accelerated blood clearance. In some embodiments, the agent inhibits production of or neutralizes natural IgM
capable of binding to the LNPs. In some embodiments, the agent inhibits binding of natural IgM to a target. In some embodiments, the agent inhibits activation of BIa cells. In some 25 cmbodiiciat, tli dgei bids CD36 un B la cells. In some embodiments, the agent is administered to the subject prior to, or currently
with the administration of the LNPs. In some embodiments, an LNP is administered to the subject at multiple doses. In some embodiments, the interval between two consecutive doses is less than 2 weeks. In some embodiments, the interval between two consecutive doses is
30 less than I week. In some embodiments, the drug response is LNP-related toxicity. In some
embodiments, the LNP-related toxicity comprises coagulopathy, disseminated intravascular
coagulation (DIC), vascular thrombosis, activation-related pseudoallergy (CARPA), acute phase response (APR), or a combination thereof.
In some embodiments, the agent is administered to the subject prior to, after, or currently with the administration of the LNPs. In some embodiments, the agent alleviates at least one symptom associated the LNP-related toxicity. In some embodiments, the agent is a nonsteroidal anti-inflammatory drug (NSAID) or an antihistamine agent. In some 5 embodiments, the NSAID is a COX-2 and/or 5-LOX inhibitor. In some embodiments, the antihistamine is a histamine receptor blocker. In some embodiments, the histamine receptor Il inverse agonist. In some embodiments, the H I blocker is an HI antagonist or an H antagonist is diphenhydramine (Benadryl), fexofenadine (Allegra) or loratadine (Claritin), N and the HI inverse agonist is cetirizine. In some embodiments, the agent inhibits CARPA or N 10 ARP. In some embodiments, the agent inhibits the classical pathway (CP). In some N embodiments, the agent inhibits the alternative pathway. In some embodiments, the agent inhibits platelet activation. In some embodiments, the agent is a platelet aggregation inhibitor. In some embodiments, the platelet aggregation inhibitor is an ADP receptor antagonist. In some embodiments, the platelet aggregation 15 inhibitor is aspirin or clopidrogrel (PLAVIX@). In some embodiments, the platelet aggregation inhibitor is selected from aspirin/pravastatin, cilostazol, prasugrel, aspirin/dipyridamole, ticagrelor, cangrelor, elinogrel, dipyridamole, and ticlopidine. In some embodiments, the agent inhibits CD36. In some embodiments, the agent inhibits a TLR receptor, CD62P, properdin, a component of the complement system, C-reactive protein, or 20 other proteins of the acute phase response. Each possibility represents a separate embodiment of the present invention. In some aspects, the invention is an accelerated blood clearance (ABC) insensitive lipid nanoparticle (LNP), comprising an ionizable cationic lipids, a PEG-lipid, a sterol, and a helper lipid, wherein the helper lipid does not comprise a phosphatidyl choline (PC). In some 25 instances, the insensitive LNP consisting essentially of the components described herein. For example, such an LNP contains the components described herein, and optionally other components that do not materially affect the basic and novel characteristics of the LNPs described herein. For example, the additional components, if any, may not substantially affect the drug delivery function of the LNP (e.g., at a very low amount such that their 30 functionality on drug delivery is insignificant). In some embodiments, the helper lipid comprises a phosphatidyl choline (PC) analog. In some embodiments, the LNP is not subject to accelerated blood clearance (ABC) when administered at least twice to a subject in a time period of 10 days or less. In some embodiments, the PC analog comprises a modified PC head group, a modified PC core group, and/or a modified PC lipid tail. In some embodiments, the LNP has no or reduced BI a stimulating activity compared to an LNP comprising phosphatidyl choline. In some embodiments, thd LNP has no or reduced binding to CD36 relative to an LNP comprising 5 phosphatidyl choline. In some embodiments, the PC analog has no or reduced binding to CD36 relative to phosphatidyl choline (PC). In some embodiments, the PC analog has no or reduced CD36 binding relative to phosphatidyl choline (PC). In some embodiments, the helper lipid comprises oleic acid or an oleic acid analog. N - In some embodiments, the LNP comprises less than 0.5% (w/w) of a PEGylated N 10. lipid. In some embodiments, the LNP comprises less than 0.25% of the PEGylated lipid. In N some embodiments, the PEGylated lipid is an alkyl-PEGylated lipid, a methoxy-PEGylated lipid, DMG-PEG, a lipid conjugated to hydroxy-PEG (hydroxy-PEGylated lipid). In some embodiments, the LNP has reduced platelet aggregation activity compared to an LNP comprising a methoxy-PEGylated lipid. In some embodiments, the cationic lipid is 15 MC3 (or DLin-MC3-DMA). In some aspects, the invention is a lipid nanoparticle (LNP) comprising or consisting essentially of a cationic lipid, a non-cationic, non-PC lipid, less than 0.5% (w/w) of a PEGylated lipid, and a sterol, and wherein the LNP is insensitive to accelerated blood clearance upon repeated administration in vivo within 10 days. In some embodiments, the 20 LNP further comprises a protein or a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is imRNA. In some aspects, the invention is a method for reducing ABC effect in a subject comprising contacting a blood sample from a subject with a lipid nanoparticle (LNP) 25 formulation, measuring reactivity of the blood sample to the LNP formulation, and administering an LNP formulation comprising a therapeutic agent to the subject, provided the subject manifests no or low reactivity to the LNP formulation. In some embodiments, the therapeutic agent is administered at intervals of 2 weeks, I week, or less. In some aspects, the invention is a method of delivering an agent to a subject, 30 comprising administering to the subject an agent formulated in a lipid nanoparticle (LNP), wherein the subject is administered a platelet inhibitor. In sorne embodiments, the platelet inhibitor is administered to the subject at the same time as the agent formulated in a LNP. In some embodiments, the platelet inhibitor is administered to the subject 1 minute to 24 hours prior to the agent formulated in a LNP. In some embodiments, the platelet inhibitor is administered to the subject 24-48 hours prior to the agent formulated in a LNP. In some embodiments, the invention further comprises administering to the subject a histamine receptor blocker. In some embodiments, the invention comprises administering to the subject 5 a non-specific inhibitor of COX enzyme. In some embodiments, the agent is a nucleic acid. In some embodiments, the nucleic acid is a RNA. In some embodiments, RNA is siRNA or mRNA. In some embodiments, the LNP comprises a cationic lipid, a PEG. In some embodiments, the subject is not administered N a corticosteroid. N 10 In some embodiments, the platelet inhibitor is an inhibitor of P2Y12 subtype receptor. N In some embodiments, the platelet inhibitor is clopidogrel. In some embodiments, the platelet inhibitor is ticagrelor. In some embodiments, is prasugrel, ticlopidine, cangrelor, or elinogrel. In some embodiments, the histamine receptor blocker is an antihistamine. In some embodiments, the antihistamine is Benadryl. In some embodiments, the non-specific inhibitor 15 of COX enzyme is aspirin. In some embodiments, the non-specific inhibitor of COX enzyme is a COX-2 inhibitor. In some embodiments, the non-specific inhibitor of COX enzyme is a COX-2 and 5-lipoxygenase (5-LOX) inhibitor.
In other aspects the invention is method for reducing dose-limiting toxicity (DLT) in a subject being treated with a therapeutic regimen involving lipid nanoparticle (LNP) 20 mediated drug delivery, by administering to the subject an LNP comprising a therapeutic nucleic acid and administering to the subject an agent that agent removes or targets B cells, such that DLT is reduced in the subject being treated with the therapeutic regimen. In some embodiments the agent removes or targets Bla cells. In other embodiments the agent is Rituximab. In yet other embodiments the agent is administered to the subject prior to, after, 25 or currently with the administration of the LNPs. The LNP may be administered to the subject at multiple doses. In other embodiments the therapeutic nucleic acid is an mRNA.
In other aspects the invention is a PEG lipid comprising a compound of Formula (V): 0 R3 O R5
30 (V), or a salts thereof, wherein: R 3 is-ORO;
Ri is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive; R 5 is optionally substituted CIo40 alkyl, optionally substituted Co40 alkenyl, or 5 optionally substituted CIO. 4 0 alkynyl; and optionally one or more methylene groups of R are 5 replaced with optionally substituted carbocyclylene, optionally substituted heterocyclyene, optionally substituted arylene, optionally substituted heteroarylene, -N(R )-,-0-,-S-,
C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, OC(O)N(RN)-, -NRNC()O-, -C(O)S-, -SC(O)-, -C(=NR N)-, -C(=NRN)N(RN
N NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S), N 10 NRNC(S)N(RN)-, -S(O)-, -OS(O))-, -S(0)0-, -OS(O)O-, N -OS(O)2-, -S(O)20-, N
N OS(O) 2 0-,-N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(R)N_, N(RN)S()O_ -S() 2-, -N(RN)S() 2 -, -S() 2N(RN)-, -N(RN)S(0) 2 N(RN, _
OS(O) 2N(RN)-, or -N(RN)S(0) 2 0-; and each instance of RN is independently hydrogen, optionally substituted alkyl, or a 15 nitrogen protecting group. In some embodiments the compound of Formula (V) is of Formula (V-OH): 0 HO O) R5
(V-OH), or a salt thereof 20 In other embodiments the compound of Formula (V) is of one of the following formulae: 0
00 (Cmpd400), or
0 25 (Cmpd40l),
(Cm pd40 1),
Cd OO
(Cmpd402), 0
(Cmpd450), H
C5 (Cmpd45 1), 0 HO
(Cmpd452), H HO N 0 10 (Cmpd453),
(Cmpd454), or a salt thereof. 15 In yet other embodiments the compound of Formula (V) is:
(Cmpd403), or a salt thereof.
20 In some aspects the invention is a lipid nanoparticle (LNP) comprising a PEG lipid as described herein and optionally further comprising a lipid of Formula (V):
R4 RN RR
Rp)V), or a salt or isomer thereof, wherein: R, is selected from the group consistingofC 5- 30 alkyl, C 5-2 0 alkenyl, -R*YR", -YR", and -R"M'R'; R 2 and R3 are independently selected from the group consisting of H, C1 4 alkyl, C 214 5 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
R 4 is selected from the group consisting of a C 3 -6 carbocycle, -(CH 2)nQ, -(CH 2)nCHQR, N -CHQR, -CQ(R) 2, and unsubstituted CI. 6 alkyl, where Q is selected from a carbcycle, N 10 heterocycle, -OR, -O(CH 2)nN(R) 2, -C(O)OR, -OC(O)R, -CX 3, -CX 2H, -CXH 2, -CN, -N(R) 2
, N -C(O)N(R) 2, -N(R)C(O)R, -N(R)S(O) 2R, -N(R)C(O)N(R) 2, -N(R)C(S)N(R) 2, -N(R)R8
, -O(CH 2 )OR, -N(R)C(=NR 9)N(R) 2, -N(R)C(=CHR 9)N(R)2, -OC(O)N(R) 2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2, -N(OR)C(S)N(R) 2
, -N(OR)C(=NR9)N(R) 2, -N(OR)C(=CHR9)N(R) 2, -C(=NR9)N(R) 2, -C(=NR9)R, 15 -C(O)N(R)OR, and -C(R)N(R) 2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; each R 5 is independently selected from the group consistingofC alkyl, C 2 -3 alkenyl, and H; each R 6 is independently selected from the group consistingof C3 alkyl, C 2. 3 alkenyl, 20 and H; M and M'are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -,
-S-S-, an aryl group, and a heteroaryl group; R 7 is selected from the group consisting ofC. alkyl, C 2 -3 alkenyl, and H;
25 R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle; R 9 is selected from the group consisting of H, CN, NO 2 , C alkyl, -OR, -S(O)2R,
-S(0) 2N(R) 2, C 2 -6 alkenyl, C 3 .6 carbocycle and heterocycle; each R is independently selected from the group consisting ofCI.3 alkyl, C 2. 3 alkenyl, and H;
30 each R' is independently selected from the group consisting of C-1 8 alkyl, C 2. 1 8
alkenyl, -R*YR", -YR", and H; each R" is independently selected from the group consisting of C 3 14 alkyl and
C3 14 alkenyl;
1 2 alkyl and each R* is independently selected from the group consisting ofCM
C 2 12 alkenyl; each Y is independently a C 3.6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. These and other embodiments and aspects will be discussed in greater detail herein.
BRIEF DESCRIPTION OF DRAWINGS The following Figures are provided in and grey scale. FIG. 1: Phycoerythrin (PE) fluorescence of CD3+ T cells and CD1 9+ B cells following incubation with PE- LNP, PE+ LNP or medium alone. The data show uptake of LNPs by splenic B cells but not T cells under ex vivo culture conditions. FIGs. 2A-2B: Phycoerythrin (PE) fluorescence of CD3+ T cells and CD19+ B cells as a function of time of incubation with PE- LNP, PE+ LNP or medium alone. The data show LNP uptake by B cells but not T cells occurs rapidly in these ex vivo culture conditions. FIG. 3: EGFP fluorescence of CD]9+ cells as a function of time of incubation with PE+ LNP comprising EGFP mRNA. No expression of EGFP by B cells is observed at any time point, despite massive LNP uptake by those cells. FIGs. 4A-4B: hEPO concentration and anti-PEG IgM levels in vivo following administration of first, second and third doses (hEPO) and first and second (IgM) of LNP carrying hEPO mRNA cargo into CD-i non-splenectomized (CD-) and splenectomized mice.
FIG. 5: PE-Rhodamine fluorescence in CD19+ circulating B cells after incubation for 2. 4, 6 and 24 hours with PE+ and PF- TNP FIGs. 6A-6B: LNP uptake (FIG. 6A) and EGFP expression (FIG. 6B) by circulating B cells as a function of incubation time. FIG. 7: CD86 expression in splenic and circulating B cells following incubation with PE- and PE+ LNP. Spleniic and circulating B cells that take up LNPs are activated. FIG. 8: CD86 expression in splenic and circulating B cells that take up or fail to take up PE+ LNPs. Splenic and circulating B cells that take up LNPs are activated, as evidenced by increased CD86 expression. FIG. 9: CD86 expression as a function of LNP uptake by splenic B cells at various incubation times.
FIG. 10: IL-6 and TNF-alpha secretion by B cells incubated with PE+ and PE- LNP for 24 hours.
FIGs. I1A-11B: Empty PE+ LNP uptake by CD19+ B cells at 24 hours (FIG. I A) 5 and the time course at 4 and 24 hours (FIG. I B). FIGs. 12A-12B: CD86 expression in B cells that have taken up empty PE+ LNP (FIG. 12A). Splenic B cells are activated by empty LNPs in a dose dependent manner. IL-6 and TNF-alpha secretion by B cells incubated with empty PE+ LNP (FIG. 12B). N FIGs.13A-13D: Uptake and cytokine secretion by B cells from wild type (WT), N 10 ApoE deficient, and LDL receptor deficient mice in PE+ LNPs (FIG. 13A) and PE- LNPs N (FIG. 13B). The percentage of CD19+ PE+ cells (FIG. 13C) and cytokine levels in pg/ml (FIG. 13D) are also given. FIG. 14: LNP uptake following pre-incubation of B cells with free PEG or anti-PEG IgG. 15 FIG. 15: Uptake of LNPs comprising PEG or PEG-OH or lacking PEG (PEGess) by B cells. FIG. 16: Uptake of LNPs comprising PEG or PEG-OH or lacking PEG (PEGess) by B cells. FIG. 17: CD86 expression in B cells incubated with PEG-less LNP or PEG-OH 20 LNP, as a function of LNP uptake. FIG. 18: LNP uptake as a function of phospholipid content ofthe LNP. FIG. 19: LNP uptake as a function of phospholipid and PEG content of the LNP in CDI9+ B cells and CD5+ B cells. FIG.20: LNP uptake by CD19+, CD19+CD5+ orCD9+CD5- B cells as a function 25 of phospholipid and PEG contciit.
FIG. 21: EUFP expression in non-conventional T (CD3-) and B (CDI 9-) cells from the spleen I and 4 hours after administration of LNPs comprising DMG-PEG or PEG-OI and PEGless LNPs. 30 FIGs. 22A-223: LNP uptake by B cells in vivo as a function of phospholipid and PEG content. FIGs. 23A-23E: CD86 expression levels in B cells after injection of LNPs comprising DMG-PEG (FIG. 23A) or Cmpd418 (FIG. 23B), or PEGless LNP (FIG. 23C).
CD86 expression level was assessed at I hour (FIG. 23D) and 4 hours (FIG. 12E) after injection of LNP. FIGs. 24A-24B: Uptake of PE+ LNP by B cells in vivo 1 hour (FIG. 24A) and 4 hours (FIG. 24B) after injection of LNP comprising DMG-PEG or PEG OH or PEGless 5 LNPs. FIG. 25: LNP uptake in B cells as a function of DMG-PEG content in LNP (intermediate content study). FIG. 26: B cell activation after LNP uptake as a function of DMG-PEG content in LNP. The data are presented in sets of 4 bars, the four bars in each set representing LNP PE i10 negative, LNP-PE positive low, LNP-PE positive intermediate, and LNP-PE positive high, N from left to right, respectively. Thus, the bars represent populations of cells that have associated or taken up an increasing amount of LNPs. FIG. 27: LNP uptake in B cells as a function of DMG-PEG content in LNP (low content study). 15 FIGs. 28: PE+ staining of CD19+ B cells contacted with Cmpd395, Cmpd404, and oleic acid comprising LNPs. FIGs. 29A-29B: B cell activation measured through an increase in activated B cell population (CD19+CD86+CD69+). FIG. 29A shows B cell activation as a function of DMG PEG mol % without Imiquimod and FIG. 29B shows B cell activation as a function of DMG 20 PEG mol% with Ioiquimud. FIGs. 30A-30B: Pro-inflammatory cytokine release (IFN-y in FIG. 30A, TNF-a in FIG. 301) in an ex vivo human B cell culture as a function of DMG-PEG mol % in the presence or absence of Imiquimod. FIGs. 31A-31D: Phospholipid designs of helper lipids that are analogs and substitutes 25 of phosphatidyl choline (PC) and DSPC. The modifications reduce association, recognition for example by receptors, and/or uptake of LNP through for example modifying the PC head group (FIG. 3IA), the PC core (FIG. 31B), or through reducing the planarity of the lipid (FIG. 31C). An oleic acid variant is also provided (FIG. 31D). FIGs. 32A-32C: Examples of PEGylated lipids comprising short lipid tails, a click 30 linker, and a hydroxy (OH) PEG end group. FIG. 32A shows Cmpd394, Cmpd395, Cmpd396, and Cmpd397. FIG. 32B shows Cmpd398 and Cmpd399. FIG. 32C shows Cmpd400-403.
FIG. 33: Anti-PEG or anti-DSPC IgM response measured by flow cytometry (beads) 96 hours after administration of the second dose. The IgM responses against DSPC or PEG are measured identically when beads are used alone or together. PEG DMG, PEG DSPE or Cmpd430 LNPs induced anti-LNP responses. Anti-PEG IgM and anti DSPC IgMs 5 were both observed suggesting that the IgM response is a natural IgM response. FIG. 34: Anti-PEG IgM measured by ELISA and anti-PEG or anti-DSPC IgM measured by flow cytometry (beads) 96 hours after administration of the second dose. The anti-PEG IgMs measured by ELISA and beads are similar and no significant differences were detected. N 10 FIG. 35: LNP association with platelets as a function of time and LNP composition. N The percent of LNP associated with platelets (as indicated by phycoerythrin (PE) fluorescence) is shown at three time points (15, 60 and 240 minutes) after administration of PBS, DMG-PEG containing LNP, PEG-O- containing LNP, and PEGless LNP (left to right for each time point). These experiments were performed on purified platelets harvested from 15 the subject at the various time points shown. FIG. 36: Platelet activation as a function of LNP composition. Platelet activation (as indicated by expression of platelet activation marker CD62P (MFI)) was measured at three time points (15, 60 and 240 minutes) after administration of PBS, DMG-PEG LNP, PEG-OHl LNP, and PEGless LNP (left to right). 20 FIGs. 37A-37B: Presence of B220+ B cells and F4/80+ macrophages in platelet aggregates in vivo as a function of time and LNP composition. Percent of B cells (as indicated by B220+ staining, FIG. 37A) and percent of macrophages (as indicated by F4/80+ staining, FIG. 37B) at three time points (15, 60 and 240 minutes) after administration of PBS, DMG-PEG LNP, PEG-OH LNP, and PEGless LNP (left to right), is shown. 25 FIG. 38: In vitro activation of platelets as assessed by upregulation of CD31 and CD62P activation markers, after contact with LNPs. FIGs. 39A-39D: In vitro activation of platelets, as indicated by increased CD31 expression relative to control (medium), at 10 minutes (FIG. 39A), 30 minutes (FIG. 3913), 60 minutes (FIG. 39C), and 120 minutes (FIG. 39D) after administration of medium, LPS and 30 LNP comprising DSPC. FIGs. 40A-40B: In vitro platelet aggregation using a whole blood assay. Aggregated cells were collected and gated based on CD41 expression (first column), high forward scatter and side scatter (second column) and F4/80 (y-axis) and CDl Ib (x-axis) expression (third column) after contact with medium (first row), LPS (second row) and DSPC LNP (third row).
Platelet aggregates were isolated based on CD4I+ expression and high FCS and high SSC, as shown in the second column for FIG. 40A. Percent of aggregated cells that are CDIlb+ F4/80+ double positive after administration of medium, LPS, and DSPC LNP (from left to 5 right) is shown (FIG. 40B). FIGs. 41A-41B: In vitro platelet aggregation with B cells (CD19+) and macrophages (CDIl b+ and F4/80+) after incubation of whole blood with medium, LPS and DSPC LNP (from left to right) for 30 minutes (FIG. 41 A) and 120 minutes (FIG. 41B). FIGs. 42A-42C: In vitro platelet aggregation with macrophages (FIG. 42A and 42B) N 10 and B cells (FIG. 42C) after incubation of whole blood with medium, LPS, and DSPC LNP N (from left to right) for 30 minutes and 120 minutes. FIGs. 43A-43B: Upregulation of platelet activation markers CD31 (FIG. 43A) and CD62P (FIG. 43B) after incubation of platelets with medium (control), DSPC LNP, DOPC LNP, DOPG LNP, DMG-PEG LNP, and LPS (from left to right) in vitro, for 0, 10, 30 and 60 15 minutes, as measured by flow cytometry, FIGs. 44A-44B: Upregulation of activation markers CD31 (FIG. 44A) and CD62P (FIG. 44B) after incubation of platelets in vitro with medium (control), DSPC LNP, DOPC LNP, DOPG LNP, DMG-PEG LNP and LPS, for 0, 10, 30 and 60 minutes, as measured by flow cytometry. 20 FIG. 45: Model of the effect of LNPs on platelets. DMG-PEG LNP physically associate with and activate platelets. DSPC LNP do not physically associate with platelets, but nevertheless are able to activate platelets. Both LNP types cause platelets to aggregate
with concomitant recruitment of B cells and macrophages. PEG-OH and PEGless LNPs do not detectably associate with platelets, suggesting a role of the DMG-PEG in the LNP 25 platelet association. Even in the absence of DMG-PEG, however there is platelet activation, suggesting an interaction between platelets and the phospholipid component, and in particular
the PC head group, in the LNP. FIG. 46A-46B: Bla and Bib cells require the spleen. FIG. 46A shows Bla (top) and B2b (bottom) cell levels following splenectomy or a sham operation. FIG. 46B shows Bl a 30 (to) and B2b (bottom) antibody levels following splenectomies. BI b cells lose the ability to secrete antibody. FIG. 47: Reticulocyte counts in intact and splenectomized non-human primates
(NHPs).
FIG. 48: Hematocrit is maintained in splenectomized NHPs.
FIGs. 49A-49D: NHP splenectomy study results. FIGs. 49A-49B show the area under the curve (AUC) results from hEPO-mRNA-MC3 (FIG. 49A) and hEPO (FIG. 4913). Cma, values for hEPO-mRNA-MC3 (FIG. 49C) and hEPO (FIG. 49D) are also presented.
5 FIGs. 50A-50B: Suppression of complement activation in splenectomized NHPs as demonstrated by levels of complement activation indicators, C3a (FIG. 50A) and C5b9 (FIG. 50B). FIGs. 51A-51B: Cytokine expression in splenectomized NHPs. Levels of IL-6 (FIG. 5 1 A) and IL-10 (FIG. 51B) are given. 10 FIGs. 52A-52B: Anti-PEG IgM (FIG. 52A) and anti-PEG IgG (FIG. 5213) levels are N greatly reduced in the absence of spleen.
FIGs. 53A-53B is a set of graphs depicting anti-PEG IgM production following administration of LNP formulations. 15 FIG. 54 is a schematic depicting the replacement of a phospholipid with a different zwitterionic group. FIGs. 55A -55B are a set of graphs depicting Luc mRNA expression (FIG. 55A) and B cell activation (FIG. 55B) following administration of LNP formulations in CD-i mice. CD-I mice were administered 0.05 mg/kg Luc mRNA and Luc expression was measured six
20 hours later.
FIGs. 56A-56E depicts effects of LNP formulation on Luc expression and B cell activation. FIG. 56B is a graph depicting CD86 expression (B cell activation) and FIG. 56C is a graph depicting expression levels of Luc as measured by total flux. The structures are shown in FIGs. 56A, 56D, and 56E. 25 FIGs. 57A-57C depicts the effects of various LNP formulations on B cells and platelets. FIG. 57A is a graph depicting activated B-cell frequencies 24 hours post dose. FIG. 57B is a graph depicting aggregation of platelets 15 minutes post dose. FIG. 57C is a graph depicting recruitment of cells in platelet aggregate. FIGs. 58A-58B depicts the positive effects of oleic acid in an LNP. FIG. 58A depicts 30 levels of luciferase expression as measured by total flux 6 hours after delivery to CD-I mice. FIG. 58B depicts in vivo B-cell activation in mouse splenocytes 24 hours following the administration.
FIG. 59 is a graph depicting hEPO concentration over time. An improved margin of expression with a particle of the invention in contrast to MC3 was demonstrated with chemically modified mRNA. FIGs. 60A-60C is a set of graphs depicting improved immune activation profile with 5 chemically modified mRNA in LNP formulations of the invention. FIG. 60A is a graph depicting in vivo B-cell activation 24 hours following administration of the hEPO loaded particles or PBS. FIG. 60B is a graph depicting in IL-6 concentration 6 hours following administration of the hEPO loaded particles or PBS. FIG. 60C is a graph depicting IP-10 N concentration 6 hours following administration of the hEPO loaded particles or PBS. N 10 FIG. 61 is a graph depicting LNP Uptake by B cells as measured by percent PE+ N CD 19+ B cells. FIG. 62 is a graph depicting B cell activation by LNPs as measured by CD86 expression on D cells. FIGs. 63A-63B are a set of graphs depicting the amount of PEG IgM produced 96 15 hours after a second dose of LNP (FIG. 63A) or 96 hours after a third dose of LNP (FIG. 63B). FIG. 64 is a graph depicting hEPO Expression 6 hours following once weekly administration by IV of hEPO mRNA - LNP formulations at weeks 1, 2, 3, and 4. FIG. 65 is a graph depicting anti-PEG IgM production 96 hours following a third 20 dose of hEPO mRNA - LNP formulations. FIG. 66 is a graph depicting data from a single dose study of LNP having various phospholipids with different headgroups injected by IV in CD-I Mice. Each phospholipid is measured at three time points (3, 6, and 24 hrs) following injection. Phospholipid structures are shown in FIG. 69. 25 FIGs. 67A-67C is a set of graphs depicting cytokine release as a measure of ex vivo human B cell activation. Levels of IFN-gamma (FIG. 67A), LI-6 (FIG. 67) and TNF-alpha (FIG. 67C) were measured. FIG. 68 is a graph depicting the amount of B cell activation as a result of various LNP formulations. LNP formulations including oleic acid demonstrated reduced splenic B 30 cell activation. FIG. 69 shows phospholipid structures.
FIG. 70 is a graph depicting Luc expression levels following administration of LUC mRNA encapsulated in various LNP formulations composed of novel PC and Oleic Acid Derivatives at 3, 6 and 24 hours following. FIGs. 71A-71B is a set of graphs depicting B cell interaction/association with various 5 LNP formulations as assessed by a percentage of CD19+PE+ cells. Several LNP formulations are depicted in FIG. 71 A. Oleic acid and Cmpd125 are depicted in FIG. 71B. FIG. 72 is a set of graphs depicting B cell activation with various LNP formulations as assessed by CD86 Expression Median Fluorescence Intensity. Several LNP formulations N are depicted in FIG. 72A. Oleic acid and Cmpd125 are depicted in FIG. 72B. N 10 FIGs. 73A-73C are a set of graphs depicting Luc expression with various LNP N formulations as assessed by a measurement of total flux at 3 hours (FIG. 73A), 6 hours (FIG. 73B), and 24 hours (FIG. 73C) following a dose, FIGs. 74A-74B are graphs depicting hEPO Expression (pg/mL) 6 hours following once weekly IV administration of hEPO mRNA-LNP formulations at weeks 1, 2, and 3. 15 FIG. 75is a graph depicting anti-PEG IgM production (ng/mL) 96 hours following administration of the second dose of LNP formulations. FIG. 76 is a graph depicting Luc expression of various LNP formulations. Luc expression was assessed by a measurement of total flux (p/s) at 3 hours following a dose, using whole body BLI imaging. 20 FIG. 77 is a graph depicting B cell activation. Percentage of activated B cells (CD86+ CD69+) in splenic CD19+ cells for LNP formulations was measured. FIG. 78 is a graph depicting EPO expression. Concentration of EPO (ng/mL) was measured at prebleed, 6 hours, and 24 hours, each of the 6 weeks. FIG. 79 is a graph depicting hEPO expression (ng/mL) at predose, 2 hours, 6 hours, 25 12 hours, 24 hours, 48 hours, and 72 hours following once weekly IV administration of hEPO mRNA-LNP formulations. FIG. 80 is a graph depicting levels of anti-PEG IgM (U/mL) following once weekly IV administration of the hEPO mRNA-LNP formulations. FIG. 81 is a graph depicting levels of anti-PEG IgG (U/mL) following once weekly 30 IV administration of the hEPO mRNA - LNP formulations. FIG. 82 is a graph depicting B cell activation. Percentage of activated B cells (CD86+ CD27+) in CD19+ cells for LNP formulations was measured. FIG.83 is a graph depicting monocyte activation measured for LNP formulations.
FIGs. 84A-84B are graphs depicting EPO expression. Concentration of EPO (ng/mL) was measured at 2 hours, 6 hours, 12 hours, 24 hours, and 48 hours following the first injection and the fifth injection. FIGs. 85A-85E are graphs depicting EPO expression. Concentration of EPO 5 (ng/mL) was measured on day I and day 29 at 2 hours, 6 hours, 12 hours, 24 hours, and 48 hours for each co-medication group. FIGs. 86A-86C are graphs depicting immune cell populations. Shows the percentage of B cells (FIG. 86A), Bla cells (FIG. 86B), and monocytes (FIG. 86C) in PBMCs for each co-medication group at day -7, 24 hours post first injection, and 24 hours post fifth injection. N 10 FIG. 87 is a graph depicting B cell activation. The percentage of activated B cells in N circulating B cells was measured for each co-medication group at day -7, 24 hours post first injection, and 24 hours post fifth injection. FIG. 88 is a graph depicting monocyte activation. The percentage of activated monocytes/macrophages in circulating PBMCs was measured for each co-medication group 15 at day -7, 24 hours post first injection, and 24 hours post fifth injection. FIGs. 89A-89B are graphs depicting anti-PEG response. Anti-PEG IgM levels (U/mL) (FIG. 89A) and anti-PEG IgG levels (U/mL) (FIG. 89B) were measured for each co medleaton group at baseline, day 9 post first injection, and day 34 post fifth injection. FIGs. 90A-90B are two graphs showing anti-PEG IgM levels (U/mL) and EPO levels 20 (ng/mL) in two of the groups from FIG. 78: C57B16J (FIG. 90A) and sIgM-/- (FIG. 90B). In FIG. 90A, anti-PEG IgM levels are graphed as light grey circles; in FIG. 90B, they are represented by black circles. EPO concentrations are indicated by black circles (FIG. 90A) and black squares (FIG. 90B). FIGs. 91A-91B: The effect of co-medication on ABC in NHP. Co-medication 25 alleviates cytokine (IL-6) production in NHPs (FIG. 91 A) and co-medication with Gilenya reduces anti-PEG IgM production, consistent with reduced ABC (FIG. 91B). FIG. 92: B cells proliferate in the presence of DSPC LNPs. Splenic cells were stained with CFSE and after washing, incubated with IL-6, anti-BCR, DOPC liposomes, DSPC LNPs containing mRNA, DSPC LNPs containing siRNA or empty LNPs for 4 days at 37C. On 30 day 4, the cells were harvested, washed and stained for surface markers, (CD19 and CD3) before they were measured on a BDFortessa flow cytometer and analyzed with ModFit 4.1 software.
FIG. 93: DSPC LNPs induce calcium release in B cells. Splenic cells were stained with Calcium Sensor Dye eFluor@ 514, CD19, and CD3. After washing, a calcium baseline was acquired for 30 seconds on a BDFortessa flow cytometer. Immediately after, the cells were incubated with DSPC LNPs containing mRNA, DOPC liposomes, oleic LNPs, or anti BCR and the calcium signal was acquired for 360 seconds. Then, the cell stimulation cocktail was added to the cells and the signal was acquired for an additional 30 seconds. The analysis was performed using ModFit 4.1 software.
DETAILED DESCRIPTION This disclosure provides lipid-comprising compounds and compositions that are not
subject to ABC and/or that have reduced toxicity, as well as methods for delivering LNPs to a subject without promoting LNP-related drug responses, including ABC and LNP-induced toxicity (e.g., coagulopathy, disseminated intravascular coagulation, vascular thrombosis, CARPA, APR, or a combination thereof). Lipid-comprising compounds and compositions are compounds and compositions that
comprise or are conjugated to one or more lipids. These agents may be referred to herein as lipid-conjugated agents or lipidated agents. Alternatively such lipids may encapsulate agents such as prophylactic, therapeutic and diagnostic agents. These agents may be referred to herein as lipid-encapsulated agents or lipid nanoparticle (LNP) encapsulated agents. Thus, it is to be understood that this disclosure provides improved compounds and compositions for reducing or eliminating ABC and toxicity upon in vivo administration. For brevity, this disclosure may however in some instances refer to compositions or formulations such as lipid nanoparticles or LNPs. This is intended for exemplary purposes and it is to be understood that the various teachings provided herein apply equally to individual compounds, such as lipid-conjugated compounds, unless explicitly stated otherwise.
Accelerated Blood Clearance The invention provides compounds, compositions and methods of use thereof for reducing the effect of ABC on a repeatedly administered active agent such as a biologically active agent. As will be readily apparent, reducing or eliminating altogether the effect of ABC on an administered active agent effectively increases its half-life and thus its efficacy. In some embodiments the term reducing ABC refers to any reduction in ABC in comparison to a positive reference control ABC inducing LNP such as an MC3 LNP. ABC inducing LNPs cause a reduction in circulating levels of an active agent upon a second or subsequent administration within a given time frame. Thus a reduction in ABC refers to less clearance of circulating agent upon a second or subsequent dose of agent, relative to a standard LNP. The reduction may be, for instance, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%. In some 5 embodiments the reduction is 10-100%, 10-50%, 20-100%, 20-50%, 30-100%, 30-50%, 40%-100%, 40-80%, 50-90%, or 50-100%. Alternatively the reduction in ABC may be characterized as at least a detectable level of circulating agent following a second or subsequent administration or at least a 2 fold, 3 fold, 4 fold, 5 fold increase in circulating N agent relative to circulating agent following administration of a standard LNP. In some i 10 embodiments the reduction is a 2-100 fold, 2-50 fold, 3-100 fold, 3-50 fold, 3-20 fold, 4-100 N fold, 4-50 fold, 4-40 fold, 4-30 fold, 4-25 fold, 4-20 fold, 4-15 fold, 4-10 fold, 4-5 fold, 5 100 fold, 5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold, 5-20 fold, 5-15 fold, 5-10 fold, 6-100 fold, 6-50 fold, 6-40 fold, 6-30 fold, 6-25 fold, 6-20 fold, 6-15 fold, 6-10 fold, 8-100 fold, 8 50 fold, 8-40 fold, 8-30 fold, 8-25 fold, 8-20 fold, 8-15 fold, 8-10 fold, 10-100 fold, 10-50 15 fold, 10-40 fold, 10-30 fold, 10-25 fold, 10-20 fold, 10-15 fold, 20-100 fold, 20-50 fold, 20 40 fold, 20-30 fuld, or 20-25 fold. The disclosure provides lipid-comprising compounds and compositions that are less susceptible to clearance and thus have a longer half-life in vivo. This is particularly the case where the compositions are intended for repeated including chronic administration, and even 20 more particularly where such repeated administration occurs within days or weeks.
Significantly, these compositions are less susceptible or altogether circumvent the observed phenomenon of accelerated blood clearance (ABC). ABC is a phenomenon in which certain exogenously administered agents are rapidly cleared from the blood upon second and subsequent administrations. This phenomenon has been observed, in part, for a 25 variety of lipid-containing compositions including but not limited to lipidated agents, liposomes or other lipid-based delivery vehicles, and lipid-encapsulated agents. Heretofore,
the basis of ABC has been poorly understood and in some cases attributed to a humoral immune response and accordingly strategies for limiting its impact in vivo particularly in a
clinical setting have remained elusive. 30 This disclosure provides compounds and compositions that are less susceptible, if at all susceptible, to ABC. In some important aspects, such compounds and compositions are lipid-comprising compounds or compositions. The lipid-containing compounds or compositions of this disclosure, surprisingly, do not experience ABC upon second and subsequent administration in vivo. This resistance to ABC renders these compounds and compositions particularly suitable for repeated use in vivo, including for repeated use within short periods of time, including days or 1-2 weeks. This enhanced stability and/or half-life is due, in part, to the inability of these compositions to activate BIa and/or BI b cells and/or 5 conventional B cells, pDCs and/or platelets. This disclosure therefore provides an elucidation of the mechanism underlying accelerated blood clearance (ABC). It has been found, in accordance with this disclosure and the inventions provided herein, that the ABC phenomenon at least as it relates to lipids and N lipid nanoparticles is mediated, at least in part an innate immune response involving Bla N 10 and/or Blb cells, pDC and/or platelets. Bla cells are normally responsible for secreting N natural antibody, in the form of circulating IgM. This IgM is poly-reactive, meaning that it is able to bind to a variety of antigens, albeit with a relatively low affinity for each. It has been found in accordance with the invention that some lipidated agents or lipid comprising formulations such as lipid nanoparticles administered in vivo trigger and are 15 subject to ABC. It has now been found in accordance with the invention that upon administration of a first dose of the LNP, one or more cells involved in generating an innate immune response (referred to herein as sensors) bind such agent, are activated, and then initiate a cascade of immune factors (referred to herein as effectors) that promote ABC and toxicity. For instance, Bla and BI b cells may bind to LNP, become activated (alone or in the
20 presence of other sensors such as pDC and/or effectors such as IL6) and secrete natural IgM
that binds to the LNP. Pre-existing natural IgM in the subject may also recognize and bind to the LNP, thereby triggering complement fixation. After administration ofthe first dose, the production of natural lgM begins within 1-2 hours of administration of the LNP. Typically by about 2-3 weeks the natural IgM is cleared from the system due to the natural half-life of 25 IgM. Natural IgG is produced beginning around 96 hours after administration of theLNP. The agent, when administered in a nafve setting, can exert its biological effects relatively unencumbered by the natural IgM produced post-activation of the BIa cells or BIb cells or natural IgG. The natural IgM and natural IgG are non-specific and thus are distinct from
anti-PEG IgM and anti-PEG IgG. 30 Although Applicant is not bound by mechanism, it is proposed that LNPs trigger ABC and/or toxicity through the following mechanisms. It is believed that when an LNP is
administered to a subject the LNP is rapidly transported through the blood to the spleen. The LNPs may encounter immune cells in the blood and/or the spleen. A rapid innate immune response is triggered in response to the presence of the LNP within the blood and/or spleen. Applicant has shown herein that within hours of administration of an LNP several immune sensors have reacted to the presence of the LNP. These sensors include but are not limited to immune cells involved in generating an immune response, such as B cells, pDC, and 5 platelets. The sensors may be present in the spleen, such as in the marginal zone of the spleen and/or in the blood. The LNP may physically interact with one or more sensors, which may interact with other sensors. In such a case the LNP is directly or indirectly interacting with the sensors. The sensors may interact directly with one another in response to recognition of N the LNP. For instance many sensors are located in the spleen and can easily interact with one N 10 another. Alternatively one or more of the sensors may interact with LNP in the blood and N become activated. The activated sensor may then interact directly with other sensors or indirectly (e.g., through the stimulation or production of a messenger such as a cytokine e.g., T1.6) In some embodiments the LNP may interact directly with and activate each of the 15 following sensors: pDC, Bla cells, Bib cells, and platelets. These cells may then interact directly or indirectly with one another to initiate the production of effectors which ultimately lead to the ABC and/or toxicity associated with repeated doses of LNP. For instance, Applicant has shown that LNP administration leads to pDC activation, platelet aggregation and activation and B cell activation. In response to LNP platelets also aggregate and are 20 activated and aggregate with B cells. pDC cells are activated. LNP has been found to interact with the surface of platelets and B cells relatively quickly. Blocking the activation of any one or combination of these sensors in response to LNP is useful for dampening the immune response that would ordinarily occur. This dampening of the immune response results in the avoidance of ABC and/or toxicity. 25 The sensors once activated produce effectors. An effector, as used herein, is an immune molecule produced by an immune cell, such as a B cell. Effectors include but are not limited to immunoglobulin such as natural IgM and natural IgG and cytokines such as IL6. Bl a and BI b cells stimulate the production of natural IgMs within 2-6 hours following administration of an LNP. Natural IgG can be detected within 96 hours. IL6 levels are 30 increased within several hours. The natural IgM and IgG circulate in the body for several days to several weeks. During this time the circulating effectors can interact with newly administered LNPs, triggering those LNPs for clearance by the body. For instance, an effector may recognize and bind to an LNP. The Fe region of the effector may be recognized by and trigger uptake of the decorated LNP by macrophage. The macrophage are then transported to the spleen. The production of effectors by immune sensors is a transient response that correlates with the timing observed for ABC. If the administered dose is the second or subsequent administered dose, and if such 5 second or subsequent dose is administered before the previously induced natural IgM and/or IgG is cleared from the system (e.g., before the 2-3 window time period), then such second or subsequent dose is targeted by the circulating natural IgM and/or natural IgG or Fc which trigger alternative complement pathway activation and is itself rapidly cleared. When LNP N are administered after the effectors have cleared from the body or are reduced in number, N io ABC is not observed. N Thus, it is useful according to aspects of the invention to inhibit the interaction between LNP and one or more sensors, to inhibit the activation of one or more sensors by LNP (direct or indirect), to inhibit the production of one or more effectors, and/or to inhibit the activity of one or more effectors. In some embodiments the LNP is designed to limit or 15 block interaction of the LNP with a sensor. For instance the LNP may have an altered PC and/or PEG to prevent interactions with sensors. Alternatively or additionally an agent that inhibits immune responses induced by LNPs may be used to achieve any one or more of these effects. It has also been determined that conventional B cells are also implicated in ABC. 20 Specifically, upon first administration of an agent, conventional B cells, referred to herein as CDI9(+), bind to and react against the agent. Unlike Bla and Bb cells though, conventional B cells are able to mount first an IgM response (beginning around 96 hours after administration of the LNPs) followed by an IgG response (beginning around 14 days after administration of the LNPs) concomitant with a memory response. Thus conventional B cells 25 react against the administered agent and contribute to IgM (and eventually IgG) that mediates ABC. The IgM and IgG are typically anti-PEG IgM and anti-PEG IgG. It is contemplated that in some instances, the majority of the ABC response is mediated through Bla cells and Bla-mediated immune responses. It is further contemplated that in some instances, the ABC response is mediated by both IgM and IgG, with both 30 conventional B cells and BIa cells mediating such effects. In yet still other instances, the ABC response is mediated by natural IgM molecules, some of which are capable of binding to natural IgM, which may be produced by activated Bl a cells. The natural IgMs may bind to one or more components of the LNPs, e.g., binding to a phospholipid component ofthe
LNPs (such as binding to the PC moiety of the phospholipid) and/or binding to a PEG-lipid component of the LNPs (such as binding to PEG-DMG, in particular, binding to the PEG moiety of PEG-DMG). Since BI a expresses CD36, to which phosphatidylcholine is a ligand, it is contemplated that the CD36 receptor may mediate the activation of BIa cells and thus 5 production of natural IgM. In yet still other instances, the ABC response is mediated primarily by conventional B cells. It has been found in accordance with the invention that the ABC phenomenon can be reduced or abrogated, at least in part, through the use of compounds and compositions (such N as agents, delivery vehicles, and formulations) that do not activate Bla cells. Compounds N i10 and compositions that do not activate Bla cells maybe referred to herein as Bla inert N compounds and compositions. It has been further found in accordance with the invention that the ABC phenomenon can be reduced or abrogated, at least in part, through the use of compounds and compositions that do not activate conventional B cells. Compounds and compositions that do not activate conventional B cells may in some embodiments be referred 15 to herein as CD19-inert compounds and compositions. Thus, in some embodiments provided herein, the compounds and compositions do not activate BI a cells and they do not activate conventional B cells. Compounds and compositions that do not activate BI a cells and conventional B cells may in some embodiments be referred to herein as Bl a/CD I 9-inert compounds and compositions. 20 These underlying mechanisms were not heretofore understood, and the role of BI a and BI b cells and their interplay with conventional B cells in this phenomenon was also not appreciated. Accordingly, this disclosure provides compounds and compositions that do not promote ABC. These may be further characterized as not capable of activating BIa and/or 25 Bib cells, platelets and/or pDC, and optionally conventional B cells also. Thesecompounds (e.g., agents, including biologically active agents such as prophylactic agents, therapeutic agents and diagnostic agents, delivery vehicles, including liposomes, lipid nanoparticles, and other lipid-based encapsulating structures, etc.) and compositions (e.g., formulations, etc.) are particularly desirable for applications requiring repeated administration, and in particular 30 repeated administrations that occur within with short periods of time (e.g., within 1-2 weeks). This is the case, for example, if the agent is a nucleic acid based therapeutic that is provided to a subject at regular, closely-spaced intervals. The findings provided herein may be applied to these and other agents that are similarly administered and/or that are subject to ABC.
Of particular interest are lipid-comprising compounds, lipid-comprising particles, and lipid-comprising compositions as these are known to be susceptible to ABC. Such lipid comprising compounds particles, and compositions have been used extensively as biologically active agents or as delivery vehicles for such agents. Thus, the ability to 5 improve their efficacy of such agents, whether by reducing the effect of ABC on the agent itself or on its delivery vehicle, is beneficial for a wide variety of active agents. Also provided herein are compositions that do not stimulate or boost an acute phase response (ARP) associated with repeat dose administration of one or more biologically active NI agents. N 10 The composition, in some instances, may not bind to IgM, including but not limited to NI natural IgM. The composition, in some instances, may not bind to an acute phase protein such as but not limited to C-reactive protein. The composition, in some instances, may not trigger a CD5(+) mediated immune 15 response. As used herein, a CD5(+) mediated immune response is an immune response that
is mediated by Bla and/or B31b cells. Such a response may include an ABC response, an acute phase response, induction of natural IgM and/or TgG, and the like. The composition, in some instances, may not trigger a CD19(+) mediated immune response. As used herein, a CD19(+) mediated immune response is an immune response that 20 is mediated by conventional CD9(+), CD5(-) B cells. Such a response may include induction of IgM, induction of IgG, induction of memory B cells, an ABC response, an anti drug antibody (ADA) response including an anti-protein response where the protein may be encapsulated within an LNP, and the like. Bl a cells are a subset of B cells involved in innate immunity. These cells are the 25 sourc of .iIulaing IgM, ieferred to as naral antibody or natural serum antibody. Natural
IgM antibodies are characterized as having weak affinity for a number of antigens, and
therefore they are referred to as "poly-specific" or "poly-reactive", indicating their ability to bind to more than one antigen. BI a cells are not able to produce IgG. Additionally, they do not develop into memory cells and thus do not contribute to an adaptive immune response.
30 However, they are able to secrete IgM upon activation. The secreted TgM is typically cleared
within about 2-3 weeks, at which point the immune system is rendered relatively nave to the
previously administered antigen. If the same antigen is presented after this time period (e.g.,
at about 3 weeks after the initial exposure), the antigen is not rapidly cleared. However, significantly, if the antigen is presented within that time period (e.g., within 2 weeks, including within I week, or within days), then the antigen is rapidly cleared. This delay between consecutive doses has rendered certain lipid-containing therapeutic or diagnostic agents unsuitable for use. 5 In humans, Bl a cells are CD19(+), CD20(+), CD27(+), CD43(+), CD70(-) and CD5(+). In mice, B I a cells are CDI9(+), CD5(+), and CD45 B cell isoform B220(+). It is the expression of CD5 which typically distinguishes Bl a cells from other convention B cells. Bl a cells may express high levels of CD5, and on this basis may be distinguished from other B-I cells such as B-l b cells which express low or undetectable levels of CD5. CD5 is a pan N 10 T cell surface glycoprotein. Bla cells also express CD36, also known as fatty acid N translocase. CD36 is a member of the class B scavenger receptor family. CD36canbind many ligands, including oxidized low density lipoproteins, native lipoproteins, oxidized phospholipids, and long-chain fatty acids. BIb cells are another subset of B cells involved in innate immunity. These cells are 15 another source of circulating natural IgM. Several antigens, including PS, are capable of inducing T cell independent immunity through BI b activation. CD27 is typically upregulated on BI b cells in response to antigen activation. Similar to Bla cells, the BIb cells are typically located in specific body locations such as the spleen and peritoneal cavity and are in very low abundance in the blood. The BI b secreted natural IgM is typically cleared within about 2-3 20 weeks, at which point the immune system is rendered relatively naTve to the previously administered antigen. If the same antigen is presented after this time period (e.g., at about 3 weeks after the initial exposure), the antigen is not rapidly cleared. However, significantly, if the antigen is presented within that time period (e.g., within 2 weeks, including within I week, or within days), then the antigen is rapidly cleared. This delay between consecutive 25 doses has rendered certain lipid-containing therapeutic or diagnostic agents unsuitable for use. In some embodiments it is desirable to block Bla and/or BIb cell activation. One strategy for blocking Bla and/or Bib cell activation involves determining which components of a lipid nanoparticle promote B cell activation and neutralizing those components. It has 30 been discovered herein that at least PEG and phosphatidylcholine (PC) contribute to BI a and Bl b cell interaction with other cells and/or activation. PEG may play a role in promoting aggregation between B Icells and platelets, which may lead to activation. PC (a helper lipid in LNPs) is also involved in activating the BIcells, likely through interaction with the CD36 receptor on the B cell surface. Numerous particles have PEG-lipid alternatives, PEG-less, and/or PC replacement lipids (e.g. oleic acid or analogs thereof) have been designed and tested. Applicant has established that replacement of one or more of these components within an LNP that otherwise would promote ABC upon repeat administration, is useful in 5 preventing ABC by reducing the production of natural IgM and/or B cell activation. Thus, the invention encompasses LNPs that have reduced ABC as a result of a design which eliminates the inclusion of B cell triggers.
Another strategy for blocking Bl a and/or Bib cell activation involves using an agent that inhibits immune responses induced by LNPs. These types of agents are discussed in 10 more detail below. In some embodiments these agents block the interaction between Bl a/Bl b N cells and the LNP or platelets or pDC. For instance the agent may be an antibody or other binding agent that physically blocks the interaction. An example of this is an antibody that binds to CD36 or CD6. The agent may also be a compound that prevents or disables the Bla/BI b cell from signaling once activated or prior to activation. For instance, it is possible 15 to block one or more components in the Bl a/BI b signaling cascade the results from B cell
interaction with LNP or other immune cells. In other embodiments the agent may act one or
more effectors produced by the Bl a/BI b cells following activation. These effectors include for instance, natural IgM and cytokines.
It has been demonstrated according to aspects of the invention that when activation of 20 pDC cells is blocked, B cell activation in response to LNP is decreased. Thus, in order to avoid ABC and/or toxicity, it may be desirable to prevent pDC activation. Similar to the strategies discussed above, pDC cell activation may be blocked by agents that interfere with
the interaction between pDC and LNP and/or B cells/platelets. Alternatively agents that act
on the pDC to block its ability to get activated or on its effectors can be used together with 25 the LNP to avoid ABC. Platelets also play an important role in ABC and toxicity. Very quickly after a first
dose of LNP is administered to a subject platelets associate with the LNP, aggregate and are activated. In some embodiments it is desirable to block platelet aggregation and/or activation. One strategy for blocking platelet aggregation and/or activation involves determining which 30 components of a lipid nanoparticle promote platelet aggregation and/or activation and
neutralizing those components. It has been discovered herein that at least PEG contribute to
platelet aggregation, activation and/or interaction with other cells. Numerous particles have
PEG-lipid alternatives and PEG-less have been designed and tested. Applicant has established that replacement of one or more of these components within an LNP that otherwise would promote ABC upon repeat administration, is useful in preventing ABC by reducing the production of natural JgM and/or platelet aggregation. Thus, the invention encompasses LNPs that have reduced ABC as a result of a design which eliminates the 5 inclusion of platelet triggers. Alternatively agents that act on the platelets to block its activity once it is activated or on its effectors can be used together with the LNP to avoid ABC.
Measuring ABCActivity and related activities N Various compounds and compositions provided herein, including LNPs, do N 10 not promote ABC activity upon administration in vivo. These LNPs may be characterized
N and/or identified through any of a number of assays, such as but not limited to those described below, as well as any of the assays disclosed in the Examples section, include the methods siihsention of th F.amplrns
In some embodiments the methods involve administering an LNP without producing 15 an immune response that promotes ABC. An immune response that promotes ABC involves
activation of one or more sensors, such as B Icells, pDC, or platelets, and one or more
effectors, such as natural IgM, natural IgG or cytokines such as IL6. Thus administration of an LNP without producing an immune response that promotes ABC, at a minimum involves administration of an LNP without significant activation of one or more sensors and
20 significant production of one or more effectors. Significant used in this context refers to an
amount that would lead to the physiological consequence of accelerated blood clearance of
all or part of a second dose with respect to the level of blood clearance expected for a second dose of an ABC triggering LNP. For instance, the immune response should be dampened such that the ABC observed after the second dose is lower than would have been expected for
25 an ABC triggering LNP.
Bla or Blb activation assay
Certain compositions provided in this disclosure do not activate B cells, such as Bla or Bib cells (CD19+ CD5+) and/or conventional B cells (CDI9+ CD5-). Activation of1B1a 30 cells, Bib cells, or conventional B cells maybe determined in a numberof ways, some of which are provided below. B cell population may be provided as fractionated B cell populations or unfractionated populations of splenocytes or peripheral blood mononuclear cells (PBMC). If the latter, the cell population may be incubated with the LNP of choice for a period of time, and then harvested for further analysis. Alternatively, the supernatant may be harvested and analyzed.
Upregulation of activation marker cell surface expression 5 Activation of Bl a cells, BI b cells, or conventional B cells may be demonstrated as increased expression of B cell activation markers including late activation markers such as
CD86. In an exemplary non-limiting assay, unfractionated B cells are provided as a splenocyte population or as a PBMC population, incubated with an LNP of choice for a particular period of time, and then stained for a standard B cell marker such as CD19 and for 10 an activation marker such as CD86, and analyzed using for example flow cytometry. A N suitable negative control involves incubating the same population with medium, and then
performing the same staining and visualization steps. An increase in CD86 expression in the
test population compared to the negative control indicates B cell activtion
15 Pro-inflammatory cytokine release B cell activation may also be assessed by cytokine release assay. For example,
activation may be assessed through the production and/or secretion of cytokines such as IL-6 and/or TNF-alpha upon exposure with LNPs of interest. Such assays may be performed using routine cytokine secretion assays well known in 20 the art. An increase in cytokine secretion is indicative of B cell activation.
LNP binding/association to and/or uptake by B cells
LNP association or binding to B cells may also be used to assess an LNP of interest and to further characterize such LNP. Association/binding and/or uptake/internalization may 25 be assessed using a detectably labeled, such as fluorescently labeled, LNP and tracking the location of such LNP in or on B cells following various periods of incubation. The invention further contemplates that the compositions provided herein may be
capable of evading recognition or detection and optionally binding by downstream mediators of ABC such as circulating TgM and/or acute phase response mediators such as acute phase 30 proteins (e.g., C-reactive protein (CRP).
Methods of usefor reducing ABC
Also provided herein are methods for delivering LNPs, which may encapsulate an agent such as a therapeutic agent, to a subject without promoting ABC. In some embodiments, the method comprises administering any of the LNPs described herein, which do not promote ABC, for example, do not induce production of 5 natural IgM binding to the LNPs, do not activate Bla and/or Bib cells. Asusedherein,an LNP that "does not promote ABC" refers to an LNP that induces no immune responses that would lead to substantial ABC or a substantially low level of immune responses that is not
sufficient to lead to substantial ABC. An LNP that does not induce the production of natural IgMs binding to the LNP refers to LNPs that induce either no natural IgM binding to the 10 LNPs or a substantially low level of the natural IgM molecules, which is insufficient to lead N to substantial ABC. An LNP that does not activate Bla and/or Bib cells refer to LNPs that
induce no response of Bla and/or BIb cells to produce natural IgM binding to the LNPs or a
substantially low level of BIa and/or BIb responses, which is insufficient to lead to substantial ABC. 15 In some embodiments the terms do not activate and do not induce production are a
relative reduction to a reference value or condition. In some embodiments the reference value
or condition is the amount of activation or induction of production of a molecule such as IgM by a standard LNP such as an MC3 LNP. In some embodiments the relative reduction is a reduction of at least 30%, for example at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 20 70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100%. In other embodiments the terms do not activate cells such as B cells and do not induce
production of a protein such as IgM may refer to an undetectable amount of the active cells or
the specific protein.
In some instances, the LNPs described herein may be free of an epitope that activates 25 Bla cells, for example, free of an epitope that activates or interacts with CD36 or CD6. Such
LNPs contain either no epitopes capable of activating Bla cells or CD36 or contain such epitopes at a substantially low amount, which is not sufficient to activate BI a or CD36 to a level high enough for inducing substantial ABC. In other embodiments, the LNPs described herein may be free of an epitope that activates BI b cells. By substantially free of, it is 30 meaclassical pnt that a LNP includes less than 99% of the recited agent. In some embodiments the LNP may include none of the recited agent. In some instances, the LNPs
described herein may contain one or more helper lipid as described herein, which may comprise at least one fatty acid chain of at least 8C and at least one polar moiety. In some examples, the helper lipid does not activate Bla and/or Bib cells. In other examples, the helper lipid does not bind or has low binding affinity to CD36. Alternatively, the helper lipid may competitively inhibit phosphatidylcholine from binding to CD36. Alternatively the LNP may be coadminstered (administered with, before or after) or 5 coformulated with an agent that removes or targets B or Bl a cells. An agent that removes or targets B or BI a cells may be Rituximab. Rituximab (RITUXAN@, Genentech/Biogen) is a monoclonal antibody against the protein CD20, which is primarily found on the surface of immune system B cells. Rituximab interacts with CD20 on the surface of B cells and destroys B cells. As shown in the Examples, the combination of Rituximab and the LNP had i 10 significantly reduced ABC upon subsequent administration of LNP. N In other embodiments the agent may bind and/or inhibit CD6 on Bla cells. An exemplary agent that binds and/or inhibits CD6 on Bla cells is an anti-CD6 antibody, such as Alzumab. Alzumab (itolizumab, Biocon) is a humanized IgGI monoclonal antibody that selectively targets CD6, a pan T cell marker involved in co-stimulation, adhesion and 15 maturation of T cells. Alzumab also binds to CD6 on the surface of Bla cells. In some instances such methods may comprise
(i) administering a first dose of an agent to a subject, (ii) administering a second or subsequent dose of the agent to the subject, wherein the second or subsequent dose is administered within 2 weeks of the first or prior dose, and (mI) repsTin8 rep (H) one or mor-etImps, wherein the agent is formulated with an LNP that dues not promote ABC.
Another method for delivering an agent to a subject involves
(i) administering a first dose of an agent to a subject, (ii) administering a second or subsequent dose of the agent to the subject, wherein the
25 second or subsequent dose is administered within 2 weeks of the first or prior dose, and (iii) repeating step (ii) one or more times,
wherein the half-life of the agent after the second and subsequent dose is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the half-life of the agent after the first dose. Still another method for delivering an agent to a subject involves 30 (i) administering a first dose of an agent to a subject, (ii) administering a second or subsequent dose of the agent to the subject, wherein the
second or subsequent dose is administered within 2 weeks of the first or prior dose, and (iii) repeating step (ii) one or more times, wherein the activity or blood concentration of the agent after the second and subsequent dose is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the activity or blood concentration of the agent after the first dose. Second or subsequent doses may be administered within I week, or within 6 days, or 5 within 5 days, or within 4 days, or within 3 days, or within 2 days, or within I day of the first or prior dose. The agent may be a biologically active agent such as a diagnostic agent or a therapeutic agent, although it is not so limited. N Agents may be administered two or more times, three or more times, four or more N 10 times, etc. Agent administration may therefore be repeated once, twice, 3, 4, 5, 6, 7, 8, 9, 10,
N or more times. The agent may be administered chronically or acutely, depending on its intended purpose.
The method may be a method of treating a subject having or at risk of having a condition that benefits from the biologically active agent, particularly if the biologically 15 active agent is a therapeutic agent. Alternatively, the method may be a method of diagnosing
a subject, in which case the biologically active agent is a diagnostic agent. The second and subsequent doses of biologically active agent may maintain an activity of at least 50% of the activity of the first dose, or at least 60% of the first dose, or at least 70% of the first dose, or at least 75% of the first dose, or at least 80% of the first dose, 20 or at least 85% of the first dose, or at least 90% of the first dose, or at least 95% of the first
dose, or more, for at least I day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days post administration ofthe second or subsequent dose. When the biologically active agent is an mRNA (a therapeutic mRNA or a mRNA encoding a vaccine antigen), a method for reducing ABC of LNPs encapsulating the mRNA 25 can be performed using a low amount of the LNPs for the first dose, and/or the second dose
(as well as the subsequent doses). The low doses can be equal to or less than 0.3 mg/kg, e.g., 0.2 mg/kg, or 0.1 mg/kg. In some instances, the first dose, the second dose, or both range from 0.1 to 0.3 mg/kg. The interval between the first dose and the second dose in any of the methods
30 described herein may be equal to or less than two weeks, for example, less than 21, 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or I days. In some instances, the subject can be administered a dose once daily, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 or 21 days in any of the methods described herein. Each possibility represents a separate embodiment of the present invention. Further, inhibiting ABC of LNPs in a subject can be achieved by the use of one or more secondary agents that inhibit immune responses induced by LNPs, e.g., inhibit the 5 binding to or activity of sensors e.g., natural IgM production, natural IgG production, activation of Bl a cells, activation of Bl b cells, and/or activation of platelets and or dendritic
cells or the activity or production of any effectors . The secondary agents are referred to
alternatively as agents that inhibit immune responses induced by LNPs. In some instances, N the secondary agent may inhibit the production of natural IgM that binds the LNPs, or N 10 neutralize such natural IgMs. In other instances, the secondary agent may inhibit activation
N of Bla cells or remove Bla cells. For example, such a secondary agent may inhibit a surface
receptor of Bl a cells, including, but not limited to CD36 . Alternatively or in addition, the secondary agent may interfere with the binding of IgM to its target. In other embodiments, the secondary agent may inhibit the production of natural IgG that binds the LNPs, or 15 neutralize such natural IgGs or may interfere with the binding of fgG to its target. In other instances, the secondary agent may inhibit activation of Bl b cells or remove Bl b cells.
Platelet effects and toxicity The invention is further premised in part on the elucidation of the mechanism 20 underlying dose-limiting toxicity associated with LNP administration. Such toxicity may
involve coagulopathy, disseminated intravascular coagulation (DIC, also referred to as
consumptive coagulopathy), whether acute or chronic, and/or vascular thrombosis. In some
instances, the dose-limiting toxicity associated with LNPs is acute phase response (APR) or complement activation-related psudoallergy (CARPA). 25 As used herein, coagulopathy refers to increased coagulation (blood clotting) in vivo.
The findings reported in this disclosure are consistent with such increased coagulation and
significantly provide insight on the underlying mechanism. Coagulation is a process that involves a number of different factors and cell types, and heretofore the relationship between and interaction of LNPs and platelets has not been understood in this regard. This disclosure 30 provides evidence of such interaction and also provides compounds and compositions that are modified to have reduced platelet effect, including reduced platelet association, reduced platelet aggregation, and/or reduced platelet aggregation. The ability to modulate, including
preferably down-modulate, such platelet effects can reduce the incidence and/or severity of coagulopathy post-LNP administration. This in turn will reduce toxicity relating to such LNP, thereby allowing higher doses of LNPs and importantly their cargo to be administered to patients in need thereof. CARPA is a class of acute immune toxicity manifested in hypersensitivity reactions 5 (HSRs), which may be triggered by nanomedicines and biologicals. Unlike allergic reactions, CARPA typically does not involve IgE but arises as a consequence of activation of the complement system, which is part of the innate immune system that enhances the body's abilities to clear pathogens. One or more ofthe following pathways, the classical complement pathway (CP), the alternative pathway (AP), and the lectin pathway (LP), may i 10 be involved in CARPA. Szebeni, Molecular Immunology, 61:163-173 (2014). N The classical pathway is triggered by activation of the Cl-complex, which contains. Cl q, CIr, C Is, or Cl qr2s2. Activation of the Cl-complex occurs when Clq binds to IgM or IgG complexed with antigens, or when C1 binds directly to the surface of the pathogen. Such binding leads to conformational changes in the Clq molecule, which leads to the 15 activation of Clr, which in turn, cleave Cls. The Clr2s2 component now splits C4 and then
C2, producing C4a, C4b, C2a, and C2b. C4b and C2b bind to form the classical pathway C3 convertase (C4b2b complex), which promotes cleavage of C3 into C3a and C3b. C3b then binds the C3 convertase to from the C5 convertase (C4b2b3b complex). The alternative pathway is continuously activated as a result of spontaneous C3 hydrolysis. Factor P
20 (properdin) is a positive regulator of the alternative pathway. Oligomerization of properdin stabilizes the C3 convertase, which can then cleave much more C3. The C3 molecules can
bind to surfaces and recruit more B, D, and P activity, leading to amplification of the complement activation. Acute phase response (APR) is a complex systemic innate immune responses for
25 preventing infection and clearing potential pathogens. Numerous proteins are involved in
APR and C-reactive protein is a well-characterized one.
It has been found, in accordance with the invention, that certain LNP are able to associate physically with platelets almost immediately after administration in vivo, while other LNP do not associate with platelets at all or only at background levels. Significantly, 30 those LNPs that associate with platelets also apparently stabilize the platelet aggregates that are formed thereafter. Physical contact of the platelets with certain LNPs correlates with the
ability of such platelets to remain aggregated or to form aggregates continuously for an extended period of time after administration. Such aggregates comprise activated platelets and also innate immune cells such as macrophages and B cells. Platelet aggregation is observed soon after LNP administration, and appears to occur at the same time as or potentially before platelet activation as evidenced through increased 5 expression of platelet activation markers such as CD31 and CD62P. LNPs that do not associate with significant numbers of platelets but which are able to activate platelets to a lesser degree than the more robust LNPs (discussed above) also cause platelet aggregation very early after administration, and presumably prior to platelet activation. Thus, in vivo, N LNP association with platelets appears to occur at about the same time as aggregation of 10 platelets, and presumably prior to the peak of platelet activation. N Also significant is the additional observation that a subset of LNPs are able to activate platelets, even without appreciable physical association with platelets. This subset is also ahleto form plateltaggregaescomprising B cells and marnphages
Certain LNPs have also been shown to stimulate early interaction between platelets
15 (whether or not activated) and macrophages and B cells, thereby activating these latter cells
as well. The effect of LNPs on B cells and macrophages is therefore both direct and indirect, but ultimately can lead to increased activation of such cells.
Activation of platelets could mediate complement activation. It is therefore contemplated that certain LNPs may induce dose-limiting toxicity such as CARPA and APR 20 via activation of platelets and subsequently the complement system. Certain lipid
components of LNPs, such as phosphatidylcholine may bind to and activate CD36 on platelets, which would trigger the TLR2/4/6 signaling, leading to aggregation and activation of the platelets. Activated platelets express CD62P (P selectin), which is a C3b-binding protein and can trigger the complement cascade. Activated platelets also recruit immune
25 cells such as macrophages and neutrophils, which lead to further immune responses including cytokine (e.g., IL-6) secretion. Further, properdin was found to bind directly to activated platelet via, e.g., CD62P and recruits C3b or C3(H 20), thus triggering the alternative pathway. Saggu et al., J. Immunol. 190:6457-6467 (2013). Accordingly, uses of LNPs that do not induce platelet activation and/or aggregation; 30 and/or do not promote the activation of the complement system (e.g., the classic pathway and/or the alternative pathway) could reduce the risk of LNP-related toxicity. Such LNPs may not induce the activation of platelets and/or the complement system at all. Alternatively, such LNPs may induce a substantially low level of platelet activation and/or complement system activation, which is not sufficient to result in substantial dose-limiting toxicity.
Alternatively or in addition, secondary agents that block the initial platelet activation/aggregation, the initial activation of the complement system, and/or the 5 downstream complement cascade, either in the classic pathway or in the alternative pathway, could be used to prevent or reduce LNP-related toxicity. In some instances, such a secondary agent may inhibit platelet activation, for example, inhibit CD36 activation triggered by LNPs. In other instances, the secondary agent may inhibit CARPA or ARP, for example, inhibit the classical pathway and/or the alternative pathway. Such a secondary agent may target at least 10 one component in the complement system or proteins involved in ARP, thereby blocking the N reaction cascade. For example, the secondary agent may be an antagonist of a TLR receptor (TLR2. TLR4, or TLR6), CD62P, CD31, properdin, a component of the complement system (e.g.,Clq,C3a,C3b,C5a,andC5b). In yet other instances, the secondary agent maybe an agent that can alleviate at least one symptom of LNP-related toxicity. Such agents include, 15 but are not limited to, nonsteroidal anti-inflammatory drug (NSAID) or an antihistamine agent, which can be a histamine receptor blocker such as an Hl antagonist or an Hl inverse agonist. In some embodiments, dose-limiting toxicity and/or ABC can be reduced in a subject
being treated with a therapeutic regimen involving LNP-mediated drug delivery by using 20 LNPs that do not activate a thrombospondin receptor (e.g., CD36), which may be expressed on the surface of immune cells (e.g., Bl a or BI b cells); or other surface receptors involved in triggering the immune responses that lead to dose-limiting toxicity and/or ABC . Such LNPs
may not activate the thrombospondin receptor at all, or could only induce a substantially low
level of its activity such that it is insufficient to induce clinically significant dose-limiting 25 toxicity and/or ABC. Alternatively or in addition, dose-limiting toxicity and/or ABC can be reduced in a subject being treated with a therapeutic regimen involving LNP-mediated drug
delivery by using one or more secondary agent that inhibits the activity of a thrombospondin
receptor (e.g., CD36) expressed on the surface of immune cells and platelets. The thrombospondins (TSP) are a family of multifunctional proteins that are expressed on the 30 surface of or secreted by cells such as blood platelets. The family consists of thrombospondins 1-5. TSP-I is a inhibitory ligand of CD36. Based on these findings, this disclosure contemplates and provides LNPs as well as
LNP-formulated active agents that have reduced platelet association and/or reduced platelet activation and/or reduced platelet aggregation activity. Use of such LNPs, for example as a delivery vehicle for an active agent, reduces the risk of developing coagulopathy, such as disseminated intravascular coagulation (DIC, also referred to as consumptive coagulopathy), whether acute or chronic, and/or vascular thrombosis, as well as any toxicity related thereto. 5 If such toxicity is dose-limiting, then use of these LNPs will enable administration of higher LNP doses and more importantly will enable the delivery of higher doses of the active agent cargo carried by such LNP. The diminution of the platelet response after LNP administration has additional desirable effects, some of which may be synergistic. LNPs have been reported to activate 10 complement shortly after administration. This activation may be direct or indirect. For N example, it has been reported that activated platelets are able to activate complement. Thus LNPs that reduce or prevent platelet activation will also indirectly reduce or prevent complement activation. Complement activation can also contribute to coagulation, for example through complement-mediated generation of thrombin. Thrombin converts 15 available fibrinogen to fibrin, which in turn forms clots together with platelets. Activated platelets have thrombin receptors on their surface and therefore are able to recruit and/or raise the local concentration of thrombin, thereby enhancing fibrin production and ultimately clot formation. The disclosure therefore contemplates and provides additional LNPs that do not activate complement or do not activate complement to the same degree as existing LNPs. 20 Yet still additional LNPs provided herewith are those that do not activate platelet and do not activate complment, Similarly, this disclosure contemplates LNPs that interfere with properdin binding to platelets. Properdin is a positive regulator of the alternative pathway of the complement system. It has been shown to bind to activated platelets, thereby activating the alternative 25 pathway in response to and in the vicinity of the activated platelet. Thus further contemplated is the use of a properdin inhibitors in combination with LNPs provided herein whether such LNPs activate or do not activate platelets, as defined below. Properdin inhibitors include DNA and sulfated glucoconjugates, both of which are bound by properdin and may interfere with properdin binding to activated platelets, 30 This disclosure therefore contemplates and provides, in some aspects, LNPs and LNP formulations that have reduced platelet effects including reduced platelet association and/or reduced platelet activation and/or reduced platelet aggregation activity. Certain LNPs may affect one, two or all three of these platelet activities. For example, some LNP may have reduced platelet association activity, or reduced platelet aggregation activity, or reduced platelet activation activity. Some LNP may have reduced platelet association activity and or reducedplateletactivationactivity,orreducedplateletassociationactivityandreduced platelet activation activity, or reduced platelet aggregation activity and reducedplatelet 5 activation activity. Some LNP may have reduced platelet association activity, reduced platelet aggregation activity, and reduced platelet activation activity. The disclosure contemplates that some LNPs may be universal LNPs, intending that they will down-modulate (or not stimulate in the first instance) one or more of the afore mentioned platelet activities upon administration in the majority of patients or in all patients. N 10 Additionally, the disclosure contemplates that some LNPs may in some instances be N defined and thus identified as patient-specific. That is, some LNPs may be effective at down regulating a platelet response, as described herein, in some but not all patients. Thus, in some instances, some LNPs and LNP formulations may be identified for particular patients and may then be used only for those particular patients. 15 In some instances, the findings provided herein may be applied directly to biologically active agents. For example, the biologically active agent that is a lipid or is conjugated to a lipid or that is conjugated to a PEG moiety directly or indirectly, may be modified as described herein to render the agent unable to stimulate a platelet response or cascade. 20
Platelet activity assays These various activities may be measured as described herein and/or as performed in the art. For example, platelet activation may be assessed by increased expression of activation markers such as CD31 and CD62P. Platelet aggregation may be assessed by flow 25 cytometry. Similarly flow cytometry may be used to detect non-platelet types such as B cells and macrophages within such aggregates. It is to be understood that the platelet effects of LNP can be assessed in vivo, for example in an animal model, as well as in vitro using for example human blood. These assays may be used to screen for and/or identify LNP having one or more of the afore-mentioned activities. 30 Compounds and Compositions, including LNP The disclosure provides lipid-comprising compositions that are less susceptible to clearance and thus have a longer half-life in vivo. This is particularly important where the compositions are intended for repeated including chronic administration, and even more particularly where such repeated administration occurs within days or weeks.
Significantly, these compositions are less susceptible or altogether circumvent the observed phenomenon of accelerated blood clearance (ABC). ABC is a phenomenon in 5 which lipid-containing exogenously administered agents are rapidly cleared from the blood upon second and subsequent administrations. This phenomenon has been observed for a variety of lipid-containing compositions, including, but not limited to, liposomes, lipid nanoparticles, and lipid-encapsulated agents. Heretofore, the basis of ABC has been poorly N understood and accordingly strategies for avoiding it have remained elusive. N 10 The lipid-containing compositions of this disclosure, surprisingly, do not experience
N or are minimally affected by ABC upon second and subsequent administration in vivo. This resistance to ABC renders these compositions particularly suitable for repeated use in vivo, including for repeated use within short periods of time, including days or 1-2 weeks. This resistance to ABC is due in part to the inability of these compositions to activate
15 Bla cells. Such compositions are therefore referred to herein as Bla inert compositions or
compositions that do not activate substantial BIa, intending that these compositions, when combined with Bla cells, do not activate Bla cells. Activation of Bla cells maybe determined in a number of ways including, but not limited to, increased expression of activation markers such as CI)86, and expression and/or secretion of cytokines. These 20 compositions may or may not bind to Bla cells, and they may or may not bind to circulating
IgM. Thus, these compositions may evade detection by circulating IgM and/or by BI a cells. BI a cells are a subset of B cells involved in innate immunity. These cells are the source of circulating IgM, referred to as natural antibody or natural serum antibody. These IgM antibodies are characterized as having weak affinity for a number of antigens, and 25 therefore they are referred to as "poly-specific" or "poly-reactive", describing their ability to bind to more than one antigen. Although able to produce such IgM, BI a cells are not capable of producing IgG. Additionally, they do not develop into memory cells and thus do not contribute to an adaptive immune response. However, they are able to secrete IgM upon
activation. The secreted IgM is typically cleared within about 12 days (half-life of IgM in 30 sera is about 5-8 days, Nature Review Drug Discovery 2, 52-62, January 2003), at which
point the immune system is rendered relatively naYve to the previously administered antigen. If the same antigen is presented after this time period (e.g., at about 2 weeks after the initial exposure), the antigen is not rapidly cleared. However, significantly, if the antigen is presented within that time period (e.g., within 2 weeks, including within I week, or within days), then the antigen is rapidly cleared. This delay between consecutive doses has rendered certain lipid-containing therapeutic or diagnostic compositions unsuitable for repeated use. The compounds, particles, and compositions described herein overcome these 5 limitations, thereby transforming a variety of lipid-containing compositions into efficacious therapeutic and diagnostic agents. The Bla lipid-compositions provided herein do not undergo accelerated blood clearance upon repeat administration and thus can be administered repeatedly to a subject, including within short periods of time, without loss of activity. Resistance to ABC may also be due in part to the inability of these compositions to io activate Bl b cells, pDC and/or platelets. Such compositions are therefore referred to herein as
N BI b pDC and/or platelets inert compositions or compositions that do not activate substantial
BI b pDC and/or platelets, intending that these compositions, when combined with Bl b cells pDC and/or platelets, do not activate BIb cells pDC and/or platelets, respectively. Activation of BIb cells, pDC and/or platelets may be determined in a number of ways including, but not 15 limited to, increased expression of activation markers such as CDl b (for BIb cells), and expression and/or secretion of cytokines, and ability to activate B cells (pDC). These
compositions may or may not bind to BI b cells, pDC and/or platelets, and they may or may not bind to circulating IgM or lgG. Thus, these compositions may evade detection by circulating IgM, IgG and/or by BI a cells pDC and/or platelets. 20 Particles, such as LNP, typically comprise one or more of the following components: lipids (which may include cationic lipids, helper lipids which may be neutral lipids, zwitterionic lipid, anionic lipids, and the like), structural lipids such as cholesterol or cholesterol analogs, fatty acids, polymers, stabilizers, salts, buffers, solvent, and the like.
Certain of the LNPs provided herein comprise a cationic lipid, a helper lipid, a 25 structural lipid, and a stabilizer which may or may not be provided conjugated to another lipid. The cationic lipid may be but is not limited to DLin-DMA, DLin-D-DMA, DLin MC3-DMA, DLin-KC2-DMA and DODMA. The cationic lipid may be an ionizable lipid. The structural lipid may be but is not limited to a sterol such as for example 30 cholesterol. The helper lipid is a non-cationic lipid. The helper lipid may comprise at least one fatty acid chain of at least 8C and at least one polar headgroup moiety.
Certain of the LNPs lack any phosphatidyl choline (PC) lipids (i.e., are free of phosphatidyl choline (PC)). Certain of the LNPs provided herein lack specific phosphatidyl choline lipids such as but not limiting to DSPC. Certain of the LNPs comprise a phosphatidyl choline analog, such analogs comprising modified head groups (e.g., a modified quaternary amine head group), modified core group, and/or modified lipid tail group. Such analogs may comprise a zwitterionic group that is a non-PC zwitterionic group. The helper lipid may be a lipid of any one or any combination of Formulae I, I-a, I-b, I-b-1, I-b-2, 1-b-3, I-b-4, I-c, I-c-1, I-c-2, 1-c-3, or 11 as provided herein. Certain of the LNP may include a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) substitute or include a minimal amount of DSPC. In certain embodiments, the DSPC substitute is a lipid that is not a phospholipid. Certain LNPs comprise other helper non-cationic lipids including for example oleic acid or oleic acid analogs. The helper lipid may be a lipid of Formula IV as provided herein. The oleic acid may be a substitute or may be in addition to another lipid in the LNP. As would be appreciated by one of skill in the art, modified versions of oleic acid or related fatty acids may be used as well. In some instances, the LNP comprise PEGylated lipids or lipids conjugated to other stabilizing moieties (or stabilizers) such as but not limited to XTEN and PAS polypeptides. Thus, the disclosure contemplates and provides LNPs or formulations thereof that do not include PEG. In certain embodiments, the LNPs include HO-PEG. In other embodiments, the LNPs include a PEG substitute such as a different polymer. When PEG is used as the stabilizer, it may be conjugated to a lipid and thus may be provided as PEG-c-DOMG or PEG-DMG, for example. The stabilizer, whether provided in a conjugated or an unconjugated form, may comprise 1.5 mol% of the LNP, or it may 23 cumprise leSS than 0.5 mol % of the LNP. For example, it may comprise less than 0.4 mol%, less than 0.3 mol %, less than 0.2 mol %, or less than 0.1 mol %. In still other embodiments, LNPs may contain less than 0.5% molar ratio of PEG lipid to the other components. Thus, an LNP may comprise at least 0.0001%, at least 0.0005%, at least 0.001%, at least 0.005%, at least 0.01%, at least 0.05%, at least 0.1%, at least 0.15%, at least 0.2%, at least 0.25%, at least 0.3%, at least 0.35%, at least 0.4%, at least 0.45%, and less than 0.5% (expressed as a molar percentage) of PEGylated lipid. Each possibility represents a separate embodiment of the present invention.
The LNP may comprise a PEGylated lipid of Formula III, including Formulae II-OH, 111-a-1, III-a-2, III-b-1, 1111-b-2, 111-b-I-OH, 111-b-2-OH, V, V-OH. Each possibility represents a separate embodiment of the present invention. Certain of the LNPs provided herein comprise no or low levels of PEGylated lipids, 5 including no or low levels of alkyl-PEGylated lipids, and may be referred to herein as being free of PEG or PEGylated lipid. Thus, some LNPs comprise less than 0.5 mol % PEGylated lipid. In some instances, PEG may be an alkyl-PEG such as methoxy-PEG. .Still other LNPs comprise non-alkyl-PEG such as hydroxy-PEG, and/or non-alkyl-PEGylated lipids such as hydroxy-PEGylated lipids. N 10 The PEGylated lipid may be a Cmpd420, a Cmpd396, a Cmpd394, Cmpd397, N Cmpd395, Cmpd417, Cmpd418, or Cmpd4l9. In some instances, the LNP may comprise about 50 mol %, 10 mol % helper lipid, 1.5 mol % PEGylated lipid, and 38.5 mol % structural lipid. In some instances, the LNP may comprise about 50 mol %, 10 mol % helper lipid, 15 less than 0.5 mol % PEGylated lipid, and 39.5 mol % structural lipid. In some embodiments, the stabilizer is a non-PEG moiety such as an XTEN peptide diat may or may not be conjugated to a lipid. Ihe XTEN peptide is capable of forming a hydrated shell around the LNP due to its hydrophilic nature. It further serves to increase the half-life of the LNP, compared to an LNP lacking (or free of) any stabilizer. Unlike PEG, 20 however, it is biodegradable and has been reported to be non-immunogenic. The XTEN peptide may have an amino acid sequence of
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS (SEQ ID NO:1)
or MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS (SEQ ID
NO:2). Other XTEN amino acid sequences are known in the art, including for example those 25 reported in U.S. Patent No. 9,062,299. Examples of XTEN conjugated lipids include but are not limited to Cmpd431, and Cmpd432 and Cmpd433. Click chemistry maybe used to conjugate the XTEN peptide to the lipid. In some embodiments, the stabilizer is a non-PEG moiety such as a PAS peptide. A PAS peptide is a peptide comprising primarily if not exclusively proline, alanine and srine. 30 Like PEG and XTEN peptides, the PAS peptide is capable of forming a hydrated shell around the LNP. It too serves to increase the half-life of an LNP, compared to an LNP lacking (or free of) a stabilizer. Unlike XTEN peptides, however, PAS peptides tend to be neutral in charge, and thus at least in this respect more similar to PEG. The PAS peptide may have an amino acid sequence of SAPSSPSPSAPSSPSPASPSSAPSSISPSA PSSPSPASPSSA PSSPSPSA PSSPSPASPS (SEQ ID NO:3 or AASPAAPSAPPAAASPAAPSAPPAAASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO:4). 5 Other PAS amino acid sequences are known in the art, including for example, those reported in WO 2008155134. Agents that inhibit immune responses induced by LNPs may also be used in the methods of the invention, together with a standard ABC inducing LNP or with a modified LNP of the invention. Agents that inhibit immune responses induced by LNPs, are N 10 compounds that inhibit the activation of a sensor, inhibit the interaction between an LNP and N a sensor or between sensors (e.g, blocks interaction between pDC and B cells), and/or inhibit the production or activity of an effector. In some instances the agent will be specific for a particular sensor or effector. In other embodiments the agent that inhibits immune responses induced by LNPs functions to prevent the activation of multiple sensors by a more general 15 mechanism, typically acting indirectly on other cellular components that can affect the seisurs. An example of an agent that tunctions on multiple sensors indirectly is a miR binding site. It has been discovered according to the invention that delivery of a miR binding site will inhibit an immune response associated with ABC and can be used to provide repeated 20 dosing of a subject with an LNP during the window of susceptibility to ABC. The miR binding site may be incorporated into a therapeutic nucleic acid that is being delivered in the LNP. Alternatively the miR binding site may separately be incorporated into the same LNP that incorporates the therapeutic nucleic acid or into a different LNP. The miR binding site may be administered to the subject in a separate vehicle at the same or different time as the 25 LNP and may or may not be incorporated into an LNP. In some embodiments the miR binding site may be a miR sponge. Although Applicant is not bound by mechanism, it is believed that the miR binding site act to soak up endogenous, targeted miRNA of interest, preventing that miRNA from functioning in the cell. It is possible to target miRNA that play a positive role in regulation of 30 immune cell function. By inhibiting the function of endogenous miRNA the miR binding site acts as an inhibitor to block the miRNA function and other downstream effects resulting from this targeting inhibition. The miRNA binding agent may also or alternatively be functioning by preventing protein translation in specific tissues or cells, such as the spleen or immune cells. By preventing translation of, for instance, an mRNA therapeutic encapsulated in the LNP, in specific tissues that are high in immune cells, the immune response in those tissues will be decreased, while not having an impact on mRNA expression in other tissues. It has been demonstrated that introduction of miR binding sites such as miR 126 5 (highly abundant in pDC) results in a reduction in B cell activation, a reduction in pDC activation, a reduction in cytokine expression, such as IL6 and IFN-gamma, and a reduction in IgM relative to the response delivered by a corresponding LNP without the miR binding site. N In some embodiments the miR binding site is a miR 126, miR 155, and/or miR 142.3p N 10 binding site. In some embodiments, the mRNA can comprise at least one miR binding site to N thereby reduce or inhibit ABC. The miR binding site may be found in, for instance, the 3' UTR of the mRNA. For example, in one embodiment, the mRNA comprises a miR- 22 binding site, to thereby allow increased expression of a polypeptide encoded by the mRNA in a liver cancer cell (e.g., a hepatocellular carcinoma cell) as compared to a normal liver cell, 15 and also comprises one or more miR binding sites, e.g., selected from the group consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, miR-27. A compound that inhibits the interaction between an LNP and a sensor as used herein will disrupt interaction between LNP and B Icell, platelet or pDC. For instance, Some interactions between LNP and B Icells are mediated through the CD36 receptor on the B1 2U cell and the PC on the LNP. The compound may block and neutralize the PC or the CD36. In some embodiments the compound is an antibody that recognizes and binds CD36, or an antigen-binding fragment or derivative thereof or a CD36 antagonist. A CD36 antagonist may be selected from the group consisting of antibodies or aptamers which bind to CD36 or fragments thereof; soluble ligands which bind to CD36 or fragments thereof; soluble CD36 25 which bind to its ligands; fusion polypeptides, peptides, small molecules, peptidomimetics inhibiting the CD36 activity; and nucleic acid molecules interfering specifically with CD36 expression. Such CD36 antagonist is preferably an antagonist which, preferably specifically, recognizes and binds to a CD36 molecule or fragment thereof, and is preferably selected from the group consisting of an antibody or an aptamer which specifically recognizes and binds to 30 CD36 or a fragment thereof, a nucleic acid molecule interfering specifically with CD36 expression, and a small molecule inhibiting the CD36 activity. More preferably, said CD36 antagonist is a function-blocking monoclonal antibody against CD36. In other embodiments, said CD36 antagonist is a small molecule selected from the group consisting of salvianolic acid B, rosmarinic acid, sodium danshensu, 3-cinnamoyl indole, 13 pentyl berberine, hexarelin, nanoblockers, statins or antioxidants such as alpha-tocopherol and SS peptides, Sulfo-N-succinimidyl oleate and Ursolic acid, and any combination thereof. Alternatively, the CD36 antagonist may comprise an antibody that recognizes and binds CD36, or an 5 antigen-binding fragment or derivative thereof. Preferably, said antibody or antigen-binding fragment or derivative thereof, is directed against the extracellular domain of CD36. Said antibody may be a full-length antibody. Preferably, said antibody is a monoclonal antibody. The antibody may be of the IgG, IgE or IgD type, preferably of the IgG type. The antibody N may be a humanized, chimeric or human antibody. The antibody may also be camelid heavy No0 chain antibody, and in particular humanized camelid heavy-chain antibody. Preferably, said
N antibody, or antigen-binding fragment or derivative thereof, is bivalent. In particular, the
antigen-binding fragment is selected from the group consisting of F(ab')2 di-scFvs, sc(Fv) 2
fragment, (VHH) 2 fragment and diabody. A compound that inhibits the activity of an effector is a compound that prevents an
15 effector from being produced by a sensor or functioning. For instance the agent may inhibit
the production ofnatural IgM that binds the LNPs by interfering with the synthesis pathway in a B Icell. Alternatively the agent may neutralize such natural IgMs or IgG. The agent may be an antibody or antigen-binding portion thereof, that binds to natural IgM or IgG and neutralizes it. Alternatively, the agent may interfere with the binding of IgM or IgG to its 20 target. In other embodiments the agent that inhibits immune responses induced by the LNPs
is an agent that inhibits IL6 activity. Agents that inhibit IL6 activity include for instance, antibodies, fragments thereof, specific humanized antibodies and fragments and variants thereof capable of binding to IL-6 and/or the IL-6/IL-6R complex. These antibodies may bind soluble IL-6 or cell surface expressed IL-6.
25 Lipid Nanoparticles,generally In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG modified lipid, a sterol and a non-cationic lipid. In some embodiments, the lipid nanoparticle carrier comprising a molar ratio ofabout 20-60% cationic lipid: 5-25% non-cationic lipid: 25 55% sterol; and 0.5-15% PEG-modified lipid. In some embodiments, the cationic lipid is an 30 ionizable cationic lipid. In some embodiments, the non-cationic lipid is a neutral lipid. In some embodiments, the sterol is a cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-
((4-(dimethylamnino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the lipid nanoparticle has a polydiversity value of less than 0.4. In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH. In some embodiments, the lipid nanoparticle has a mean diameter of 50-200nm. Each possibility represents a separate 5 embodiment of the present invention. Lipid nanoparticles may comprise one or more lipid species, including, but not limited to, cationic/ionizable lipids, non-cationic lipids, structural lipids, phospholipids, and helper lipids. Any of these lipids may be conjugated to polyethylene glycol (PEG) and thus may be referred to as PEGylated lipids or PEG-modified lipids. N 10 The formation of the lipid nanoparticle (LNP) may be accomplished by methods N known in the art and/or as described in U.S. Pub. No. 2012/0178702, herein incorporated by reference in its entirety. A lipid nanoparticle formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the selection 15 of the non-cationic lipid component, the degree ofnon-cationic lipid saturation, the selection of the structural lipid component, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In certain non-limiting examples, a LNP comprises four basic components: () a cationic lipid; (2) a non-cationic lipid (e.g., a phospholipid such as DSPC); (3) a structural lipid (e.g., a sterol such as cholesterol); and (4) PEG or a PEG 20 modified lipid. In one example by Semple et al. (Nature Biotech. 2010 28:172-176; herein incorporated by reference in its entirety), the lipid nanoparticle formulation is composed of molar ratios as follows: 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA. As another example, changing the composition of the cationic lipid can more effectively deliver siRNA to various antigen presenting cells (Basha 25 et al., Mol Ther. 2011 19:2186-2200; herein incorporated by reference in its entirety). The lipid nanoparticles described herein comprise one or more lipids. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid and a non-cationic lipid. In certain embodiments, the LNP formulation comprises a cationic lipid and a DSPC substitute. In certain embodiments, the LNP formulation comprises a cationic lipid and a fatty 30 acid. In certain embodiments, the LNP formulation comprises a cationic lipid and oleic acid. In certain embodiments, the LNP formulation comprises a cationic lipid and an analog of oleic acid.
Cationic lipids are positively charged lipids that may associate with nucleic acids in the lipid/LNP-based delivery systems. A positive charge on the LNP promotes association with the negatively charged cell membrane to enhance cellular uptake. Cationic lipids may also combine with negatively charged lipids to induce nonbilayer structures that facilitate 5 intracellular delivery. Suitable cationic lipids for use in making the LNPs disclosed herein can be ionizable cationic lipids, for example, amino lipid, and those disclosed herein. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a non-cationic lipid, and a structural lipid. In certain embodiments, the LNP formulation N comprises a cationic lipid, a fatty acid, and a structural lipid. In certain embodiments, the N i LNP formulation comprises a cationic lipid, oleic acid, and a structural lipid. In certain N embodiments, the LNP formulation comprises a cationic lipid, an analog of oleic acid, and a structural lipid. In certain embodiments, the LNP formulation comprises a cationic lipid, a fatty acid, and a sterol. In certain embodiments, the LNP formulation comprises a cationic lipid, oleic acid, and a sterol. In certain embodiments, the LNP formulation comprises a 15 cationic lipid, oleic acid, and cholesterol. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a non-cationic lipid, and PEG or a PEG lipid. In certain embodiments, the LNP formulation comprises a cationic lipid, a non-cationic lipid, and a PEG lipid. In certain embodiments, the LNP formulation comprises a cationic lipid, a non-cationic lipid, and a PEG-O- lipid. In 20 certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a non cationic lipid, and a PEG-OH lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a fatty acid, and a PEG-OH lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, oleic acid, and a PEG-OH lipid. In certain embodiments, the lipid nanoparticle formulation comprises a 23 atiuiiic lipid, aii analog of oleic acid, and a PEG-OH lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a non-cationic lipid (e.g., a phospholipid or fatty acid), a structural lipid, and a PEG lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a non cationic lipid (e.g., phospholipid or fatty acid), a structural lipid, and a PEG-OH lipid. In 30 certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a non cationic lipid (e.g., phospholipid or fatty acid), and structural lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a fatty acid (e.g., oleic acid or an analog thereof), a structural lipid, and a PEG lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a fatty acid (e.g., oleic acid or an analog thereof), a structural lipid, and a PEG-OH lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, oleic acid, a structural lipid (e.g., a sterol), and a PEG-OH lipid. In certain embodiments, the lipid nanoparticle formulation 5 comprises a cationic lipid, oleic acid, and a structural lipid (e.g., cholesterol). In certain embodiments, the lipid nanoparticle formulation comprises one or more cationic or non cationic lipids, a fatty acid (e.g., oleic acid), and a PEG lipid. In certain embodiments, the lipid nanoparticle formulation comprises one or more cationic or non-cationic lipids, a fatty acid (e.g., oleic acid), and a PEG-OH lipid. N 10 In some embodiments, the LNP comprises a fatty acid. In certain embodiments, the N fatty acid is a monounsaturated fatty acid. In certain embodiments, the fatty acid is a polyunsaturated fatty acid. In some embodiments, the LNP comprises oleic acid. In certain embodIments, the lipid nanoparticle formulation comprises one or more cationic or non cationic lipids, and a fatty acid (e.g., oleic acid). In certain embodiments, the lipid 15 nanoparticle formulation comprises one or more cationic or non-cationic lipids, and oleic acid. In certain embodiments, when the LNP includes oleic acid, the LNP does not include a phospholipid. In certain embodiments, when the LNP includes oleic acid, the LNP does not include DSPC. In certain embodiments, when the LNP includes a fatty acid, the LNP does not include a phospholipid. In certain embodiments, when the LNP includes a fatty acid, the 20 T NP does not include DSPC. In some embodiments, the LNP comprises PEG-OH lipids. In certain embodiments, the lipid nanoparticle formulation comprises one or more cationic or non-cationic lipids, and a PEG-OH lipid. In some embodiments, lipid nanoparticle formulations may comprise, in molar 25 percentages, 35 to 45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipid and/or 55% to 65% cationic lipid. In some embodiments, the ratio of lipid to nucleic acid (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1 and/or at least 30:1.
In some embodiments, the ratio of PEG in the lipid nanoparticle formulations may be 30 increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the lipid nanoparticle
formulations. In certain embodiments, lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0%, and/or 3.0% to 6.0% of the lipid molar ratio of PEG lipid to the other components. As a non-limiting example, lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to
4.5%, 2.5% to 5.0%, and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(o methoxy-poly(ethyleneglycol)2000)carbamoyl)]-I,2-dimyristyloxypropyl-3-amine) (also 5 referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC, and cholesterol. In some embodiments, the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG- DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), DMG-PEG (1,2-dimyristoyl-sn-glycerol) and/or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in i10 the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2 N DMA. In certain embodiments, the lipid nanoparticle does not contain a PEG lipid. In certain
embodiments, the lipid nanoparticle contains a PEG lipid substitute such as a PEG-OH lipid. Incorporation of PEG-O1 lipids in the nanoparticle formulation can improve the pharmacokinetics and/or biodistribution of the lipid nanoparticle formulations. For example, 15 incorporation of PEG-OH lipids in the nanoparticle formulation can reduce the ABC effect. In certain embodiments, lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to
3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0%, and/or 3.0% to 6.0% of the lipid molar ratio of PEG-OH lipid to the other components (e.g., the cationic, neutral, and structural lipids). Each possibility represents a separate embodiment of the present invention. 2U In some embodiments, a LNP formulation is a nanoparticle that comprises at least one
lipid. In certain embodiments, the lipids is selected from cationic/ionizable lipids, non cationic lipids (e.g., fatty acids and phospholipids), PEG lipids, structural lipids (e.g., sterols), and PEG-OH lipids. The lipid may be selected from, but is not limited to, DLin-DMA, DLin K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, 25 PEG-DMG, PEGylated lipids, and amino alcohol lipids. In some embodiments, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and amino alcohol lipids. The amino alcohol cationic lipid may be the lipids described in and/or made by the methods described in US Patent Publication No. US2013/0150625, herein incorporated by reference in its entirety. As a non-limiting 2 30 example, the cationic lipid may be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]- {[(9Z,2Z)-octadeca-9,2-dien-1-yloxy]methyl}propan--ol (Compound I in US2013/0150625); 2-amino-3-[(9Z)-octadec-9-en-I-yloxy]-2-{[(9Z)-octadec-9-en-l yloxy]methyl}propan-1-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,I2Z)- octadeca-9,12-dien- I -yloxy]-2-[(octyloxy)mnethyl]propan-I-ol (Compound 3 in US2013/0150625); and 2-(dimethylam ino)-3-{(9Z,I2Z)-octadeca-9,12-dien-1 -yoxy]-2 {[(9Z,I2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-I-ol (Compound 4 in US2013/0150625); or any pharmaceutically acceptable salt or stercoisomer thereof. Each 5 possibility represents a separate embodiment of the present invention. Lipid nanoparticle formulations can comprise a lipid, in particular, an ionizable cationic lipid, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2 DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en I -yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further comprise a N 10 non-cationic lipid (e.g., phospholipid or fatty acid), a structural lipid (e.g., a sterol such as N cholesterol), and a molecule capable of reducing particle aggregation, for example, a PEG or PEG-modified lipid (e.g., PEG-OH lipid). In certain embodiments, the formulation does not contain the PEG lipid. In some embodiments, the LNP formulation consists essentially of a molar ratio of 15 20-60% cationic lipid; 5-25% non-cationic lipid; 25-55% sterol; 0.5-15% PEG lipid. In some embodiments, the LNP formulation consists essentially of a molar ratio of 20-60% cationic lipid; 5-25% non-cationic lipid; 25-55% sterol; 0.5-15% PEG-OH lipid. In some embodiments, the LNP formulation consists essentially of in a molar ratio of 20-60% cationic lipid; 5-25% non-cationic lipid; and 25-55% sterol. In certain embodiments, the 20 non-cationic lipid is a fatty acid. In certain embodiments, the non-cationic lipid is oleic acid or an analog thereof. In certain embodiments, the PEG lipid is a PEG-OH lipid. In some embodiments, a lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl
[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3 25 DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoy)oxy)heptadecanedioate (L319); (ii) a non-cationic lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic lipid; 5-25% non-cationic lipid; 25-55% sterol; 0.5-15% PEG-lipid. Each possibility represents a separate embodiment of the present invention. 30 In some embodiments, a lipid nanoparticle formulation consists essentially of (i) at
least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl
[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3 DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate
(L319); (ii) a non-cationic lipid as a DSPC substitute (e.g., a different phospholipid, or a fatty acid); (iii) a structural lipid (e.g., a sterol such as cholesterol); and (iv) a PEG-lipid or a PEG OH lipid (e.g., PEG-DMG or PEG-cDMA), in a molar ratio of 20-60% cationic lipid; 5-25% DSPC substitute; 25-55% structural lipid; 0.5-15% PEG-lipid. Each possibility represents a 5 separate embodiment of the present invention. In some embodiments, a lipid nanoparticle formulation includes 25% to 75% on a molar basis of a cationic lipid. The cationic lipid may be selected from 2,2-dilinoleyl-4 dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4 dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4 10 (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., 35 to 65%, 45 to 65%, 60%, N 57.5%, 50% or 40% on a molar basis. Each possibility represents a separate embodiment of the present invention. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of the non-cationic lipid, e.g., 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a 15 molar basis. In certain embodiments, the non-cationic lipid is a phospholipid. In certain embodiments, the non-cationic lipid is a DSPC substitute (e.g., a phospholipid other than DSPC, or a fatty acid). In certain embodiments, the non-cationic lipid is a fatty acid (e.g., oleic acid or an analog thereof). Other examples of non-cationic lipids include, without limitation, POPC, DPPC, DOPE and SM. In some embodiments, a lipid nanoparticle 20 formulation includes 0.5% to 15% on a molar basis of a fatty acid, e.g., 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of oleic acid, e.g., 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of an analog of oleic acid, e.g., 3 to 12%, 5 to 10% or 15%, 25 10%, or 7.5% on a molar basis. In some embodiments, the formulation includes 5% to 50% on a molar basis of the structural lipid, e.g., 15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 3 1% on a molar basis. In some embodiments, the formulation includes 5% to 50% on a molar basis of a sterol, e.g., 15 to 45%, 20 to 40%, 40%,38.5%,35%, or 31% on a molar basis. Anon-limitingexampleofa 30 sterol is cholesterol. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 20% on a molar basis of the PEG or PEG-modified lipid, e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%, 1.5%,3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEG-modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example, around 1,500 Da, around 1,000 Da, or around 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearoyl 5 glycerol (PEG-DMG) (also referred herein as Cmpd422), PEG-cDMA (further discussed in Reyes et al. J. ControlledRelease, 107, 276-287 (2005) the contents of which are herein incorporated by reference in its entirety). As described herein, any PEG lipids or PEG modified lipids may be PEG-OH lipids. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 20% on a molar basis of a PEG-OH lipid, e.g., 0.5 to 10%, 0.5 i10 to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis. N In some embodiments, lipid nanoparticle formulations include 25-75% of a cationic lipid, 0.5-15% of the non-cationic lipid; 5-50% of the structural lipid, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 25-75% of a cationic lipid, 0.5-15% of the non-cationic lipid; 5-50% of 15 the structural lipid, and 0.5-20% of a PEG-OH lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 25-75% of a cationic lipid, 0.5-15% of the non cationic lipid, and 5-50% of the structural lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 25-75% of a cationic lipid selected from 2,2-dilinoleyl-4 dimethylaminoethyl-[I,3]-dioxolane (DLin-KC2-DMA), dilinoley-methyl-4 20 dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4 (dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, lipid nanoparticle formulations include 35-65% of a cationic lipid, 3-12% of the non-cationic lipid, 15-45% of the structural lipid, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle 25 formulations include 35-65% of a cationic lipid, 3-12% of the non-cationic lipid, 15-45% of the structural lipid, and 0.5-10% of the PEG-OH lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 35-65% of a cationic lipid, 3-12% of the non-cationic lipid, and 15-45% of the structural lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 35-65% of a cationic lipid selected 30 from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinolcyl methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4 (dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Each possibility represents a separate embodiment of the present invention.
In some embodiments, lipid nanoparticle formulations include 45-65% of a cationic lipid, 5-10% of the non-cationic lipid, 25-40% of the structural lipid, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 45-65% of a cationic lipid, 5-10% of the non-cationic lipid, 25-40% of 5 the structural lipid, and 0.5-10% of a PEG-OH lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 45-65% of a cationic lipid, 5-10% of the non-cationic lipid, and 25-40% of the structural lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 45-65% of a cationic lipid selected from 2,2-dilinoleyl-4 dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4 10 dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4 N (dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Each possibility represents a separate embodiment of the present invention. In some embodiments, lipid nanoparticle formulations include 60% of a cationic lipid, 7.5% of the non-cationic lipid, 31 % of a structural lipid, and 1.5% of the PEG or PEG 15 modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 60% of a cationic lipid, 7.5% of the non-cationic lipid, 31% of a structural lipid, and 1.5% of a PEG-OH lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 60% of a cationic lipid, 9% of the non-cationic lipid, and 31% of a structural lipid on a molar basis. In some embodiments, lipid nanoparticle formulations 20 include 60% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3] dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3 DMA), and dl((Z)-non-2-en-1-yl) 9-((4-(dimethylamno)butanoyl)oxy)heptadecanedioate (L319). Each possibility represents a separate embodiment of the present invention. In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid, 25 10% of the non-cationic lipid, 38.5 % of the structural lipid, and 1.5% of the PEG or PEG modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid, 10% of the non-cationic lipid, 38.5 % ofa structural lipid, and 1.5% of a PEG-OH lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid, 10% of the non-cationic lipid, and 40% of a 30 structural lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3] dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-
DMA), and di((Z)-non-2-en-I-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Each possibility represents a separate embodiment of the present invention. In some embodiments, lipid nanoparticle formulations include 40% of a cationic lipid, 15% of the non-cationic lipid, 40% of the structural lipid, and 5% of the PEG or PEG 5 modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 40% of a cationic lipid, 15% of the non-cationic lipid, 40% of the structural lipid, and 5% of a PEG-OH lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 40% of a cationic lipid, 20% of the non-cationic lipid, 40% of the structural lipid on a molar basis. In some embodiments, lipid nanoparticle formulations 1o include 40% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3] N dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3 DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Each possibility represents a separate embodiment of the present invention. In some embodiments, lipid nanoparticle formulations include 57.2% of a cationic 15 lipid, 7.1% of the non-cationic lipid 34.3% of the sterol, and 1.4% of the PEG or PEG modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 57.2% of a cationic lipid, 7.1% of the non-cationic lipid, 34.3% of the structural lipid, and 1.4% of the PEG-Ol lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 57.2% of a cationic lipid, 8.5% of the non-cationic lipid, and 34.3% of 20 the structural lipid on a molar basis. In some embodiments, lipid nanoparticle formulations
include 57.2% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3J dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3 DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Each possibility represents a separate embodiment of the present invention. 25 In some embodiments, lipid nanoparticle formulations consists essentially of a lipid
mixture in molar ratios of 20-70% cationic lipid; 5-45% non-cationic lipid; 20-55% structural lipid; 0.5-15% PEG-modified lipid. In some embodiments, lipid nanoparticle formulations consists essentially of a lipid mixture in molar ratios of 20-70% cationic lipid; 5-45% non cationic lipid (e.g., phospholipid or fatty acid); 20-55% structural lipid; and 0.5-15% PEG 30 OH lipid. In some embodiments, lipid nanoparticle formulations consists essentially of a lipid
mixture in molar ratios of 20-70% cationic lipid; 5-45% non-cationic lipid (e.g., phospholipid or fatty acid); 20-55% structural lipid (e.g., sterols); and 0.5-15% PEG-OH lipid. In some embodiments, lipid nanoparticle formulations consists essentially of a lipid mixture in molar ratios of 20-70% cationic lipid; 5-45% non-cationic lipid (e.g., phospholipid or fatty acid); and 20-55% structural lipid (e.g., sterols). In some embodiments, lipid nanoparticle formulations consists essentially of a lipid mixture in molar ratios of 20-70% cationic lipid; 5-45% fatty acid (e.g., oleic acid or analog thereof); 20-55% structural lipid (e.g., sterols); 5 and 0.5-15% PEG-OH lipid. In some embodiments, lipid nanoparticle formulations consists essentially of a lipid mixture in molar ratios of 20-70% cationic lipid; 5-45% fatty acid (e.g., oleic acid or analog thereof); and 20-55% structural lipid (e.g., sterols). In some embodiments, lipid nanoparticle formulations consists essentially of a lipid mixture in molar ratios of 20-70% cationic lipid; 5-45% oleic acid; 20-55% structural lipid (e.g., sterols); and i10 0.5-15% PEG-OH lipid. In some embodiments, lipid nanoparticle formulations consists N essentially of a lipid mixture in molar ratios of 20-70% cationic lipid; 5-45% oleic acid; and 20-55% structural lipid (e.g., sterols). In some embodiments, the molar lipid ratio is 50/10/38.5/1.5 (mol% cationic lipid/non-cationic lipid/structural lipid/PEG lipid). In some embodiments, the molar lipid 15 ratio is 57.2/7.1134.3/1.4 (mol% cationic lipid/non-cationic lipid/structural lipid/PEG lipid). In some embodiments, the molar lipid ratio is 40/15/40/5 (mol% cationic lipid/non-cationic lipid/structural lipid/PEG lipid). In some embodiments, the molar lipid ratio is 50/10/35/4.5/0.5 (mol% cationic lipid/non-cationic lipid/structural lipid/PEG lipid). In some embodiments, the molar lipid ratio is 50/10/35/5 (mol% cationic lipid/non-cationic 20 lipid/structural lipid/PEG lipid). In some embodiments, the molar lipid ratio is 40/10/40/10 (mol% cationic lipid/non-cationic lipid/structural lipid/PEG lipid). In some embodiments, the molar lipid ratio is 35/15/40/10 (mol% cationic lipid/non-cationic lipid/structural lipid/PEG lipid). In some embodiments, the molar lipid ratio is 52/13/30/5(mol% cationic lipid/non cationic lipid/structural lipid/PEG lipid). As described herein, any non-cationic lipid may be a 25 DSPC substitute such as a non-DSPC phospholipid or a fatty acid (e.g., oleic acid). As described herein, any PEG lipid may be a PEG-OH lipid. Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Bioechnol. 28:172-176; Jayarama el al. (2012), Angew. Chen. Int. Ed., 51: 8529-8533; and Maier et al. (2013) 30 Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, lipid nanoparticle formulations may comprise a cationic lipid, a PEG lipid (e.g., PEG-OH lipid) and optionally comprise a non-cationic lipid (e.g., phospholipid or fatty acid). In some embodiments, lipid nanoparticle formulations may comprise a cationic lipid, a PEG lipid (e.g., PEG-OH lipid) and a structural lipid (e.g., a sterol) and optionally comprise a non-cationic lipid (e.g., phospholipid or fatty acid). Lipid nanoparticles described herein may comprise 2 or more components (e.g., 5 lipids), not including the payload. In certain embodiments, the LNP comprises two components (e.g., lipids), not including the payload. In certain embodiments, the lipid nanoparticle comprises 5 components (e.g., lipids), not including the payload. In certain embodiments, the LNP comprises 6 components (e.g., lipids), not including the payload. N In some embodiments, the lipid nanoparticle formulations described herein may be N 10 four component lipid nanoparticles. A 4 component LNP may comprise four different lipids N selected from any described herein. The four components do not include the payload. The lipid nanoparticle may comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid nanoparticle comprises a cationic lipid, a fatty acid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid nanoparticle 15 comprises a cationic lipid, a fatty acid, a PEG-OH lipid, and a structural lipid. Each possibility represents a separate embodiment of the present invention. In some embodiments, the lipid nanoparticle formulations described herein may be three component lipid nanoparticles. A three component LNP may comprise three different lipids described herein. The lipid nanoparticle may comprise a cationic lipid, a non-cationic
20 lipid (e.g., phospholipid or fatty acid), and a structural lipid. In certain embodiments, the lipid nanoparticle comprises a cationic lipid, a fatty acid, and a structural lipid. In certain
eribudiments, the lipid nanoparticle comprises a cationic lipid, a phospholipid, and a structural lipid. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter
25 of 10-500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm. or 80 200 nm. In one embodiment, the LNP formulation may be formulated by the methods described in International Publication Nos. W02011127255 or W02008103276, the contents of each of 30 which is herein incorporated by reference in their entirety. As a non-limiting example, LNP
formulations as described in W02011127255 and/or W02008103276; each of which is herein incorporated by reference in their entirety.
In one embodiment, the lipid nanoparticle may be formulated by the methods described in US Patent Publication No US2013/0156845 or International Publication No W02013/093648 or W02012024526, each of which is herein incorporated by reference in its entirety. The lipid nanoparticles described herein may be made in a sterile environment by the 5 system and/or methods described in US Patent Publication No. US20130164400, herein incorporated by reference in its entirety. In one embodiment, the LNP formulation may be formulated in a nanoparticle such as a
nucleic acid-lipid particle described in US Patent No. 8,492,359, the contents of which are herein incorporated by reference in its entirety. 10 As a non-limiting example, the lipid particle may comprise one or more active agents or
N therapeutic agents (e.g., RNA); one or more cationic lipids comprising from about 50 mol% to about 85 mol% of the total lipid present in the particle; one or more non-cationic lipid lipids comprising from about 13 mol% to about 49.5 mol% of the total lipid present in the particle; and one or more structural lipids that inhibit aggregation of particles comprising from about 15 0.5 mol% to about 2 mol% of the total lipid present in the particle. n one embodiment, the LNP formulation may be formulated by the methods described
in International Publication Nos. W0201127255 or W02008103276, the contents of each of which are herein incorporated by reference in their entirety. As a non-limiting example, LNP formulations as described in W02011127255 and/or W02008103276; the contents of each of 20 which are herein incorporated by reference in their entirety. In one embodiment, LNP
formulations described herein may comprise a polycationic composition. As a non-limiting example, the polycationic composition may be selected from formula 1-60 of US Patent
Publication No. US20050222064; the content of which is herein incorporated by reference in its entirety.
25 In some embodiments, LNPs comprise the lipid KL52 (an amino-lipid disclosed in U.S. Application Publication No. 2012/0295832 expressly incorporated herein by reference in its entirety). Activity and/or safety (as measured by examining one or more of ALT/AST, white blood cell count and cytokine induction) of LNP administration may be improved by incorporation of such lipids. LNPs comprising KL52 may be administered intravenously and/or 30 in one or more doses. In some embodiments, administration of LNPs comprising KL52 results in equal or improved mRNA and/or protein expression as compared to LNPs comprising
MC3.
As a non-limiting example, the LNP may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in International Pub. No. W02012013326 or US Patent Pub. No. US20130142818; each of which is herein incorporated by reference in its entirety. In some 5 embodiments, the lipid nanoparticle includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolarnine (DOPE). In some embodiments, the lipid nanoparticles described herein can have a diameter from below 0.1 um up to 100 nm such as, but not limited to, less than 0.1 um, less than 1.0 N um, less than 5 um, less than 10 urn, less than 15 um, less than 20 um, less than 25 um, less N 10 than 30 um, less than 35 um, less than 40 um, less than 50 um, less than 55 um, less than 60 N um, less than 65 um, less than 70 um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less than 95 urn, less than 100 um, less than 125 um, less than 150 um, less than 175 um, less than 200 urn, less than 225 urn, less than 250 um, less than 275 um, less than 300 um, less than 325 um, less than 350 um, less than 375 um, less than 400 um, less than 425 urn, 15 less than 450 um, less than 475 um, less than 500 um, less than 525 um, less than 550 um, less than 575 um, less than 600 um, less than 625 um, less than 650 um, less than 675 um, less than 700 um, less than 725 um, less than 750 um, less than 775 um, less than 800 um, less than 825 um, less than 850 um, less than 875 um, less than 900 um, less than 925 um, less than 950 um, less than 975 um. 20 In another embodiment, LNPs may have a diameter from about I nm to about 100 nm, from about I nm to about 10 nm, about I nm to about 20 nm. from about I nrn to about 30 nm. from about I nm to about 40 nm, from about I nm to about 50 nm, from about I nm to about 60 nm, from about I nm to about 70 nm, from about I nm to about 80 nm, from about I nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to 25 about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about-20 to 30 about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and/or from about
70 to about 90 nm. Each possibility represents a separate embodiment of the present invention.
A nanoparticle composition may be relatively homogenous. A polydispersity index 5 may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 10 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle N composition may be from about 0.10 to about 0.20. Each possibility represents a separate embodiment of the present invention.
The zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the 15 surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may
interact undesirably with cells, tissues, and other elements in the body. In some
embodiments, the zeta potential of a nanoparticle composition may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, 20 from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +IU m V, trom about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about
25 +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV. Each
possibility represents a separate embodiment of the present invention. The efficiency of encapsulation of a therapeutic agent describes the amount of therapeutic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation
30 efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic agent in a solution
containing the nanoparticle composition before and after breaking up the nanoparticle
composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic agent (e.g., nucleic acids) in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic agent may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the 5 encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%. Each possibility represents a separate embodiment of the present invention.
A nanoparticle composition may optionally comprise one or more coatings. For example, a nanoparticle composition may be formulated in a capsule, film, or tablet having a N 10 coating. A capsule, film, or tablet including a composition described herein may have any
N useful size, tensile strength, hardness, or density. In some embodiments, such LNPs are synthesized using methods comprising microfluidic mixers. Exemplary microfluidic mixers may include, but are not limited to a slit interdigitial micromixer including, but not limited to those manufactured by Microinnova 15 (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published (Langmuir. 2012. 28:3633-40; Belliveau, N.M. et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 20 2012. I:e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51; each of which is herein incorporated by reference in its entirety). In some embodiments, methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure-induced chaotic
25 advection (MICA). According to this method, fluid streams flow through channels present in a
herringbone pattern causing rotational flow and folding the fluids around each other. This
method may also comprise a surface for fluid mixing wherein the surface changes orientations
during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Application Publication Nos. 2004/0262223 and 2012/0276209, each of which is expressly 30 incorporated herein by reference in their entirety. In one embodiment, the lipid nanoparticles may be formulated using a micromixer such as,
but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit
Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM)from the Institute fur Mikrotechnik Mainz GmbH, Mainz Germany). In one embodiment, the lipid nanoparticles are created using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 5 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-65 1; each of which is herein incorporated by reference in its entirety). As a non-limiting example, controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (See e.g., Abraham et al. N Chaotic Mixer for Microchannels. Science, 2002 295: 647651; which is herein incorporated 10 by reference in its entirety). N In one embodiment, the mRNA ofthe present invention may be formulated in lipid nanoparticles created using a micromixer chip such as,.but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism. 15 In one embodiment, the lipid nanoparticles may have a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, 20 about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm., about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nni, about 50 to about 70 nm about 50 to 25 about 80 no, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm. In one embodiment, the lipid nanoparticles may have a diameter from about 10 to 500 nm. In one embodiment, the lipid 30 nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm. Each possibility represents a separate embodiment of the present invention.
In some embodiments, the lipid nanoparticle has a polydiversity value of less than 0.4. In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH. In some embodiments, the lipid nanoparticle has a mean diameter of 50-200nm.
Lipids As generally defined herein, the term "lipid" refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing 1o metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids leads them to form liposomes, vesicles, or membranes in aqueous media. Cationiclionizable lipids A nanoparticle composition may include one or more cationic and/or ionizable lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH). Cationic and/or ionizable lipids may be selected from the non-limiting group consisting of 3 (didodecylamino)-N1,N1,4-tridodecyl-I-piperazineethanamine (KL10), N 1 -[2 (didodecylamino)ethyl] N 1,N4.,N4-tridodecyl-1,4-piperazinediethanamine (K L22), 14,25 2u dltrldecyl-15,18,21,24-tetraaza-octatiacontane (K L25), 1,2-dilinoleyloxy-N,N dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-I9-yl-4-(dimethylamino)butanoate (DLin MC3-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8 [(3)-cholest-5-en-3 yloxy]octyl}oxy) N,N dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-I-yloxy]propan-l-amine (Octyl-CLinDMA), (2R)-2-({8-[(3 P)-cholest-5-en-3-yloxy]octyl} oxy)-NN-d imethyl-3
[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan--amine (Octyl-CLinDMA (2R)), and (2S) 2 ({8-[(3p)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-I yloxy]propan-1-amine (Octyl-CLinDMA (2S)). In addition to these, a cationic lipid may also be a lipid including a cyclic amine group. Each possibility represents a separate embodiment of the present invention. Each possibility represents a separate embodiment of the present invention.
In one embodiment, the cationic lipid may be selected from, but not limited to, a
cationic lipid described in International Publication Nos. W02012040184, W02011153120, W02011149733, W02011090965, W02011043913, W02011022460, W02012061259, W02012054365, WO2012044638, W02010080724, W0201021865, W02008103276, 5 W02013086373 and W02013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent Publication No. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and S20130225836; the contents of each of which are herein incorporated by reference in their entirety. N In another embodiment, the cationic lipid may be selected from, but not limited to, N 10 formula A described in International Publication Nos. W02012040184, W02011153120, N W02011149733, W02011090965, W02011043913, W02011022460, W02012061259, W02012054365, W02012044638 and W02013116126 or US Patent Publication No. US20130178541 and US20130225836; the contents of each of which is herein incorporated by reference in their entirety. In yet another embodiment, the cationic lipid may be selected 15 from, but not limited to, formula CLI-CLXXIX of International Publication No.
W02008103276, formula CLI-CLXXIX of US Patent No. 7,893,302, formula CLI CLXXXXII of US Patent No. 7,404,969 and formula I-VI of US Patent Publication No. US20100036115, formula I of US Patent Publication No US20130123338; each of which is herein incorporated by reference in their entirety. 20 As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)-NN dimethylnonacosa-20,23-dien-10-amine, (I7Z,20Z)-NN-dimemylhexacosa-I7,20-dien-9 amine, (1Z,I9Z)-N5N-dimethylpentacosa-l 6, 19-dien-8-amine, (I3Z, I6Z)-N,N dimethyldocosa- 3,16-dien-5-amine, (12Z,I5Z)-N,N dimethylhenicosa-12,15- dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa 25 15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z) N,N-dimethyltetracosa-15,18-dien-5-amine, (I4Z,I7Z)-N,N-dimethyltricosa- 4,17-dien-4 amine, (19Z,22Z)-N,N-dimeihyloctacosa-l9,22-dien-9-amine, (18Z,21 Z)-N,N dimethylheptacosa- 18 ,21 -dien-8 -amine, (17Z,20Z)-N,N-dimethylhexacosa- 17,20-dien-7 amine, (16Z,I9Z)-N,N-dimethylpentacosa-I 6,19-dien-6-amine, (22Z,25Z)-N,N 30 dimethylhentriaconta-22,25-dien-I0-amine, (21 Z ,24Z)-N,N-dimethyltriaconta-21,24-dien-9 amine, (18Z)-N,N-dimetylheptacos-18-en-10-amine, (I7Z)-N,N-dimethylhexacos- 7-en-9
amine, (I 9Z,22Z)-N,N-dimethyloctacosa-I 9,22-dien-7-amine, N,N-dimethylheptacosan-10 amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, I-[(] I Z, 4Z)-l- nonylicosa-11,14-dien-1-y] pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-I 0-amine, (I5Z)-N,N-dimethyl eptacos-15-en-l 0-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-NN-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-I 0-amine, (22Z)-N,N-dimethylhentriacont-22-en-0 5 amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,I 5Z)-N,N-dimethyl-2 nonylhenicosa-12,15-dien-l-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-l amine, N,N-dimethyl-I-[(IS,2R)-2-octylcyclopropyl] eptadecan-8-amine, I-[(1S,2R)-2 hexylcyclopropyl]-N,N- dimethylnonadecan-10-amine,N,N-dimethyl-I-[(IS ,2R)-2 octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(lS,2R)-2 10 octylcyclopropyllhenicosan-10-amine,N,N-dimethyl-I-[(IS,2S)-2-{[(IR,2R)-2 N pentylcyclopropyl]methyl}cyclopropyl]nonadecan-I0-amine,N,N-dimethyl-1-[(1S,2R)-2 octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(IR,2S)-2 undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1 S,2R)-2 octylcyclopropyl]hepLyl} dodecan-i-amine, 1-{(1R,2S)-2-hepty lcyclopropylJ-N,N 15 dimethyloctadecan-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6 amine, N,N-dimethyl-I-[(S,2R)-2-octylcycIopropyljpentadecan-8-amine, R-N,N-dimethyl-I
[(9Z,I2Z)-octadeca-9,12- dien-I-yloxy]-3-(octyloxy)propan-2-amine, S-N,N-dimethyl-I
[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z) octadeca-9,12-dien-I-yloxy]- -[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-I 20 [(9Z,12Z)-octadeca-9,12-dien-I-yloxy]-3-[(5Z)-oct-5-cn-I-yloxy]propan-2-amIne, 1-{2
[(9Z,12Z)-octadeca-9,12-dien-I -yloxy]-I-[(octyloxy)methyl]ethyl.azetidine, (2S)-1 (hexyloxy)-N,N-dimethyl-3-[(9Z,I2Z)-octadeca-9,12-dien-I -yloxy]propan-2-amine, (2S)-1 (heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N
dimethyl-I-(nonyloxy)-3-[(9Z,I2Z)-octadeca-9,I2-dien-I-yloxy]propan-2-amine, N,N 2 25 dimethyl-I-[(9Z)-octadec-9-en-I-yloxy]-3-(octyloxy)propan- -amine; (2S)-N,N-dimethyl-1
[(6Z,9Z, I2Z)-octadeca-6,9,12-trien-I -yloxy]-3- (octyloxy)propan-2-am ine, (2S)-I
[(1I Z,14Z)-icosa-1 1, 14-dien- I -yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-l (hexyloxy)-3-[(1 IZ,14Z)-icosa-11,14-dien-I -yloxy]-N,N-dimethylpropan-2-amine, I
[( lIZ,I4Z)-icosa-11,14-dien-I-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1
30 [(13Z,I6Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-am ine, (2S)-I
[(I3Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1
[(13Z)-docos-13-en-I-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos
13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-cn-1-yloxy]-
N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-HI(1-metoyloctyl)oxy]-3
[(9Z,I2Z)-octadeca-9,12-dien-I -yloxy]propan-2-amine, (2R)-1-[(3,7- dimethyloctyl)oxy] N,N-dimethyl-3-[(9Z,I2Z)-octadeca-9,12-dien-I-yloxy]propan-2-amine, N,N-dimethyl-1
(octyloxy)-3-({8-[(] S,2S)-2-{[(I R,2R)-2
pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1 -{[8-(2
ocIylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (IIE,20Z,23Z)- N,N dimethylnonacosa-l,20,2-trien-I0-amine or a pharmaceutically acceptable salt or stereoisomer thereof. Each possibility represents a separate embodiment of the present invention. Additional examples of cationic lipids include the following:
ClC0 HONH 3 H NH N N 0HH O 0
NH 3 0
O 0 0 O 0 O
D C1 o O NH, 0
HO 0 0
0 H0 HO 0 O NH 3 O
CI 1 NHJH 0 HO 0 O O O
O Cd0H 0 HO N ® NH 3 O 'ICI 8 , and
Cl G Cl 0 NH3 O
HO N 0 In one embodiment, the lipid may be a cleavable lipid such as those described in International Publication No. W02012170889, herein incorporated by reference in its entirety. In another embodiment, the lipid may be a cationic lipid such as, but not limited to, Formula (1) of U.S. Patent Application No. US20130064894, the contents of which are herein incorporated by reference in its entirety. In one embodiment, the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. W02013086354; the contents of each of which are herein incorporated by reference in their entirety. In another embodiment, the cationic lipid may be a trialkyl cationic lipid. Non limiting examples of trialkyl cationic lipids and methods of making and using the trialkyl cationic lipids are described in International Patent Publication No. W02013126803, the contents of which are herein incorporated by reference in its entirety. In some embodiments, additiohal lipids may comprise a compound of Formula (X):
R 4~ NR 1 R N R
RRs m M R3 m, M
or a salt or isomer thereof, wherein: Ri is selected from the group consisting ofCs.30 alkyl, C5 -2 0 alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R 3 are independently selected from the group consisting of H, CI. 14 alkyl, C 2- 14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C 36 carbocycle, -(CH 2 )nQ, -(CH 2),CHQR,
-CHQR, -CQ(R) 2, and unsubstituted C 1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH 2 )nN(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2, -CN, -N(R) 2
, -C(O)N(R) 2, -N(R)C(O)R, -N(R)S(O) 2R, -N(R)C(O)N(R) 2, -N(R)C(S)N(R) 2, -N(R)Rs, -O(CH 2)nOR, -N(R)C(=NR 9)N(R) 2, -N(R)C(=CHR 9)N(R) 2, -OC(O)N(R) 2, -N(R)C(O)OR, 5 -N(OR)C(O)R, -N(OR)S(O) 2R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2, -N(OR)C(S)N(R) 2
, -N(OR)C(=NR 9)N(R) 2, -N(OR)C(=CHR 9)N(R) 2, -C(=NR 9)N(R) 2, -C(=NR 9)R, -C(O)N(R)OR, and -C(R)N(R) 2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; 1 - alkyl, C 2. 3 alkenyl, each R 5 is independently selected from the group consistingof C i10 and H; N each R 6 is independently selected from the group consistingofC 1-3 alkyl, C 2. 3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OlH)-, -P(O)(OR')O-, -S(O) 2 -, 15 -S-S-, an aryl group, and a heteroaryl group; R 7 is selected from the group consisting ofC. 3 alkyl, C 2 -3 alkenyl, and H; R 8 is selected from the group consistingofC 3. 6 carbocycle and heterocycle;
R 9 is selected from the group consisting of H, CN, NO 2 , C1 . 6 alkyl, -OR, -S(O) 2 R,
-S(O) 2 N(R) 2 , C 2 - 6 alkenyl, C 3. carbocycle and heterocycle; 20 each R is independently selected from the group consistingofC3 alkyl, C 2 -3alkenyl, and H; each R' Is Independently selected trom the group consisting ofCi 8 alkyl, C2 z 8
alkenyl, -R*YR", -YR", and H; each R" is independently selected from the group consisting ofC 3 .14 alkyl and 25 C 3 14 alkenyl; each R* is independently selected from the group consisting of C1 1 2 alkyl and C 2 12 alkenyl; each Y is independently a C 3 . 6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and 30 m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. In some embodiments, a subset of compounds of Formula (X) includes those in which when R 4 is -(CH 2),Q, -(CH2)nCIQR, -CHQR, or -CQ(R) 2, then (i) Q is not -N(R)2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is I or 2.
In some embodiments, another subset of compounds of Formula (X) includes those in which R, is selected from the group consistingof C5 . 30 alkyl, C5 - 20 alkenyl, -R*YR", -YR", and -R"M'R'; 5 R 2 and R 3 are independently selected from the group consisting of 1, C-1 4 alkyl, C2. 14
alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3, together with the atom to which they are attached, form a heterocycle or carbocycle; R 4 is selected from the group consisting of a C 3. 6 carbocycle, -(CH 2)nQ, Nl -(CH 2)nCHQR, Ni10 -CHQR, -CQ(R) 2, and unsubstituted C 1 .alkyl, where Q is selected from a C 3-6 carbocycle, a N 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, 0, and S, OR, -O(CH 2)nN(R) 2, -C(O)OR, -OC(O)R, -CX 3, -CX 2H, -CXH 2 , -CN, -C(O)N(R) 2
, -N(R)C(O)R, -N(R)S(O) 2R, -N(R)C(O)N(R) 2, -N(R)C(S)N(R) 2, -CRN(R) 2C(O)OR, -N(R)Rs, 15 -O(CH 2)nOR, -N(R)C(=NR9)N(R) 2, -N(R)C(=CHR 9)N(R) 2, -OC(O)N(R) 2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2, -N(OR)C(S)N(R) 2
, -N(OR)C(=NR 9)N(R) 2, -N(OR)C(=CHR 9)N(R) 2, -C(=NR 9)N(R) 2, -C(=NR 9)R, -C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or more heteroatorns selected from N, 0, and S which is substituted with one or more substituents selected from 20 oxo (=O), OH, amino, mono- or di-alkylamino, and C1 3 alkyl, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C3 alkyl, C .2 3 alkenyl, and H; each R 6 is independently selected from the group consistingofC alkyl, C 2 -3 alkenyl,
25 and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(0) 2 -, -S-S-, an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C.3 alkyl, C 2-3 alkenyl, and H; 30 Rs is selected from the group consisting ofC3 -6 carbocycle and heterocycle; R 9 is selected from the group consisting of H, CN, NO 2 , CI. 6 alkyl, -OR, -S() 2R,
-S(O) 2N(R) 2 , C 2-6 alkenyl C 3 -6 carbocycle and heterocycle; each R is independently selected from the group consisting ofC1 . 3 alkyl, C2-3 alkenyl, and H; each R' is independently selected from the group consistingofCI-18 alkyl, C2- 18 alkenyl, -R*YR", -YR", and H; 5 each R" is independently selected from the group consisting ofC 3 - 14 alkyl and C 3- 14 alkenyl; each R* is independently selected from the group consistingofC1 -12 alkyl and C2- 12 alkenyl; N each Y is independently a C 3 6 carbocycle; N 10 each X is independently selected from the group consisting of F, Cl, Br, and 1; and N m is selected from 5, 6, 7, 8, 9, 10, I1, 12, and 13, or salts or isomers thereof.
In some embodiments, another subset of compounds of Formula (X) includes those in which 15 R, is selected from the group consistingofC 5-3 0 alkyl, C5 -20 alkenyl, -R*YR", -YR", and -R"M'R'; R, and R3 are independently selected from the group consisting of H, CI. 14 alkyl, C2- 14 alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; 20 R 4 is selected from the group consisting of a C 3 -6 carbocycle, -(CH2)nQ,
-(CH 2).CHQR, -CHQR, -CQ(R)2, and unsubstituted CI-6 alkyl, where Q is selected from a C3-6 carbocycle, a
5- to 14-membered heterocycle having one or more heteroatoms selected from N, 0, and S, OR, 25 -O(C H2)nN(R)2, -C(O)O'R, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R) 2, -CRN(R)2C(O)OR, -N(R)Rs, -O(CH2),,OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CH4R9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, 30 -N(OR)C(=NR 9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR 9)R, -C(O)N(R)OR, and -C(=NR 9)N(R)2, and each n is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle and (i) R4 is -(CH,)nQ in which n is I or 2, or (ii) R4 is
-(CH 2)nCHQR in which n is 1, or (iii) R 4 is -CHQR, and -CQ(R) 2, then Q is either a 5- to 14 membered heteroaryl or 8- to 14-membered heterocycloalkyl; each R 5 is independently selected from the group consisting ofC1 3 alkyl, C2 -3 alkenyl, and H; 5 each R 6 is independently selected from the group consisting ofC 3 alkyl, C 2 -3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(0)2-, -S-S-, an aryl group, and a hetcroaryl group; 10 R 7 is selected from the group consisting ofCI. 3 alkyl, C2 -3 alkenyl, and H;
N R 8 is selected from the group consisting ofC3. 6 carbocycle and heterocycle; R 9 is selected from the group consisting of H, CN, NO2 , C1 - alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2, C 2 - 6 alkenyl, C3. 6 carbocycle and heterocycle; each R is independently selected from the group consisting ofC1 3 alkyl, C2 -3 alkenyl, 15 and H; each R' is independently selected from the group consisting of CI.1 8 alkyl, C2. 18 alkenyl, -R*YR", -YR", and H; each R" is independently selected from the group consistingofC .3 14 alkyl and C .3 14
alkenyl; 20 each R* is independently selected from the group consistingofC 1 2 alkyl and C 2- 12 alkenyl; each Y is independently a Cj carbocycle; each X is independently selected from the group consisting ofF, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, I1, 12, and 13, 25 or Salts or Isomers thereof. In some embodiments, another subset of compounds of Formula (X) includes those in which R, is selected from the group consistingof C5 . 30 alkyl, C5 - 2 0 alkenyl, -R*YR", -YR", and -R"M'R'; 30 R 2 and R3 are independently selected from the group consisting of H, C1 i4 alkyl, C 2-14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
R4 is selected from the group consisting of a C 3 .6 carbocycle, -(CH 2 )nQ, -(CH 2)nCHQR, -CHQR, -CQ(R) 2, and unsubstituted Ca alkyl, where Q is selected from a C 3.- carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, 0, and S, 5 OR, -O(CH 2).N(R) 2, -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2, -CN, -C(O)N(R) 2
, -N(R)C(O)R, -N(R)S(O) 2R, -N(R)C(O)N(R) 2, -N(R)C(S)N(R) 2, -CRN(R) 2C(O)OR, -N(R)R8
, -O(CH 2)nOR, -N(R)C(=NR 9)N(R) 2, -N(R)C(=CHR 9)N(R) 2, -OC(O)N(R) 2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O) 2R, -N(OR)C(O)OR, -N(OR)C(O)N(R) 2, -N(OR)C(S)N(R) 2
, N 10 -N(OR)C(=NR 9)N(R) 2, -N(OR)C(=CHR 9)N(R) 2, -C(=NR9)R, -C(O)N(R)OR, and N -C(=NR 9)N(R) 2, and each n is independently selected from 1, 2, 3, 4, and 5; each R 5 is independently selected from the group consisting of C alkyl, C 2 -3alkenyl, and H; each R 6 is independently selected from the group consisting ofC.3 alkyl, C 2 .3 alkenyl, 15 and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(O-I)-, -P(O)(OR')O-, -S(O) 2-, -S-S-, an aryl group, and a heteroaryl group; R 7 is selected from the group consisting of C1 3 alkyl, C 2 -3 alkenyl, and H; 20 Rs is selected from the group consisting of C 3 -6carbocycle and heterocycle; R 9 is selected from the group consisting of H, CN, NO 2 , Ci. 6 alkyl, -OR, -S(O) 2 R,
-S(O) 2N(R) 2, C2 6alkenyl, C3 2carbocycle and heterncycle: each R is independently selected from the group consistingofC3 alkyl, C2 . 3 alkenyl, and H; 25 each R' is independently selected from the group consistingofCI1 8 alkyl, C2 18
alkenyl, -R*YR", -YR", and H; each R" is independently selected from the group consistingof C.31 4 alkyl and C 314 alkenyl; each R* is independently selected from the group consisting ofC 12 alkyl and C2 1 2 30 alkenyl; each Y is independently aC3. bcarbocyclc;
each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
(N2 or salts or isomers thereof. In some embodiments, another subset of compounds of Formula (X) includes those in which R, is selected from the group consistingof C 5 .30 alkyl, C5 -20 alkenyl, -R*YR", -YR", 5 and -R"M'R'; R 2 and R 3 are independently selected from the group consisting of-, C 21 4 alkyl, C2 14
alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3, together with the atom to which they are attached, form a heterocycle or carbocycle; N R4 is -(CH 2 )Q or -(CH )nCHQR, 2 where Q is -N(R)2, and n is selected from 3, 4, and 10 5;
N each R 5 is independently selected from the group consistingof C1 . 3 alkyl, C 2-3 alkenyl, and H;
each R 6 is independently selected from the group consisting of C. 3 alkyl, C 2-3 alkenyl, and 11; 5 M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(0) 2 -, -S-S-, an aryl group, and a heteroaryl group; R 7 is selected from the group consisting of C. 3 alkyl, C2-3 alkenyl, and H;
each R is independently selected from the group consistingof C. 3 alkyl, C 2-3.alkenyl,
20 and H;
each R' is independently selected from the group consisting of C 18 alkyl, C 18
alkenyl, -R*YR", -YR", and H: each R" is independently selected from the group consistingof C 314 alkyl and C 3 14
alkenyl; 25 each R* is independently selected from the group consistingof C1 2 alkyl and C 12
alkenyl; each Y is independently a C 3. 6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and 1; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, 30 or salts or isomers thereof.
In some embodiments, another subset of compounds of Formula (X) includes those in which
R, is selected from the group consistingofC 5 .30 alkyl, C- 20 alkenyl, -R*YR",-YR", and -R"M'R'; R2 and R 3 are independently selected from the group consistingof C4 alkyl, C 2 - 14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3, together with the atom to which they are 5 attached, form a heterocycle or carbocycle; R4 is selected from the group consistingof -(CH 2)nQ, -(CH 2)nCHQR, -CHQR, and -CQ(R) 2 , where Q is -N(R) 2, and n is selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting ofC1 . 3 alkyl, C 2 -3alkenyl, NI and H; N 10 each R 6 is independently selected from the group consisting ofCI. 3 alkyl, C 2. 3 alkenyl, NI and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2 -, -S-S-, an aryl group, and a heteroaryl group; 15 R7 is selected from the group consisting ofC1 . 3 alkyl, C2 -3 alkenyl, and H; each R is independently selected from the group consisting ofC3 alkyl, C 2 .3 alkenyl, and H; each R' is independently selected from the group consistingofC 18 alkyl, C .21 8 alkenyl, -R*YR", -YR", and H; 20 each R" is independently selected from the group consistingof C.3 14 alkyl and C 3 14
alkenyl; each R* is independently selected from the group consistingofCM2 alcyl and C 1 2
alkenyl; each Y is independently a C 3. 6 carbocycle; 25 each X is independently selected from the group consisting of F, Cl, Br, and 1; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof. In some embodiments, a subset of compounds of Formula (X) includes those of Formula (XA): M1'R' R2 R4N M
30 R3 (XA), or a salt or isomer thereof, wherein I is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M, is a bond or M'; R 4 is unsubstituted C3 alkyl, or -(CI- 2 )nQ, in which Q is OH, -NHC(S)N(R) 2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O) 2R, -N(R)R8
, -NHC(=NR 9)N(R) 2, -NHC(=CHR 9)N(R) 2, -OC(O)N(R) 2, -N(R)C(O)OR, heteroaryl or 5 heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -S-S-, an aryl group, and a heteroaryl group; and R 2 and R 3 are independently selected from the group consisting of H, C 1 4 alkyl, and C 2. 14 alkenyl. In some embodiments, a subset of compounds of Formula (X) includes those of Formula (XI): N M1 -R'
R4'N R2 M
10 R3 (II) or a salt or isomer thereof, wherein I is selected from 1, 2, 3, 4, and 5; M, is a bond or M'; R4 is unsubstituted C 3 alkyl, or -(CH 2)nQ, in which n is 2, 3, or 4, and Q is OH, -NC(S)N(R) 2, -NHC(O)N(R) 2
, -N(R)C(O)R, -N(R)S(O) 2R, -N(R)Rs, -NHC(=NR 9)N(R) 2, -NHC(=CHR 9)N(R) 2, -OC(O)N(R) 2, -N(R)C(O)OR, heteroaryl or 15 heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -S-S-, an aryl group, and a heteroaryl group; and R 2 and R 3 are independently selected from the group consisting of H, C 1 4 alkyl, and C 2 .14 alkenyl. In some embodiments, a subset of compounds of Formula (X) includes those of Formula (Xla), (XIb), (XIc), or (XIe): 0
20 0 (Xla),
0
01'O0 (Xlb),
R"N R4'N 0 (XIc), or 0
04'0O(XIe),
or a salt or isomer thereof, wherein R4 is as described herein. In some embodiments, a subset of compounds of Formula (X) includes those of Formula (Xld):
HO n N R5 R3
0 R2 (lid),
or a salt or isomer therco I, wherein n is 2, 3, or 4; and m, R', R", and R2 through R6 are as described 5 . 14 alkyl herein. For example, each of R2 and R 3 may be independently selected &om the group consistingof C and C 51 4 alkenyl. In some embodiments, a subset of compounds of Formula (X) includes those of Formula (Xa), (Xb), (XIc), or (Xle): 0
O4'0O(Xla), 0
O R4'N
0 0 (Xlb),
R4'N0 O (Xic), or 0
N0 R4N O 00N (X~c), or a salt or isomer thereof, wherein R 4 is as described herein. In some embodiments, a subset ofcompounds ofFormula (X) includes those ofFormula (Xd):
O O1R'
',R" HO n N (R 5 R3
0 R2 (XId), or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R', R", and R 2 through R6 are as described herein. For example, each ofR2 and R 3 maybe independently selected from the group consistingofC52 4 alkyl and C5 1 4 alkenyl. In some embodiments, the compound ofFormula (X) is selected from the group consisting of:
0 O (Compound 500),
0 0 (Compound 501),
HO~N
00 (Compound 502),
0 0 (Compound 503), cd HO N
O O = (Compound 504),
O (Compound 505), HO N
H NO (Compound 506),
N 0
N NO (Compound 507),
5 0O (Compound 508), O
HO 00 0 (Compound 508), N
HO U' O' O(Compound 509), 0
HO ' O (Compound 511), cd O
H N O (Compound 512), 0
o 0 (Compound 513). 0
0 0 (Compound 514),
O O (Compound 515), 0
5 0 O (Compound 516), N-0
N N 0
0 (Compound 517), N-N
0 N
0 (Compound 518),
114I
ci 0
cd Hl 0,,, c~OK=
O 0( (Compound520)
0
H0 N
0 0( (Compound519 ), 0
& H00 0 (Comp (ound522). 0)
NC~~
50 0 = (Comod523), 1) 0
0 0 (Compound 524), cd
0 H HO (Compound 525), 0
0 0 (Compound 526), 0
0 O (Compound 527). 0
HO~~~O H O,- N
0 0 (Compound 528),
0
HO~~O
5 0 O (Compound 529), 0
(Compound 530),
o N O (Compound 531), 0
HO ~~NO H0 N0 (Compound 532), cd
O= (Compound 533), 0I 0
0 O (Compound 534), O
0 N HO
o O (Compound 535),
0
H O (Compound 536), 0
5 0 0 (Compound 537), 0
H O 0 N N
0 0 (Compound 538), 0
H Ny
N0 0 (Compound 539), 0
00 (Compound 540),
H H O N yN NO
0 0 (Compound 541), 0
0 N0N (Compound 542),
O0
0 0 (Compound 543), 0 H2 N
0 0 O (Compound 544),
N NU (Compound 545),
H NH 2 N- 0 N
0 O (Compound 546),
H0 O (Compound 547),
0
0
0H0 (Compound 548),
H O,- N 0 O (Compound 549),
H N(Compound 550),
HOO (Compound 55 1),
0 0= (Compound 552),
0
HO~~~O HO' N
O O (Compound 553),
0
o 0 (Compound 554),
0
0 0N (Compound 555),
O'C H O N0 (Compound 556),
OIO(Compound 557), HO~~O 0
0 O (Compound 558),
HO~SN 0
0
o 0 (Compound 559),
0
0 0O (Compound 560), and
0 O (Compound 561).
[00011 in further embodiments, the compound of Formula (X) is selected from the group consisting of: 0
O(m n6 HO N0 (Compound 562),
HO N0 0 (Compound 563), and
0
HOO H0N
0 0 (Compound 564).
in some embodiments, the compound of Formula (X) is selected from the group consisting of: Cl~ 0 0
0 (Compound 565), HO N 0 0
o (Compound 566), 0
HO0 N O 0 0 0
o (Compound 567),
HO -NNO 0 0 0 (Compound 568),
0O (Comnpound %9),
0(Compound 570), c-I HO N 0O
0
(Compound 571),
~N O (Compound 572),
0OC (Compound 573), 0
O (Compound 574),
0 0=(Compound 755),
0 (Compound 576),
0 (Compound 577),
o (Compound 578),
HO N 0
0 (Compound 579),
0 Nl O 0
(Compound 580),
O 0
O0 (Compound 581), HO,-- N 0
0
5 (Compound 583), 0 0
(Compound 584),
0 0
(Compound 585), HO,, N 0
0 (Compound 586),
H0 Cl 0
(Compound 587), 'I HO N 0
ClO 0 0
O (Compound 588),
0
0 (Compound 589),
0 (Compound 590),
0
0 HO0 N O 50 (Compound 591),
~O~c
0
o 0 (Compound 592), - N 0
0 (Compound 593),
0
0 (Compound 5 94),
-~ 0 cd MeOj~
0 ~(Compound 595),
0
0 (Compound 596),
0
0 0= (Compound 597),
0
0,
001 (Compound 599), 0 X-
NN 0 K0
0 (Compound 600), N
NN K 0
0 (Compound 601),
N MeO N MO 0
O O(Compound 602).
0 ON 0 N 0
(Compound 603), cnI
0
O (Compound 604),
0
O (Compound 605), NH 2
OH 0
5 0 (Compound 606), F N F O O
0 (Compound 607), 0
H Of 0 0 (Compound 609),
WO 2017/099823 PCTUS2O16OOO129 126
0
H 0
I (Compound 609), 0
H0
0 (Compound 610),
I H 0
0
H H 0
0 (Compound 612),
0
0
.HNyN,.- N, 0 O 0 (Compound 614), 0
H2N 0
0 (Compound 615),
H 2N / N r
N (Compound 616), 0 H NH 2 O N PN (Compound 617),
0
0
N (Compound 618), 0
HO NO (Compound 619), 0
HO N 0 (Compound 620), 0
0 HO (Compound 621),
0
0 (Compound 622),
0 0 0- =(Compound 623).
S N 0 0
O (Compound 624), 0
HO NO 0 (Compound 625),
ClNO 0
0 (Compound 626),
HON 0
O O (Compound 627), 0
0
(Compound 628), HO
NN 1 0
0 (Compound 629),
HO INN 0
0 (Compound 630),
00 0 0 N0 (Compound 631),
0 0
(Compound 632), 0
0 HO N O 11
(Compound 633), O HO -''N~
0
0 (Compound 634),
0 O' (Compound 636),
0 O^" (Compound 636),
0 HO N (Compound 637),
0
HO0 (N
0 O (Compound 638), cd HOO o O (Compound 639), 0
0 O (Compound 640),
Nl O
0 O (Compound 641), 0 0~
0 0 (Compound 642), 0
5 0 (Compound 643), 0
0 N (Compound 644),
HO N O 0
0 (Cuinpuund 645),
0O
0 (Compound 646),
HO 0
0 (Compound 647),
0
0
O0 (Compound 648),
0 (Compound 649),
N0 0
0 (Compound 650), 0 HO
O (Compound 651),
K 0
(Compound 652),
HO N 0
(Compound 653),
HO -N 0
0 OI (Compound 654),
0
HO N 0 (Compound 655),
HO 0
O (Compound 656),
HO NO 0
O (Compound 657),
HON 0
O (Compound 658),
HO 0 N
O0 Ayo" = (Compound 659),
0Z
O HO N 0 0 M (Compound 660), 0 HO NO
0 (Compound 661), 0
O HON (Compound 662), 00
66)
HO N 0
5 O 0-(Compound 664),
1 101
0
0 (Compound 665),
WO 2017/099823 PCTUS2O16OOO129 134
HO~~~0
0I (Compound 666),
0
(~10 (Compound 667),
Nl NA 'N ltN"'N 0 IH L 0 0- ~ ~
O (Compound 668), 0 0 N 0
0 (Compound 669).
Q2 N,
N (0 H H 0 NN~
0 (Compound 670), OH
0 (Compound 67 1)
0
O = (Compound 672),
WO 2017/099823 PCTUS2O16OOO129 135
00
I 0
0 (Compound 673), 0
0
Cl0 (Compound 674),
c~K1 0
N O0k 'N H 0
0 (Compound 675),
0
N N 0
0 (Compound 676),
0, 0
0 (Compound 677),
0 0 H 0
0 (Compound 678),
NU 0
0 (Compound 679),
H~NHO -'NH 0"0
0 'I (Compound 680), 0
Cl 0 N'J "
(Compound 681),
N N HN H 0 OC
0 (Compound 682),
0
(COiMpuuid 683),
0 H 0
0 (Compound 684),
0
0
HO N- t(Compound 685),
N 0
0 (Compound 686),
0 O
OdO = (Compound 687), 0 H0 N0
0 (Compound 688),
0
0 (Compound 689),
0
0 (Compound 690),
H O'-N-O'O 0 Qo 0
0 (Compound 693),
N 00 HO HO
00 HN O O
0
0 (Compound 694),
WO 2017/099823 PCTUS2O16OOO129 138
00 (N N0
0 ~(Compound 695), 0 Nl N 0
Cl0 (Compound 696),
I 0 H 0,
0 (Compound 697).
0 N- - 0 H0
0 (Compound 698), 0
0
50 (Compound 699), 02N, N
N N ~N0 H H
0 (Compound 700), 0 0
0
0 (Compound 701),
WO 2017/099823 PCTUS2O16OOO129 139
00
0I (Compound 702), 0
0
Cl0 (Compound 703),
OH 0
O (Compound 704), 0
O 0
O (Compound 705), 0 11
OH 0
0 (Compound 706), NH H 2N N ""N N H 0
0
0 (Compound 707),
WO 2017/099823 PCTUS2O16OOO129 140
02N, N JIN N0 I H 0
'I0 (Compound 709),
UN N ~'~ H H 0
0 (Compound 710),
N N'j, 0 I H 0
0 (Compound 71 1),
0SN
H H 0
0 (Compound 712),
NN ' N~ H0
50 (Compound 713),
0,0 ~0
0 (Compound 714),
0 (Compound 715),
WO 2017/099823 PCTUS2O16OOO129 141
HCl 0
0 0-( =(Compound 716),
N 00 00 HO- 0 Cl 0(Compound 717), N0
0l (Compound 718), H 2N /0p
0
O (Compound71), H 2N, N 0 0~
50 (Compound 722J), H 0
00
0 ~(Compound 723),
N NO 0
0 0
O (Compound 724), H HO'N N0O Cl0 0
0 (Compound 725), H O' N N0
N O O (Compound 726),
0 HO'N 0 N
o (Compound 727),
'N O 0 00
0 (Compound 728).
o (Compound 729),
0 (Compound 730), and salts and isomers thereof. In some embodiments, a nanoparticle comprises the following compound:
NI 0
(Compound 73 1) or salts and isomers thereof. In some embodiments, the disclosure features a nanoparticle composition including a lipid component comprising a compound as described herein (e.g., a compound according to Formula (X), (XA), (XI), (Xia), (XIb), (Xlc), (XId) or (XIe)).
Non-cationic lipids, including non-cationic helper lipids The lipid component of the nanoparticle may include any neutral and/or non-cationic lipid (e.g., lipids that are neutral or non-cationic lipid at physiological pH). Non-cationic lipid lipids may include, but are not limited to, fatty acids, glycerolipids, and prenol lipids. In certain embodiments, the non-cationic lipid is a fatty acid. The fatty acid may be saturated or unsaturated. Examples of unsaturated fatty acids include, but are not limited to, myristoleic acid, palmitoleic acid sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linoelaidic acid arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexanoic acid, or any cis/trans double-bond isomers thereof. In certain embodiments, the lipid is oleic acid. In certain embodiments, the lipid is an isomer of oleic acid (e.g., the double bond is in a different location along the aliphatic chain relative to oleic acid). In certain embodiments, the lipid is an analog of oleic acid (e.g., the aliphatic chain is 1-10 carbons longer or 1-10 carbons shorter than the aliphatic chain of oleic acid). Examples of saturated fatty acids include, but are not limited to, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, and cerotic acid. In certain embodiments, the non-cationic lipid is a glycine derivative of a fatty acid (e.g., N-palitoylglycine or N-oleoglycine) In certain embodiments, the non-cationic lipid is a glycerolipid (e.g., monoglyceride, diglyceride, triglyceride). In certain embodiments, the non-cationic lipid is a monoglyceride. In certain embodiments, the non-cationic lipid is a diglyceride. In certain embodiments, the non-cationic lipid is a triglyceride. In certain embodiments, the non-cationic lipid comprises a sugar moeity (e.g., saccharide, disaccharide, polysaccharide). Examples of non-cationic lipids include, but are not limited to, the following:
(Cmpd183)
l 0
Cl (Cmpd3125) 00 HOkl
00 HO N
50 0
0 HO N
0 HOt"
0 HO-I- N
5 H 0
HO0 N
0
0
0
~0-NN0'OH 10 H3lC0 H 0 O 0 OH, 0
0 0
OH OH OH 0 OH OH ,and
N0
Examples of non-cationic lipids comprising sugars include, but are not limited to the following: HO HOH OHO
C5
HC~'
OH , and 0I0
10
0 o o o
The replacement with adifferent zwitterionic group is depicted in FIG. 73. In certain embodiments, the different zwitterionic group is not aphosphocholine group. Inlcertain embodiments,anon-cationiclipidusefulinthepresentinventionisa compound ofFormula (II). Provided herein are compounds of Formula (II):
e (II), or a salts thereof, wherein: Z is a zwitterionic moiety, 0 N~ P wherein the zwitterionic moiety is not of the formula: 0 m is0,1,2,3,4,5,6,7,8,9,or 10;
L-R 2 (R 2 )p A is of the formula: or each instance of L2 is independently a bond or optionally substituted C1 .6 alkylene, wherein one methylene unit of the optionally substituted C 1.6 alkylene is optionally replaced with -0-, -N(RN)-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C(O)0-, -OC(O)-, OC(O)0-, -OC(O)N(RN)-, -NRNC(0)0-, or -NRNC(O)N(RN)_; each instance of R 2 is independently optionally substituted C- 30 alkyl, optionally substituted C 30 alkenyl, or optionally substituted CI 3 0 alkynyl; optionally wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -0-, -S-, -C(O)-, -C(O)N(RN) NRNC(O)-N(RN), -C(0)O-, -OC(O)-, -OC()0-, -OC(O)N(R)-, NRNC(O)0-, -C(O)S-, -SC(O)-, -C(=NR)-, -C(=NRN)N(R'N)-, -NRNC(=NRN) NRNC( =NR)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-,-S()-, OS(O)-, -S(0)O-, -OS(O)0-, -OS(O) 2-, -S(O) 20-, -OS(0) 2 0-, -N(RN)S(0)_, S(O)N(RN)-, -N(R N)S (O)N(R N)-, -OS(O)N(RN)-, -N(R N)S(O)O-, -S(0)2-, -N(R N)S (O)2-,
-S(O) 2N(RN)-, -N(RN)S(0) 2N(RN)~, -OS(0) 2N(RN)-, or -N(RN)S(0)20-; each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is I or 2. In certain embodiments, Z is an amino acid or a derivative thereof. In certain embodiments, Z is of one of the following formulae:
N (RN) 3 RN 0 O® (0 N
Cd 0 OlO R )N3
0 RN3N ON)3 O 0- 0 0 O /,
(RN) 3N O (R 3ON NO O O(RN)N (R )3
, 0 (RN) 3N RN (RN) 3N RN embodim Nts a D copon o Formul (N) s of on0fth oloigfomle 0, 0 ,or 00
0(N) 0'0 wherein R4is hydrogen, optionally substituted alkyl oran oxygen protecting group. In certain
(RN) 3N( RN 0 0 R3O-RYN
0O 0 A NOAO A 90 80NN (N3 0 A (RN OOR Ay ORA 0 ( N 0 DA0 -0 0,' 0 (RN)m N (RN) 3N R S 0l 0 %ROO11 0 6'(6 ONDR) 3 m RoN N^ 00 I-0 N RN 00 (RN 3 AN(N) N(t 0 (R03 RN N Icranembodiments, a compound of Formula (11) is of one of the followingfomle
0( RN )3N RN 00
A0 R
10 0 H R 3NR
or asalt thereof. In certain embodiments, acompound of Formula (11)is ofone of the following formulae:
0 Y0 R2 0 yR 2
(RN) 3NO RN 0 Q®0 N 0" kR '& -O'R2 (ED) 00
0 R2 00 R2 II y 00 0 R 0 0-R.. ON(RN )30/00
0D 0 R' 2 6N(RN2 R 0 R 0fl
00 NR) 3
0 R2 0 R2
N 0 0 0 (R) 3 RN 00
R 3N (RN) (B N P' -K0OR O) 2 Go' NKR OR 0 0 0
0 R2 0 R2
0o 0R 3 RN 0 (RN)N0RN 00
2 1 0
0 Go 0
OyR (DOyH
orasaltth Rf RN 0 FoeapeIcraiebo)ensacmoudfJrul(I snefh following:N" JO e Nme3 lp, 0 MI 0 0 00
NqMe3
E) 0
H 0 3 10 0
00 '
0
0.N 10 .0
Cd~ NH 3 OeNH 0 0 00 0 O
00 0
00 0 O
eQ
H3 O0 H
HN3 0 H N "H 0 H 3N H 0 0 0
or salts thereof.
Oleic Acid Analogs As described herein, non-cationic lipids useful in the present invention include analogs of oleic acid. As described herein, an oleic acid analog can comprise a modified olce acid tail, a modified carboxylic acid moiety, or both. In certain embodiments, an analog of oleic acid is a compound of Formula (IV). Provided herein are compounds of Formula (IV): 0 HO R4
(IV), or a salt thereof, wherein: R4 is optionally substituted,CIO 40 alkyl; optionally substituted, CIO-o alkenyl; 4 optionally substituted,CIO-40 alkynyl; wherein at least one methylene group of R is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN) , -0-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, OC(O)0-, -OC(O)N(RN)-, -NRNC()_,_C(O)S_ -SC()-, C(=NRN _
C(=NRN)N(RN), -NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN' NRNC(S)-, -NRNC(S)N(RN)-, -S(O)-, -OS(O)-, -S(0)0-, -OS(O)O-, -OS(O) 2 -,
S(0)2 0-, -OS(0) 2 0-,-N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN._ N(RN)S(O)0, -S(0)2-, -N(RN)S(O) 2 -, -S(0) 2N(RN)-, -N(RN)S(0) 2 N(R N)
OS(O) 2N(RN)-, or -N(R N)S(0) 2 0-; and each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In certain embodiments, the compound of Formula (IV) is one of the following: HO
0 HO O
(Cmpd148) HO
O 0 (Cmpd149) 0 HO
0 HO
(Cmpd159),
or salts thereof.
In certain embodiments, an oleic acid analog is a compound wherein the carboxylic acid moiety of oleic acid replaced by a different group. In certain embodiments, an oleic acid analog useful in the present invention is one of the following: H 'I'I0lN 0 0 (Cmpdl57) H 0 N
(Cmpdl58) H N
0 D H CF 3CO2 H3N S,N
0Yb0 | H N N
00 H N
0
0
H0 H3S
or salts thereof. In certain embodiments, an oleic acid analog useful in the present invention is:
OH NaO
0
PEGylated lipids The lipid component of a nanoparticle composition may include one or more PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG DPPC, or a PEG-DSPE lipid. In some embodiments the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure: 0 0
0
In one embodiment, PEG lipids useful in the present invention can be PEGylated
lipids described in International Publication No. W02012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also referred to herein as "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodimetns, a PEG-OiH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.
PEG and PEG-OFT Lipids In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (III). Provided herein are compounds of Formula (III): R3 Li-DA or salts thereof, wherein: R 3 is -OR'; Ro is hydrogen, optionally substituted alkyl, or an oxygen protecting group; 5 r is an integer between I and 100, inclusive; Ll is optionally substituted Ci-o alkylene, wherein at least one methylene of the optionally substituted C 1io alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, N optionally substituted heteroarylene, -0-, -N(RN)-, -S-, -C(O)-, -C(O)N(RN)_, N o NRNC(O)-, -C(O)O-, -OC(O)-, -OC(0)O-, -OC(O)N(RN)-, -NRNC()-, or Nl NRNC(O)N(RN D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is0,1,2,3,4,5,6,7,8,9,or10;
B (R 2)p L 2 -R 2 or \ ; 15 A is of the formula: each instance of L2 is independently a bond or optionally substituted CI. alkylene, wherein one methylene unit of the optionally substituted CI. alkylene is optionally replaced with 0 , N(RN)-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC()_ __C(0)0_,-OC(0)-,
OC(Q)O-. -OC(0)N(RN). -NRNC(0)-.or -NRNC(O)N(RN)-; 20 each instance of R2 is independently optionally substituted C- 30 alkyl, optionally substituted CI_3o alkenyl, or optionally substituted C- 3 0 alkynyl; optionally wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -O-, -S-, -C(O)-, -C(O)N(RN_,_ 25 NRNC(O)-, -NRC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(0)O-, -OC(O)N(RN)-, NRNC(O)O-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=NRN)- NRNC(=NR)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN)-,-(0)_, OS(O)-, -S(0)0-, -OS(O)O-, -OS(0)2-, -S(O)20-, -OS(0)20-, -N(RN)S(0)-, S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN)-, -N(RN)S(0)0-, -S(O)- -N(RN)S(O)2-,
30 -S(0) 2N(RN)-, -N(RN)S(0) 2N(RN)-, -OS(0) 2N(RN)-, or -N(RN)S(0)20-; each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;
Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is I or 2. 3 In certain embodiments, the compound of Fomula (III) is a PEG-OH lipid (i.e., R is -OR°, and Ro is hydrogen). In certain embodiments, the compound of Formula (II) is of Formula (III-OH): HO \-LI-D A
(III-OH), or a salt thereof. In certain embodiments, D is a moiety obtained by click chemistry (e.g., triazole). In certain embodiments, the compound of Formila (III) is of Formula (III-a-1) or (II-a-2):
R3 L N R3 O N
(III-a-1) (III-a-2), or a salt thereof. In certain embodiments, the compound of Formula (III) is of one of the following formulae:
0 N==N L2 2 N N L2 2 R3 ' r "'' L ,RI R3 N11_A L?, 2 ~N),R ,R2o;~N.2{U
O N=N L2 R2 O N N L 2 HO ANO. L' HOL2
or a salt thereof, wherein s is 0, 1, 2, 3, 4, 5, 6, 7, 8 9, or 10. In certain embodiments, the compound of Formula (III) is of one of the following formulae: 0 R2 O R2
O1 N=N 00 0 N 0 R3 ON O R2 R3 O s 0 R2
N 0 R2 0 R2
0 N=N 00 0 N!N 0 HO )O %}O 0" 'R2 HO o O R2
or a salt thereof In certain embodiments, a compound of Formula (III) is of one of the following formulae: R2 O R2 O
N N OR N R2 0 N/ N O R2 O
5 R3/ R3
OR2 2 OOR 0 N N R2 N:=N 0 0 O R0 N
or a salt therenf In certain embodiments, a compound of Formula (III) is of one of the following formulae:
N N ,0
10 (Cmpd394), 0
(Cmpd396),
N=N 0 0 __ /0
(Cmpd395),
N==N 0 N
O0
(Cmpd397),
or a salt thereof. In certain embodiments, D is a moiety cleavable under physiological conditions (e.g., ester, amide, carbonate, carbamate, urea). In certain embodiments, a compound ofFormula (III) is of Formula (III-b-1) or (III-b-2): 3 0 R3 OL ,O 1 m R3 O L1,O m- A
(III-b-i) (III-b-2), or a salt thereof. In certain embodiments, a compound of Formula (III) is of Formula (I-b-1-O1) or (111-b 2-OH): 0 >OL,0 _A 0 HO+ HrL O m HO OyL O mA 0r (III-b-1-OH) (III-b-2-OH), or a salt thereof. In certain embodiments, the compound of Formula (III) is of one of the following formulae:
L2'R2 21R2
R3 O L Og L2 R3 LO1 2'
0r
, R2 L2'R2 R 0 L2 HO OL O L H L L'R2
0 or a salt thereof. In certain embodiments, a compound of Formula (III) is of one of the following formulae: OyR 2 O R 2
0/ R3 O L O O AR,2 R3LL O R 0 0 5 0 0 R2 O R2
00 0 00 HO }..LV O O R2 HO O R2 LJ 0r or a salt thereof. In certain embodiments, a compound of Formula (III) is of one of the following formulae: 0 R2 OyR2 0 n 2 3 '0 R R O O R2 0 ,r
O R2 OyR 2
HO 0 O R2 HO OO O R2 0r
or a salt thereof. In certain embodiments, a compound of Formula (III) is of one of the following formulae:
O0 0 00
0+- 0 0a
or salts thereof. In certain embodiments, a PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (V). Provided herein are compounds of Formula (V): 0 3 R O R5
(V), CMl or a salts thereof, wherein: R 3 is-OR0 ; Ro is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between I and 100, inclusive; R 5 is optionally substituted Clo-o alkyl, optionally substituted Co.40 alkenyl, or optionally substituted CIo 4 0alkynyl; and optionally one or more methylene groups of R5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(R)N-,-0-,, C(O)-,-C(O)N(R_ NRC(O),-NRC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)0-, 1 OC(O)N(R")-, -NR"C(0)O-, -C(O)S-, -SC(O)-, -C(=NR)-, -C(=NR )N(R"J)-, NRNC(=NRN)-, -NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)_, NRNC(S)N(RN)-, -S(O)-, -OS(O)-, -S(0)0-, -OS(O)O-, -OS(0) 2-, -S(0)20-, OS(O) 20-, -N(RN)S(O)-, -S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN)- N(RN)S()_ -S(0) 2-, -N(RN)S() 2 -, -S() 2 N(RN)-, -N(RN)S(0) 2N(RN) OS(O)2N(RN)-, or-N(RN)S(O) 20-;and each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In certain embodiments, the compound ofFormula (V) is of Formula (V-Ol): 0 HO -O R5
(V-OH), or a salt thereof.
In certain embodiments, a compound of Formula (V) is of one of the following formulae:
0
N 5 (Cmpd400), 0 10 O
(Cmpd4OI1), Or .0 (Cmpd4021),
(Cmpd4O1), 0
100
'H O, 00 0 r H HO N
15HO N
or a salt thereof. Numerous LNP formulations having different PEG-lipids were prepared and tested for activity, as demonstrated in the Examples included below.
20 Phospholipids,includinghelperphospholipids Phospholipids, as defined herein, are any lipids that comprise a phosphate group. Phospholipids are a subset of non-cationic lipids. The lipid component of a nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated
CN lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic 5 acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species including natural N 10 species with modifications and substitutions including branching, oxidation, cyclization, and N alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions 15 may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful cuunponent such as a Largeting or imaging moiety (e.g., a dye). Eachpossibility represents a separate embodiment of the present invention. Phospholipids useful in the compositions and methods may be selected from the non 20 limiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), I,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2 dioleoyl sn glycero 3 phosphocholine (DOPC), 25 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), I-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), I-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 30 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, I,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, M 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamie, I,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 5I 1,2-diarahidnoyl-sn-glycero-3-phosphoethanolamine, 5 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, I,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(-glycerol) sodium salt (DOPG), and sphingomyelin. Each possibility represents a separate embodiment of the present invention. In some embodiments, a nanoparticle composition includes DSPC. In certain 10 embodiments, a nanoparticle composition includes DOPE. In some embodiments, a N nanoparticle composition includes both DSPC and DOPE. Examples of phospholipids include, but are not limited to, the following:
0 H 0
O 0 O
O 0
.n - O O
O O o
ISd o
200 H0
O 200
H 0
20 0
O 0
0 00
00 O 0 0
0
0 0 I O
N OO0 and
0 00 P, N
In certain embodiments, aphospholipid useful in the present invention is ananalog or variant of DSPC. In certain embodiments, aphospholipid useflulin the present invention is a compound of Formula (1):
ke 0 R1-N 0 10 A / -n 'P' T1r 0 or asalt thereof, wherein: each R'is independently optionally substituted alkyl; or optionally two R1arejoined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R' arejoined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1,2,3,4,5, 6, 7,8, 9,or 10; mis0, 1,2, 3, 4, 5,6,7,8, 9,or 10;
B (R 2)p L2-R2 d A is of the formula: or
each instance of L 2 is independently a bond or optionally substituted C1 6- alkylene, wherein one methylene unit of the optionally substituted C1. 6 alkylene is optionally replaced with -0-,N(RN)-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C(O)O-, -OC(O)-, 5 OC(O)0-, -OC(O)N(RN)-, -NRNC(0)0-, or -NRNC(O)N(RN)-; each instance of R 2 is independently optionally substituted C 30 alkyl, optionally substituted C 30 alkenyl, or optionally substituted CI-30 alkynyl; optionally wherein one or more methylene units of R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene,
10 optionally substituted heteroarylene, -N(RN)-, -0-, -S-, -C(O)-, -C(O)N(RN)-, NRNC(O)-, -NRNC(O)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(RN)_, NRNC(O)0, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN)-, -NRNC(=NR N) NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(RN_,-_(0)_, OS(O)-, -S(0)0-, -OS(0)0-, -OS(0)2-, -S(0) 20-, -OS(0)20-, -N(RN)S(),_
15 S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(RN)-, -N(RN)S(0)0- -S(O) 2 -, -N(RN)S(0) 2-, -S(O) 2N(RN)-, -N(R N)S(0)2N(RN)-, -OS() 2N(R N)-, or -N(RN)S(O) 2 -; each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;
Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, 20 optionally substituted aryl, or optionally substituted heteroaryl; and p is I or 2;
provided that the compound is not of the formula: O R2 10 _ 0 '
O O O R2
wherein each instance of R 2 is independently unsubstituted alkyl, unsubstituted alkenyl, or 25 unsubstituted alkynyl.
Phospholipid Head Modifications In certain embodiments, a phospholipid useful in the present invention comprises a modified phospholipid head (e.g., a modified choline group). In certain embodiments, a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine. For example, in embodiments of Formula (I), at least one of R is not methyl. In certain embodiments, at least one of R is not hydrogen or methyl. In certain embodiments, the compound of Formula (I) is of one of the following formulae:
0 (le 00 D e lo O0 °N A 0 0 AOQ N00 0 A 500
0 OT ~ .00 N 0 10 A ( nO0 A RN 0 0
or a salt thereof, wherein: each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each v is independently 1, 2, or 3. In certain embodiments, the compound of Formula (I) is of one of the following formulae: E) 0 0-N 0 0 0.,
N0 N 010CQ I,)-, X'-Y. C 00
00 0 0 r-N .K0 OA 0 A+ NA 10 0
00 0 o lO 0 N 0 10 A NPO O010 A '(-f'l"P' 1>Tm II >1.f 0 0 1 N 0 01 0 RN O
or a salt thereof. In certain embodiments, a compound of Formula (I) is one of the following: 0
0oE) 0 O 0
(Cmpd150)
0 D 0 N O O 0 (Cmpd160)
0 p '1I 0 0
(Cmpd151)
E)K 00
00
(Cmpd165)
0
Ne 0 0
(Cmpd152)
Co 0
p 0 0 (Cmpd61)
0(
((JmpdI153)
C1l O 0 0
0
0 eo 0 N<NO 00O 0 O
Nl O 0 (Cmpdl55)
OE) 0 N 0
(Cmpd166). or a salt thereof.
PhospholipidCore Modifications 10 In certain embodiments, a compound of Formula (I) is of Formula (I-a): 2 R 0 L 2-R
Ri 1
(I-a), or a salt thereof. In certain embodiments, phospholipids useful in the present invention comprise a 15 modified core (see, e.g., FIG 47B). In certain embodiments, a phospholipid with a modified core described herein is DSPC, or analog thereof, with a modified core structure. For example, in certain embodiments of Formula (I-a), group A is not of the following formula: 0.R 2 .0
0 R2
In certain embodiments, the compound of Formula (I-a) is of one of the following 20 formulae:
02 R2 1 0 \~ 0 CdR e- R'0 K(1TN P11 m R2 R1-N 01-110 R
'I0 0
0- R1 e NRN RD 0 R-N o~o 0 R2 R 1-N 00 / -n P m 0 0 0 RN
c-i 0 R2 R1 ~ N-RNN
R1 00
or asalt thereof. In certain embodiments, acompound of Formula (1)is one of the following:
N o e 0,
00 o0 0
O 0 0
'H 0 0
0e
N -o00 N I H
insalts ther eof. In certain embodiments, a phospholipid useful in the present invention comprises a cyclic moiety in place of the glyceride moiety. In certain embodiments, a phospholipid useful in the present invention is DSPC, or analog thereof, with a cyclic moiety in place ofthe glyceride moiety. In certain embodiments In certain embodiments, the compound of Formula (I) is of Formula (I-b): R1 e \@ 0 B (R 2)p R-N 0 -0 Ri 11
(I-b), or a salt thereof, In certain embodiments, the compound of Formula (I-b) is of Formula (1-b-1):
R1 e 0 )0 P 0 1 R O 010 O (R2)p R -N Ri
(I-b-1), or a salt thereof, wherein: w is 0, 1, 2, or 3. In certain embodiments, the compound of Formula (I-b) is of Formula (I-b-2):
M ~R1 G O R1 0 O O3(R2), R- 0
(I-b-2), or a salt thereof. In certain embodiments, the compound of Formula (I-b) is of Formula (I-b-3):
R eD O 2) 1 R -N 0
R 0 0 (I-b-3), or a salt thereof. In certain embodiments, the compound of Formula (I-b) is of Formula (I-b-4):
R (E) 0 R2 -Nn® Ri , 0 e1O R?( I "ln "
0
is (1-b-4), or a salt thereof. In certain embodiments, the compound of Formula (I-b) is one of the following: 0 0
0
0 O 0
2 01
H3N O O 0 0
or salts thereof.
Phospholipid Tail Modifications Incertainembodiments,aphospholipidusefulinthepresentinventioncomprisesa modifiedtail.In certainembodiments, a phospholipid useful in the present entintion is 5 DSPC, or analog thereof, with a modified tail. As described herein, a "modified tail" may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof. For example, in certain embodiments, the compound of (I) is of Formula (I-a), or a salt thereof, 2 10 wherein at least one instance of R2 is each instance of R is optionally substituted C 3 0 alkyl, 2 N wherein one or more methylene units of R are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -0-, -S-, -C(O)-, -C(O)N(RN-_, NRNC(O)-, -NRNC(O)N(RN)-, -C(0)0-, -OC(O)-, -OC(O)0-, -OC(O)N(RN)-, 15 NReC(O)0-, -C(O)S-, -SC(O)-, -C(=NRN)-, -C(=NRN)N(RN), -NRNC(=NRN)_, NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, NRNC(S)N(RN)-,-()-,
OS(O)-, -S(0)0-, -OS(0)0-, -OS(0)2-, -S(0)20-, -OS(0)20-, -N(R)S(O)-, S(O)N(R N)-, -N (R N)S(O)N(R N)-, -OS(O)N(RN)-, -N(RN)S(0)O-, -S(0)2-, -N(RN) S(O)r-,
-S(O) 2N(RN)-, -N(RN)S(0) 2N(RN)-, -OS(0) 2 N(RN)-, or -N(RN)S(O) 2 0-.
20 In certain embodiments, the compound of Formula (I-a) is of Formula (I-c):
G x~ R1 (D L2 1 \ 0"G |) )x R -N O m L Ri H
(I-c),
or a salt thereof, wherein: each x is independently an integer between 0-30, inclusive; and 25 each instance is G is independently selected from the group consisting of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN)-, -0-, -S-, -C(O)-, -C(O)N(R)-,
NRNC(O)-, -NR NC (O)N(RN)-C(00-, -OC(O)-, -OC(0)0-, -OC(O)N(R)-, NRC(O)0, -C(O)S-, -SC(O)-, -C(=NR N)-, -C(=NR)N N)-, -NR NC(=NR)-,
30 NRNC(=NRN)N(RN)-, -C(S)-, -C(S)N(RN)-, -NRNC(S)-, -NRNC(S)N(R)N,(0), OS(O)-, -S(0)0-, -OS(0)0-, -OS(O)2-, -S(0) 2 0-, -OS(0)20-, -N(RN)S(O)-, -
S(O)N(RN)-, -N(RN)S(O)N(RN)-, -OS(O)N(R (0)2
-S(O) 2N(RN)-, -N(RN)S(0) 2N(RN)-, -OS(O) 2 N(RN)-, or -N(RN)S(0) 2 0-. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the compound of Formula (I-c) is of Formula (I-c-1):
)x
R1 S L2 ) )X R1-NRi-- 0,10 O Om 'L2 Ri 0
(I-e-1), or salt thereof, wherein: each instance of v is independently 1, 2, or 3. In certain embodiments, the compound of Formula (I-c) is of Formula (I--2):
)X R 8 0 L2 )x R 1-NR/ , 01 02 P O m 'L 2 0 (I-c-2), or a salt thereof. In certain embodimenrs, the compound of Formula (I-c) is of the following formula:
0 00 k )
RI-N OO R 0
or a salt thereof. In certain embodiments, the compound of Formula (I-c) is the following: 0 0
N P 0 0
or a salt thereof. In certain embodiments, the compound of Formula (I-c) is of Formula (I-c-3):
Q e 1.2) 0®
(I-c-3), 0
or asalt thereof. In certain embodiments,RIthe compound of Formula (I-c) isof the following formulae: P 0 O2
R 'e0O)O N 1 \ 0 0 R -N 010
or a salt thereof. \ D0 P 1- the compound In certain embodiments, of Formula (I-c) is the following: 0 1' 0 0O
R 0
or a salt thereof. 10 Phiosphocholine Linker Modifications In certain embodiments,0aphospholipid 0 useful in the present0 invention comprises a modified phosphocholine moiety, wherein 1I the alkyl chain linking the quaternary 0 A amine to the phosphoryl group is not ethylene (e.g., nis not 2). Therefore, in certain embodiments, a is phospholipid useful in the present invention is acompound of Formula (I), wherein nisI1,3, 4, 5,6, 7,8, 9,or 10.For example, in certain embodiments, acompound of Formula (I) is of one of the following formulae:
R0 - A R O -Om 0R R R1 0
or a salt thereof. 20 In certain embodiments, a compound ofFormula (I) is oneofthefollowing:
M 00 p 0
0
H30 - -I 0 0 p- 0
0
0
I® 0 0
p 0 0
0 0
0 p11
0 o '0
0 0I
0 (Cmpd 162)
N 0 O P O N,8( N H 0
0"
0 o P N e1 H 0 N Ho c-KI 0 00
D o P- 0 0 11 0 (Cmpdl 54)
0
0 8 '0
0 5 (Cmpdl6)
or salts thereof. Numerous LNP formulations having phospholipids other than DSPC were prepared and tested for activity, as demonstrated in the examples below. Exemplary phospholipids are shown in the Figures, including Figs. 75A, 75D and 75E. The following Table provides a summary of the phospholipids and indicates which examples include data on the phospholipids.
Compound Common Example Formulation Name Name testing lipid Oleic acid OL 23 MC3:OL:Chol:PEG-DMG
Cmpd393 Trialkyl 25 MC3:PC:Chol:PEG-DMG
Ml Cmpd125 Dialkyl 23 MC3:PC:Chol:PEG-DMG
Cmpd-148 OL 25 MC3:OL:Chol:PEG-DMG
Cmpd-149 OL 23 MC3:OL:Chol:PEG-DMG
Cmpd-150 PC 23 MC3:PC:Chol:PEG-DMG
Cmpd-151 PC 23 MC3:PC:Chol:PEG-DMG
Cmpd-152 PC 23 MC3:PC:Chol:PEG-DMG
Cmpd-153 PC 23 MC3:PC:Chol:PEG-DMG
DOPC PC 22 MC3:PC:Chol:PEG-DMG
Oleic acid FA 22 MC3:PC:Chol:PEG-DMG DOCP PC 22 MC3:PC:Chol:PEG-DMG
DOCPe PC 22 MC3:PC:Chol:PEG-DMG
DOPE PE 22 MC3:PC:Chol:PEG-DMG
DOPG PG 22 MC3:PC:Chol:PEG-DMG
DOPA PA 22 MC3:PC:Chol:PEG-DMG
DOPS 0.1% PS 22 MC3:OL:Chol:PEG-DMG
DOPS1% PS 22 MC3:OL:Chol:PEG-DMG
DOPS 1% PS 22 MC3:PC:Chol:PEG-DMG
Cmpd-279 PC 23 MC3:PC:Chol:PEG-DMG
Cmpd-280 PC 23 MC3:PC:Chol:PEG-DMG
Cmpd-281 PC 23 MC3:PC:Chol:PEG-DMG
Cmpd-160 PC 25 MC3:PC:Chol:PEG-DMG
Cmpd-161 PC 25 MC3:PC:Chol:PEG-DMG
Cmpd-162 PC 25 MC3:PC:Chol:PEG-DMG
Cmpd-163 PC 25 MC3:PC:Chol:PEG-DMG
Cmpd-157 OL 25 MC3:OL:Chol:PEG-DMG
Cmpd-158 OL 25 MC3:OL:Chol:PEG-DMG
Cmpd-159 OL 25 MC3:OL:Chol:PEG-DMG
Cmpd-164 PC 25 MC3:PC:Chol:PEG-DMG
Cmpd-165 PC 25 MC3:PC:Chol:PEG-DMG
Cmpd-166 PC 25 MC3:PC:Chol:PEG-DMG
DSPC PC 24 Cmpd18:PC:Chol:PEG-DMG (50:1-:38.5:1.5) DPPC PC 24 Cmpd18:PC:Chol:PEG-DMG (50:1-:38.5:1.5) DMPC PC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:38.5:1.5) SMPC PC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:38.5:1.5) DMPC PC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:38.5:1.5) SPPC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:38.5:1.5) OPPC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:30.5:1.5) PSPC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:38.5:1.5) POPC PC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:38.5:1.5) PLPC PC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:38.5:1.5) PMPC PC 24 Cmpdl8:PC:Cho:PEG-DMG (50:1-:38.5:1.5) MSPC PC 24 Cmpdl8:PC:Chol:PEG-DMG (50:1-:38.5:1.5) Steric acid CL 24
Oleic Acid OL 24
Linoleic Acid CL 24
Structural lipids The lipid component of a nanoparticle composition may include one or more structural lipids. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group 5 consisting of, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids,
phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a
sterol. As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. N In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural Ni10 lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In N certain embodiments, the structural lipid is alpha-tocopherol. Examples of structural lipids include, but are not limited to, the following:
0 HF H 0 Hand
15
Chemical Definitions Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of 20 the Elements, CAS version, Handbook of Chemistry and Physics, 7 5th Ed., inside cover, and
specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are
described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;
Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons,
25 Inc., New York, 2001; Larock, Comprehensive Organic Transformations,VCH Publishers,
Inc., New York, 1989; and Carruthers, Some Modern Methods of'OrganicSynhesis, 3 Edition, Cambridge University Press, Cambridge, 1987. Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers. For 5 example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can
be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; 10 or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et N al., Enantiomers, Racemates andResolutions (Wiley Interscience, New York, 1981); Wilen
ei al., Tetrahedron 33:2725 (1977); Eliel, E.L.Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H., Tables of Resolving Agents andOptical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).The 15 invention additionally encompasses compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
In a formula, ~~ is a single bond where the stereochemistry of the moieties immediately attached thereto is not specified, --- is absent or a single bond, and or
is a single or double bond. 20 Unless otherwise stated, structures depicted herein are also meant to include
compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by 9 2 C with "C or '4C deuterium or tritium, replacement of F with "F, or the replacement of
are within the scope of the disclosure. Such compounds are useful, for example, as analytical 25 tools or probes in biological assays. When a range of values is listed, it is intended to encompass each value and sub-range
within the range. For example "C 1 -6 alkyl" is intended to encompass, C 1, C 2 , C 3 , C 4, C 5 , C6 ,
C 1-6 , C 1 -5 , C 1 4 , C1 3 , C1 2 , C 2 .6 , C 2-5 , C 24 , C 2 .3 , C 3 -6 , C 3. 5 , C34, C 4. 6 , C 4 .5 , and C 5 .6 alkyl. The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. 30 Likewise, the term "heteroaliphatic" refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups. The term "alkyl" refers to a radical of a straight-chain or branched saturated
hydrocarbon group having from I to 10 carbon atoms ("C 1. 10 alkyl"). In some embodiments, an alkyl group has I to 9 carbon atoms ("C-9 alkyl"). In some embodiments, an alkyl group has 1 to 8 carbon atoms ("C.s alkyl"). In some embodiments, an alkyl group has I to 7 carbon atoms ("C7 alkyl"). In some embodiments, an alkyl group has I to 6 carbon atoms ("C. 6alkyl"). In some embodiments, an alkyl group has I to 5 carbon atoms ("Cs alkyl"). 5 In some embodiments, an alkyl group has I to 4 carbon atoms ("C4 alkyl"). In some embodiments, an alkyl group has I to 3 carbon atoms ("C3 alkyl"). In some embodiments, an alkyl group has I to 2 carbon atoms ("C2 alkyl"). In some embodiments, an alkyl group has I carbon atom ("Cl alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C 2 . 6 alkyl"). Examples ofC 1 .6 alkyl groups include methyl (C1), ethyl (C 2), propyl N 10 (C 3) (e.g., n-propyl, isopropyl), butyl (C 4 ) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), N pentyl (C 5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C 6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C 7), n octyl (C 8), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") 15 with one or more substituents (e.g., halogen, such as F). In certain embodiments, the alkyl group is an unsubstituted C1 10 alkyl (such as unsubstituted C 1. 6 alkyl, e.g., -CH 3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu),-unsubstituted sec-butyl (sec-Bu), unsubstituted
20 isobutyl (i-Bu)). In certain embodiments, the-alkyl group is a substituted CI.10 alkyl (such as substituted C 1.6 alkyl, e.g., -CF 3, Bn). The term "haloalkyl" is a substituted alkyl group, wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkyl moiety has I to 8 carbon atoms ("Cs haloalkyl"). In 25 some embodiments, the haloalkyl moiety has I to 6 carbon atoms ("C 1 . 6 haloalkyl"). In some embodiments, the haloalkyl moiety has I to 4 carbon atoms ("CI. 4 haloalkyl"). In some embodiments, the haloalkyl moiety has I to 3 carbon atoms ("C.. haloalkyl"). In some embodiments, the haloalkyl moiety has I to 2 carbon atoms ("C2 haloalkyl"). Examples of haloalkyl groups include -CHF 2, -CH 2F, -CF 3, -CH 2CF 3, -CF 2CF3, -CF 2CF 2 CF3 , -CC3, 30 -CFCl 2, -CF 2CI, and the like. The term "heteroalkyl" refers to an alkyl group, which further includes at least one
heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within
(i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from I to 10 carbon atoms and I or more heteroatoms within the parent chain ("heteroCIio alkyl"). In some embodiments, a heteroalkyl group is a saturated group having I to 9 carbon atoms and I or more heteroatoms within the parent chain 5 ("heteroC In some embodiments, a heteroalkyl group is a saturated group having I 19 alkyl").
to 8 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroCis alkyl"). In some embodiments, a heteroalkyl group is a saturated group having I to 7 carbon atoms and 1 or more heteroatoms within the parent chain ("heteroC 1 7 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 1to 6 carbon atoms and I or more heteroatoms 10 within the parent chain ("heteroCI 6 alkyl"). In some embodiments, a heteroalkyl group is a N saturated group having I to 5 carbon atoms and I or 2 heteroatoms within the parent chain ("heteroCI 5 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having I to 4 carbon atoms and Ior 2 heteroatoms within the parent chain ("heteroC4 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having I to 3 carbon atoms and 1 15 heteroatom within the parent chain ("heteroCI3 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having I to 2 carbon atoms and I heteroatom within the parent chain ("heteroCI2 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having I carbon atom and I heteroatom ("heteroC1 alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms 20 within the parent chain ("heteroC 2-6 alkyl"). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an "unsubstituted heteroalkyl") or substituted (a "substituted heteroalkyl") with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC. 1 o alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroCo 10 alkyl.
25 The term "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 2 to 9 carbon atoms ("C 2-9 alkenyl"). In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("C. 8 alkenyl"). In some embodiments, an alkenyl group has 2 to 7 carbon atoms ("C27 alkenyl"). 30 In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("C 2 -6 alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C2 .5 alkenyl"). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C2-4 alkenyl"). In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("C2 .3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). The one or more carbon carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in I-butenyl). Examples of C 24 alkenyl groups include ethenyl (C2 ), 1-propenyl (C 3), 2-propenyl (C3 ), butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), and the like. Examplesof C 2-6 alkenyl groups 5 include the aforementionedC 2-4 alkenyl groups as well as pentenyl (Cs), pentadienyl (C), hexenyl (C 6), and the like. Additional examples of alkenyl include heptenyl (C), octenyl (C 8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an "unsubstituted alkenyl") or substituted (a N "substituted alkenyl") with one or more substituents. In certain embodiments, the alkenyl N i10 group is an unsubstituted C2 10 alkenyl. In certain embodiments, the alkenyl group is a N substitutedC 2 0 alkenyl. In an alkenyl group, a C=C double bond for which the stereochemistry is not specified (e.g., -CH=CHCH 3 or )may be an (E)- or (Z) double bond. The term "heteroalkenyl" refers to an alkenyl group, which further includes at least 15 one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and I or more heteroatoms within the parent chain ("heteroC 2-o alkenyl"). In some embodiments, a heteroalkenyl group 20 has 2 to 9 carbon atoms at least one double bond, and I or more heteroatoms within the parent chain ("heteroC 2 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and I or more heteroatoms within the parent chain ("heteroC 2 .s alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and I or more heteroatoms within the parent chain ("heteroC 2-7 25 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and I or more heteroatoms within the parent chain ("heteroC 2-6 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain ("heteroC 2-s alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and I or 2 30 heteroatoms within the parent chain ("heteroC 2 4 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and I heteroatom within the parent chain ("heteroC 2 3 alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain ("heteroC 2-6 alkenyl"). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an "unsubstituted heteroalkenyl") or substituted (a "substituted heteroalkenyl") with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC 2-a alkenyl. In certain embodiments, the 5 heteroalkenyl group is a substituted heteroC 21 o alkenyl. The term "alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) ("C2 10 alkynyl"). In some embodiments, an alkynyl group has 2 to 9 carbon atoms ("C 2.9 alkynyl"). In some embodiments, an alkynyl group has 2 to 8 carbon N 10 atoms ("C 2.-8 alkynyl"). In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C2 N In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C 2-6 alkynyl"). 7 alkynyl"). In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C2-5 alkynyl"). In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C 24 alkynyl"). In some embodiments, an alkynyl group has 2 to 3 carbon atoms("C 2 -3 alkynyl"). In some 15 embodiments, an alkynyl group has 2 carbon atoms ("C2 alkynyl"). The one or more carbon carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in I-butynyl). Examples of C24 alkynyl groups include, without limitation, ethynyl (C 2 ), 1-propynyl (C 3), 2 propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C4 ), and the like. Examples ofC 2 -6 alkenyl groups include the aforementioned C 2 .4 alkynyl groups as well as pentynyl (C5 ), hexynyl (C 6), and 20 the like. Additional examples of alkynyl include heptynyl (C 7 ), octynyl (C 8 ), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C 2 1 0 alkynyl. In certain embodiments, the alkynyl group is a substituted C 2 1 0 alkynyl. 25 The term "heteroalkynyl" refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms 30 within the parent chain ("heteroC 2 1o alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and I or more heteroatoms within the parent chain ("heteroC 2-9 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and I or more heteroatoms within the parent chain ("heteroC 2 .
8 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and I or more heteroatoms within the parent chain ("heteroC 2.7 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and I or more heteroatoms within the parent chain ("heteroC 2-6 alkynyl"). In some 5 embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and I
or 2 heteroatoms within the parent chain ("heteroC 2-s alkynyl"). In some embodiments, a
heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and Ior 2 heteroatoms within the parent chain ("heteroC 2.4 alkynyl"). In some embodiments, a heteroalkynyl group N has 2 to 3 carbon atoms, at least one triple bond, and I heteroatom within the parent chain N 1o ("heteroC 2 3. alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms,
N at least one triple bond, and I or 2 heteroatoms within the parent chain ("heteroC 2.6 alkynyl"). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an "unsubstituted heteroalkynyl") or substituted (a "substituted heteroalkyny") with one or more substituents. In certain embodiments, the heteroalkynyl
15 group is an unsubstituted heteroC 2 1 0 alkynyl. In certain embodiments, the heteroalkynyl
group is a substituted heteroC 2 -o alkynyl.
The term "carbocyclyl" or "carbocyclic" refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms ("C 3 -1 4 carbocyclyl") and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has
20 3 to 10 ring carbon atoms ("C 3-1 0 carbocyclyl"). In some embodiments, a carbocyclyl group
has 3 to 8 ring carbon atoms("C38- carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms ("C3. 7 carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C3. 6 carbocyclyl"). In some embodiments, a carbocycly group has 4 to 6 ring carbon atoms ("C 4 -6 carbocyclyl"). In some embodiments, a carbocyclyl group
25 has 5 to 6 ring carbon atoms ("C5. 6 carbocyclyl"). In some embodiments, a carbocyclyl group
has 5 to 10 ring carbon atoms ("C5lo carbocyclyl"). Exemplary C 3. 6 carbocyclyl groups
include, without limitation, cyclopropyl (C 3), cyclopropenyl (C3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4), cyclopentyl (Cs), cyclopentenyl (C), cyclohexyl (C6), cyclohexenyl (C6 ), cyclohexadienyl (C 6), and the like. Exemplary C 3. carbocyclyl groups include, without 30 limitation, the aforementioned C 3 -6 carbocyclyl groups as well as cycloheptyl (C7 ),
cycloheptenyl (C 7 ), cycloheptadienyl (C 7), cycloheptatrienyl (C 7), cyclooctyl (C8 ), cyclooctenyl (C), bicyclo[2.2.l]heptanyl (C 7), bicyclo[2.2.2]octanyl (C 8), and the like. Exemplary C3 - 10 carbocyclyl groups include, without limitation, the aforementioned C .3 8 carbocyclyl groups as well as cyclononyl (C), cyclononenyl (C), cyclodecyl (CIO), cyclodecenyl (Cio), octahydro-1H-indenyl (C 9 ), decahydronaphthalenyl (Cio), spiro[4.5]decanyl (CIO), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic ("monocyclic carbocyclyl") or 5 polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic carbocyclyl") or tricyclic system ("tricyclic carbocyclyl")) and can be saturated or can contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or N heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such N 10 instances, the number of carbons continue to designate the number of carbons in the N carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted carbocyclyl") with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C 3 .14 carbocyclyl. In certain embodiments, the carbocyclyl group is a 15 substituted C 3.14 carbocyclyl. In some embodiments, "carbocyclyl" is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms ("C 3 . 14 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms ("C 3 10 cycloalkyl"). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C3.- cycloalkyl"). In some embodiments, a 20 cycloalkyl group has 3 to 6 ring carbon atoms ("C3 -6cycloalkyl"). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms ("C 4. 6 cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms ("Cs. cycloalkyl"). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms ("C 5 . 10 cycloalkyl"). Examplesof C 5 .6 cycloalkyl groups include cyclopentyl (C 5 ) and cyclohexyl (C).Examples of C 3 - cycloalkyl 25 groups include the aforementioned C5 . cycloalkyl groups as well as cyclopropyl (C 3) and cyclobutyl (C 4 ). Examples ofC 3.. cycloalkyl groups include the aforementioned C 36 cycloalkyl groups as well as cycloheptyl (C 7) and cyclooctyl (Cg). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl") or substituted (a "substituted cycloalkyl") with one or more substituents. In 30 certain embodiments, the cycloalkyl group is an unsubstituted C 3-14 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C 314 cycloalkyl. The term "heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and I to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("3-14 membered heterocyclyl"). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a fused, 5 bridged or spiro ring system such as a bicyclic system ("bicyclic heterocyclyl") or tricyclic system ("tricyclic heterocyclyl")), and can be saturated or can contain one or more carbon carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems wherein the N heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the N 10 point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein
N the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently 15 unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocycly")
with one or more substituents. In certain embodiments, the heterocyclyl group is an
unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl. In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring 20 system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is
independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heterocyclyl"). In
some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heterocyclyl"). In some 25 embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring
carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected
from nitrogen, oxygen, and sulfur ("5-6 membered heterocyclyl"). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms 30 selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered
heterocyclyl has I ring heteroatom selected from nitrogen, oxygen, and sulfur. Each possibility represents a separate embodiment of the present invention.
Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without
limitation, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing I heteroatom include, without 5 limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and
dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered N 10 heterocyclyl groups containing I heteroatom include, without limitation, piperidinyl, N tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl,
dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl 15 groups containing I heteroatom include, without limitation, azepanyl, oxepanyl and
thiepanyl. Exemplary 8-membered heterocyclyl groups containing I heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups
include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, 20 tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8 naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, I H-benzo[e][l,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-41 25 thieno(2,3-c]pyranyl, 2,3-dihydro-IH-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3 b]pyridinyl, 4,5,6,7-tetrahydro-IH-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2 c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-,6-naphthyridinyl, and the like. The term "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or 30 tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 n electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring
system ("C 6- 14 aryl"). In some embodiments, an aryl group has 6 ring carbon atoms ("C6
aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("Cio aryl"; e.g., naphthyl such as I-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 aryl"; e.g., anthracyl). "Aryl" also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, 5 the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C6 .14 aryl. In certain N embodiments, the aryl group is a substituted C6- 14 aryl. N 10 The term "heteroaryl" refers to a radical of a 5-14 membered monocyclic or N polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 x electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). In heteroaryl groups that 15 contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring 20 members continue to designate the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (ary/heteroaryl) ring system. Polycyclic heteroaryl 25 groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, 30 wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatorns provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5 5 6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each N instance of a heteroaryl group is independently unsubstituted (an "unsubstituted heteroaryl") N 10 or substituted (a "substituted heteroaryl") with one or more substituents. In certain N embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl. Exemplary 5-membered heteroaryl groups containing I heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups 15 containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, 20 without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing I heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6 25 bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, 30 isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.
The term "unsaturated bond" refers to a double or triple bond. The term "unsaturated"
or "partially unsaturated" refers to a moiety that includes at least one double or triple bond. The term "saturated" refers to a moiety that does not contain a double or triple bond, i.e., the moiety only contains single bonds. 5 Affixing the suffix "-ene" to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the N divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, N 10 heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl,
N and heteroarylene is the divalent moiety of heteroaryl. A group is optionally substituted unless expressly provided otherwise. The term "optionally substituted" refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, 15 aryl, and heteroaryl groups are optionally substituted. "Optionally substituted" refers to a
group which may be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl,
"substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl, "substituted" or "unsubstituted" carbocyclyl, 20 "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl or
"substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted" means
that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a
substituent which upon substitution results in a stable compound, e.g., a compound which
does not spontaneously undergo transformation such as by rearrangement, cyclization,
25 elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a
substituent at one or more substitutable positions of the group, and when more than one
position in any given structure is substituted, the substituent is either the same or different at each position. The term "substituted" is contemplated to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described 30 herein that results in the formation of a stable compound. The present invention contemplates
any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety. The invention is not intended to be limited in any manner by the exemplary substituents described herein.
Exemplary carbon atom substituents include, but are not limited to, halogen, -CN, -NO 2 , -N 3, -SO 2 H, -SO 3 H, -OH, -ORaa, -ON(Rb) 2, -N(R) 2, -N(Rb)3+X-, -N(ORc)R, 5 -SH, -SRaa, -SSRcc, -C(=O)Raa, -CO 2H, -CHO, -C(ORcc) 2, -COIRaa, -OC(=O)Raa -OCO 2 Raa, _C(=O)N(Rbb) 2, -OC(=O)N(Rbb) 2, -NRC(=0)Raa, -NRbbCO 2 Raa,
-NRbbC(=O)N(Rbb) 2, -C(=NRbb)Raa, -C(=NR )OR aa, OC(=NRbb)Raa, -OC(=NR )ORLa,
-C(=NR )N(Rb)2, -OC(=NRb)N(Rbb)2, -NRbbC(=NR )N(R b)2, -C(=O)NR bbSO 2 Raa,
N -NR SO2 Raa, -SO 2N(R )2, -SO 2 Raa, _SO 2 ORaa, -OSO 2Raa, -S(=O)Raa, -OS(=O)Raa, Ni10 -Si(Raa) 3 , -OSi(Raa )3 C(=S)N(Rb) 2 , -C(=O)SRaa, C(=S)SRaa, -SC(=S)SRaa
N -SC(=O)SRaa, -OC(=O)SRaa, SC(=O)ORaa, -SC(=O)R", -P(=O)(Raa) 2, P(=O)(OR") 2
, -OP(=O)(R") 2, -OP(=o)(OR") 2, -P(=O)(N(Rbb) 2) 2 , -O)(=O)(N(Rbb) 2 ) 2 , -NR P(=O)(R-a) 2
, -NRbbP(=O)(ORcc) 2, -NRbbP(=O)(N(R )2)2, -P(Rcc) 2 , -P(ORcc) 2, -P(Rcc) 3 )'X, -P(ORcc) 3 X, -P(Rcc) 4, -P(ORcc) 4, -OP(Rc)2, -OP(Rcc) 3 +X , -OP(ORcc) 2, -OP(ORcc)X-, is -OP(Rcc) 4 , -OP(ORcc) 4 , -B(Raa) 2, -B(ORc) 2 , -BRaa(ORec), C. 10 alkyl, Ci- o perhaloalkyl, C 2 10 alkenyl, C 2-10 alkynyl, heteroCi 10 alkyl, heteroC 2-o alkenyl, heteroC 2-1o alkynyl, C 31 0 carbocyclyl, 3-14 membered heterocyclyl, C 6. 14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R 20 groups; wherein X- is a counterion; or two geminal hydrogens on a carbon atom are replaced with the group =0, =S,
=NN(RIb) 2 , =NNRbbC(=O)Raa, =NNR C(=O)ORaa, =NNR S(=O) 2 Raa, =NRbb, or =NOR";
each instance ofRaa is, independently, selected from C1 1 0 alkyl, C1 10 perhaloalkyl, C 2-10 alkenyl, C2 1 0 alkynyl, heteroCI 10 alkyl, heteroC2 calkenyl, heteroC 2-oalkynyl, C3 10 25 carbocyclyl, 3-14 membered heterocyclyl, C 6 .14 aryl, and 5-14 membered heteroaryl, or two
Ra groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl
ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rld groups; 30 each instance ofRbb is, independently, selected from hydrogen, -OH, -ORaa,
-N(R) 2, -CN, -C(=O)Raa, -C(=O)N(R)2, -CO 2 Raa, -SO 2 Raa, _C(=NR)ORaa -C(=NRc")N(Rcc) 2, -S0 2N(R") 2, -SO 2 Rc, -SO 2ORc, -SORaa, -C(=S)N(R") 2, -C(=O)SR", -C(=S)SRC, -P(=O)(Raa) 2, -P(=O)(ORcc) 2, -P(=O)(N(Rcc) 2 ) 2 , CiIo alkyl, CI-o perhaloalkyl,
C- C 2 - 10 alkenyl, C 2 1 0 alkynyl, heteroCI.oalkyl, heteroC 2 1oalkenyl, heteroC 2 Ioalkynyl, C3 . 10 carbocyclyl, 3-14 membered heterocyclyl, C6 14 aryl, and 5-14 membered heteroaryl, or two Rbb groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, 5 carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dgroups; wherein X is a counterion; each instance of RCC is, independently, selected from hydrogen, CI-o alkyl, C 10
perhaloalkyl, C 2 I0 alkenyl, C 2 - 10 alkynyl, heteroC 11 o alkyl, heteroC 2-1o alkenyl, heteroC 2 0 alkynyl, C 3 10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered 10, heteroaryl, or two Rc groups arejoined to form a 3-14 membered heterocyclyl or 5-14 N membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; each instance of R d is, independently, selected from halogen, -CN, -NO 2, -N 3
, 15 -SO2H, -SO3H, -OH, -OR'e, -ON(R E)2, -N(R ')2, -N(Rrr +3-X--N(OR )R", -S H, -S R' -SSRee, -C(=O)R e, -CO 2 H, -CO 2Rc, OC(=O)R'e, -OCO2R',-C(=O)N(R"):, -OC(=O)N(RE) 2, -NRfiC(=O)Re, -NR"'CO 2Rc, -NR""C(=O)N(R) 2, -C(=NR)ORcc 1 -OC(=NR )Rcc -OC(=NR")OR'", -C(=NR)N(R') 2, -OC(=NR)N(R") 2 , -NRIC(=NR")N(R') 2, -NR"SO 2R', -SO 2N(R) 2 , -SO 2R", -SO 2OR", -OSO 2Re, 20 -S(=O)Ree, -Si(R'") 3, -OSi(R'') 3, -C(=S)N(Rrr) 2, -C(=O)SRee, -C(=S)SR"`, -SC(=S)SR, -P(=O)(OR'") 2, -P(=O)(Re) 2, -OP(=O)(Re") 2, -OP(=O)(ORee)2, C, 6 alkyl, C. perhaloalkyl, C2-6 alkenyl, C 2-6 alkynyl, heteroCi-6 alkyl, heteroC 26- alkenyl, heteroC 26- alkynyl, C 3-1 0
carbocyclyl, 3-10 membered heterocyclyl, C6 . 1 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, 25 heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R" groups, or two geminal R d substituents can bejoined to form =0 or =S; wherein X is a counterion; each instance of Ree is, independently, selected from C1 -6 alkyl, C1 . 6 perhaloalkyl, C 2 -6 alkenyl, C 2-6alkynyl, heteroC,. alkyl, heteroC 2-6 alkenyl, heteroC 2.6 alkynyl, C 3 10 30 carbocyclyl, Co aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R8 groups; each instance of Rf is, independently, selected from hydrogen, C1 .6 alkyl, C1. 6 perhaloalkyl, C 2 -6 alkenyl, C2 -6 alkynyl, heteroCI. 6 alkyl, heteroC .26 alkenyl, heteroC 2-6 alkynyl, C3 _10 carbocyclyl, 3-10 membered heterocyclyl, C 6 .io aryl and 5-10 membered heteroaryl, or two R groups are joined to form a 3-10 membered heterocyclyl or 5-10 membered 5 heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R" groups; and each instance of RII is, independently, halogen, -CN, -NO 2 , -N 3, -SO H, -SO 2 3H,
-OH, -OC6 alkyl, -ON(CI-6 alkyl) 2 , -N(CI-6 alkyl) 2 , -N(CI-6 alkyl)3 X , -NH(CI.. 6 i10 alkyl) 2 X , -NH 2 (CI-6 alkyl) X~-, -NH 3+X~, -N(OC. 6 alkyl)(C. 6 alkyl), -N(OH)(C. 6 alky), N -NH(OH), -SH, -SC -6 alkyl, -SS(CI-6 alkyl), -C(=O)(C.. alkyl), -CO 2 1, -C0 2 (CI-6 alkyl), -OC(=O)(C -6 alkyl), -OC 2 (CI-6 alkyl), -C(=)NH 2, -C(=)N(CI.. alkyl) 2
, -OC(=O)NH(C1-6 alkyl), -NHC(=0)( CI. alkyl), -N(C.. alkyl)C(=0)( C 6- alkyl), -NHCO 2 (CI.. alkyl), -NHC(=O)N(CI. 6 alkyl) 2, -NHC(=O)NH(CI-6 alkyl), -NHC(=0)N1 2
, 15 -C(=NH)O(C,.6 alkyl), -OC(=NH)(CI-6 alkyl), -OC(=NH)OCI.6 alkyl, -C(=N)N(C 6
alkyl) 2, -C(=NH)NH(C 1 -6 alkyl), -C(=NH)NH 2, -OC(=NH)N(C-6 alkyl) 2 , -OC(NH)NH(C
6 alkyl), -OC(NH)NHl 2, -NHC(NH)N(CI- 6 alkyl) 2 , -NHC(=NH)NH 2, -NHSO 2 (Ci-6 alkyl), -SO 2 N(CI. 6 alkyl) 2 , -SO 2NH(CI. 6 alkyl), -SO 2NH 2, -SO 2 CI.. alkyl, -SO 2 OCI.. alkyl, -OS0 2 CI- 6 alkyl, -SOCI.. alkyl, -Si(C 1 .. alkyl) 3, -OSi(C,.. alkyl) 3 -C(=S)N(C alkl)2
, 20 C(=S)NH(CI. alkyl), C(=S)NH 2 , -C(=O)S(C 1 .. alkyl), -C(=S)SCI-6 alkyl, -SC(=S)SCI-6 alkyl, -P(=O)(OCI6 alkyl) 2, -P(=0)(CI-6 alkyl) 2 , -OP(=O)(CI-6 alkyl) 2, -OP(=0)(OC-6 alkyl) 2 , C1 . alkyl, C,. perhaloallcyl, C2 6 alkenyl, C2 -6 alkynyl, heteroCI- 6alkyl, heteroC 2..
6 alkenyl, heteroC 2-alkynyl, C 3-10 carbocyclyl, C 6 .10 aryl, 3-10 membered heterocyclyl, 5-10 membered heternaryl; or two geminal R99 substituents can be joined to form =0 or =S;
25 wherein X is a counterion. The term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -C), bromine (bromo, -Br), or iodine (iodo, -I). The term "hydroxyl" or "hydroxy" refers to the group -OH. The term "substituted hydroxyl" or "substituted hydroxyl," by extension, refers to a hydroxyl group wherein the 30 oxygen atom directly attached to the parent molecule is substituted with a group other than hydrogen, and includes groups selected from -ORaa, -ON(R) 2 , -OC(=O)SRaa -OC(=O)Raa, -OCO 2 Raa, -OC(=O)N(Rb) 2 , -OC(=NR b)Rau, -OC(=NR ')ORaa, -OC(=NR b)N(R ')2, -OS(=O)Ra", -OSO 2 Raa _OSi(Raa) 3 , -OP(Rcc) 2, -OP(R"c) 3*X~
-OP(ORCC) 2 , -OP(ORCC) 3 +)C, -OP(=O)(Raa) 2 , -OP(=O)(OR") 2, and -OP(=)(N(R")) 2
, wherein X, Raa, Rb, and RC are as defined herein. The term "amino" refers to the group -NH 2. The term "substituted amino," by extension, refers to a monosubstituted amino, a disubstituted amino, or a trisubstituted amino.
5 In certain embodiments, the "substituted amino" is a monosubstituted amino or a disubstituted amino group.
The term "monosubstituted amino" refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with one hydrogen and one group bb a N other than hydrogen, and includes groups selected from -NH(R ), -NHC(=O)Raa N0 -NHCO2Raa, -NHC(=O)N(Rb) 2, -NH C(=NRb')N(Rbb)2, -NH SO 2Raa, -NHP(=O)(ORc) 2
, N and -NHP(=O)(N(Rb) 2) 2, wherein R", Rband R' are as defined herein, and whereinRbbof
the group -NH(Rb) is not hydrogen. The term "disubstituted amino" refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with two groups other than hydrogen, 15 and includes groups selected from -N(R) 2, -NRbbC(=O)Raa, NRhbCO 2 Raa, -NR"C(=)N(Rbb) 2, -NRbbC(=NRbb)N(Rb)2, -NR SO 2Raa, -NR P(=O)(ORc) 2, and -NRebP(=O)(N(R")2)2, wherein R", Rb, and R" are as defined herein, with the proviso that the nitrogen atom directly attached to the parent molecule is not substituted with hydrogen. The term "trisubstituted amino" refers to an amino group wherein the nitrogen atom
20 directly attached to the parent molecule is substituted with three groups, and includes groups
selected from -N(Rb) 3 and -N(Rb),'X-, wherein Rbb and X~ are as defined herein. The term "sulfonyl" refers to a group selected from -SO 2N(R b)2, -SO 2 Raa, and SO2 ORaa, wherein Raa and R bare as defined herein. The term "sulfinyl" refers to the group -S(=O)R", wherein Raa is as defined herein. 25 The term "acyl" refers to a group having the general formula -C(=O)Rx,
-C(=O)ORx, -C(=O)-O-C(=O)Rx, -C(=O)SRxl, -C(=0)N(RxI)2, -C(=S)RxI, -C(=S)N(RxI) 2, and -C(=S)S(Rx), -C(=NRe)Rx, -C(=NRxl)ORx, -C(=NRx)SRxl and -C(=NRx)N(Rx) 2, wherein Rx is hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or
30 unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched
aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di- aliphaticamino, mono- or di- heteroaliphaticamino, 5 mono- or di- alkylamino, mono- or di- heteroalkylamino, mono- or di-arylamino, or mono- or di-heteroarylamino; or two R groups taken together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-CO 2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents N include, but are not limited to, any of the substituents described herein, that result in the i 10 formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, N heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyoxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, 15 heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted). The term "carbonyl" refers a group wherein the carbon directly attached to the parent molecule is sp 2 hybridized, and is substituted with an oxygen, nitrogen or sulfur atom, e.g., a group selected from ketones (-C(=O)Raa), carboxylic acids (-CO2H), aldehydes (-CHO), 20 esters (-CO 2 Raa, -C(=O)SRaa, -C(=S)SRaa), amides (-C(=O)N(Rbb) 2 , -C(=O)NR SO 2R aa -C(=S)N(Rbb) 2), and imines (-C(=NRbb)Raa, -C(=NRbb)ORaa), -C(=NRbb)N(R b) 2), wherein Raa and Rbb are as defined herein.
Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom
25 substituents include, but are not limited to, hydrogen, -OH, -ORaa, -N(R') 2 , -CN, -C(=O)Raa, -C(=O)N(Rc)2, CO2Raa _SO2 Raa, -C(=NR)Raa _C(=NRe;)ORaa -C(=NR'c)N(R') 2, -SO 2N(Rcc)2, -SO2R'c, -SOORec, SORaa, -C(=S)N(RC) 2 , -C(=O)SR", -C(=S)SRcc, -P(=O)(ORcc) 2, -P(=O)(Raa) 2, -P(=O)(N(R") 2) 2 , CI 1o alkyl, Cio perhaloalkyl, C 2 -10 alkenyl, C 2 10 alkynyl, heteroCI-oalkyl, heteroC 2-oalkenyl, heteroC 21oalkynyl, C 310o 30 carbocyclyl, 3-14 membered heterocyclyl, C 6 14 aryl, and 5-14 membered heteroaryl, or two R" groups attached to an N atom are joined to form a 3-14 membered heterocyclyl or 5-14
membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R d groups, and wherein Raa, Rb, Rc' and R d are as defined above. In certain embodiments, the substituent present on the nitrogen atom is an nitrogen protecting group (also referred to herein as an "amino protecting group"). Nitrogen protecting 5 groups include, but are not limited to, -OH, -ORa, -N(Rcc) 2, -C(=O)Raa, -C(=O)N(Rcc) 2
, -C0 2 Raa, _SO 2 Raa, _C(-NR)Raa, C(=NR"c)ORaa, -C(=NRcc)N(Rcc) 2, -SO 2N(Rc) 2
, -SO 2Rc, -SO 2OR", -SORaa, C(=S)N(Rc) 2, -C(=O)SRcc, -C(=S)SRcc, C 1 o alkyl (e.g., aralkyl, heteroaralkyl), C 2 - 10 alkenyl, C 2 -10 alkynyl, heteroCa 1 0 alkyl, heteroC 2-10 alkenyl,
N heteroC 2-o alkynyl, C 3 10 carbocyclyl, 3-14 membered heterocyclyl, C6 -14 aryl, and 5-14 Ni10 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, N heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R d groups, and wherein Raa, Rbb, R and Rdd
are as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in ProtectingGroups in Organic Synthesis, T. W. Greene and P. G. M. 15 Wuts, 3' edition, John Wiley & Sons, 1999, incorporated herein by reference. For example, nitrogen protecting groups such as amide groups (e.g., -C(=)Raa)
include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3 pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o 20 nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'
dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide and o 25 (benzoyloxymethyl)benzamide. Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)ORaa) include, but
are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tnoc), 30 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2 trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), I-(I-adamantyl)-I methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2 dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate
(TCBOC), 1-methyl-I-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-I methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), I-isopropylallyl 5 carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, N 10 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3 N dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4 dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2 triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5 15 benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4 dimethoxy-6-nitrobenzyl carbamare, phehyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p 20 decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N dimethylcarboxamido)benzyl carbamate, I,1-dimethyl-3-(N,N-dimethylcarboxamido)propy carbamate,,I1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoboryni carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, I-methylcyclobutyl carbamate, 1 25 methylcyclohexyl carbamate, 1-methyl-i-cyclopropylmethyl carbamate, 1-methyl-1-(3,5 dimethoxyphenyl)ethyl carbamate, 1-methyl-I-(p-phenylazophenyl)ethyl carbamate, I methyl-I-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4 (trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate. 30 Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=0) 2 R") include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4 methoxybenzenesulfonamide (Mtr), 2.,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6 dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6 trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8' 5 dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoronethylsulfonamide, and phenacylsulfonamide.
Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(0) acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N'-phenylaminothioacyl N derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3 N i0 oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleinide, N-2,5 N dimethylpyrrole, N-1, 1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5 substituted 1,3-dimethyl-I,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5 triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(]-isopropyl 15 4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4 methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N
[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N 2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2 picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p 20 methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2 pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N' isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5 chlorosalicylidenearnine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneam ine, N cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-I -cyclohexenyl)amine, N-borane derivative, 25 N-diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,
diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphorarnidate, benzenesulfenarnide, o-nitrobenzenesulfenamide (Nps), 2,4 30 dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4
methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).
In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an "hydroxyl protecting group"). Oxygen ,a bb aa protecting groups include, but are not limited to, -Raa, -N(R )2, -C(=O)SRaa ,C(=O)Raa
-CO 2 Raa, -C(=O)N(Rbb) 2, -C(=NRbb)Raa C(=NRbb)ORaa, -C(=NRbb)N(Rbb) 2, -S(=O)Raa,
5 -S0 2 Raa, -Si(Raa) 3 , -P(R'c) 2 , -P(R°) 3 *X-, -P(ORc) 2 , -P(ORcc) 3 +X~, -P(=)(Raa) 2
, -P(=)(ORc) 2 , and -P(=O)(N(Rbb )2)2, wherein X-, Raa, Rbb, and Rc are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in ProtectingGroups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference. 10 Exemplary oxygen protecting groups include, but are not limited to, methyl, N methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymnethyl (SMOM), benzyloxymethyl (BOM), p methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmCthyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2 15 methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2 (trimcthyl3ilyl)cthoxymethyl (SLMOR), tetrahydropyranyl (THP), 3 bromotetrahydropyranyl, tetrahydrothiopyranyl, I-methuxycycluhexyl, 4 methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4 methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4 20 methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran- -yl, 1-ethoxyethyl, I (2-chloroethoxy)ethyl, 1-methyl-i-methoxyethyl, 1-methyl-I-benzyloxyethyl, 1-methyl-I benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p 25 methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6 dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a naphthyldiphenylmethyl, p-nethoxyphenyldiphenylmethyl, di(p methoxyphenyl)phenylmethyl, tri(p-rnethoxyphenyl)rnethyl, 4-(4' 30 bromophenacyloxyphenyl)diphenylnethyl, 4,4',4"-tris(4,5 dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4" tris(benzoyloxyphenyl)methyl, 3-(imidazol-I-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1 bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-I0- oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, 5 diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4 oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6 Ni o trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl N carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC or Boc), p nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl 15 carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4 ethoxy-I-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4 nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonatc, 2
(methylthiomcthoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2 (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4 20 (1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o (methoxyacyl)benzoate, -naphthoate, nitrate, alkyl N,N,N',N' tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and 25 tosylate (Ts). In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting
group (also referred to as a "thiol protecting group"). Sulfur protecting groups include, but are not limited to, -Raa, -N(R ')2, -C(=O)SRaa, -C(=O)Raa, -CO 2Raa, -C(=O)N(R )2,
-C(=NR )Raa, C(=NRb)ORaa, -C(=NRbb)N(R bb) 2 , -S(=O)Raa, SO 2 Raa, -Si(Raa) 3 ,
30 -P(Rcc) 2, -P(Rcc) 3 +X, -P(ORcc) 2 , -P(ORcc) 3+X~, -P(=O)(Raa) 2, -P(=O)(ORc°) 2, and
-P(=O)(N(R b) 2 ) 2 , wherein Ra, Rbb, and Rc are as defined herein. Sulfur protecting groups
are well known in the art and include those described in detail in Protecting Groups in
Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference. A "counterion" or "anionic counterion" is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. An anionic counterion 5 may be monovalent (i.e., including one formal negative charge). An anionic counterion may
also be multivalent (i.e., including more than one formal negative charge), such as divalent or
trivalent. Exemplary counterions include halide ions (e.g., F-, Cl~, Br, F), N0 3 ~, C10 4-, OH~, H 2 PO4-, HCO3 HS0 4 -, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p N toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, N 10 naphthalene--sulfonic acid-5-sulfonate, ethan-I-sulfonic acid-2-sulfonate, and the like),
N carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, and the like), BF 4 , PF 4 -, PF~, AsF 6 , SbF6~, B[3,5-(CF 3) 2C 6H 3] 4]~, B(C6 s) 4
, BPhJ, AI(OC(CF 3)3)4-, and carborane anions (e.g., CB 1 H 12 or (HCB Me5 Br6 )). , Exemplary counterions which may be multivalent include C03 , HP0 4 2 -, P0 4 3 B 4 0 7 ~, 15 SO4~,2 S 30 ~, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate,
gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate,
phthalates, aspartate, glutamate, and the like), and carboranes. As used herein, use of the phrase "at least one instance" refers to 1, 2, 3, 4, or more instances, but also encompasses a range, e.g., for example, from I to 4, from I to 3, from 1 to 20 2, from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive. These and other exemplary substituents are described in more detail throughout. The
invention is not intended to be limited in any manner by the above exemplary listing of substituents.
As used herein, the term "salt" refers to any and all salts, and encompasses
25 pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well
30 known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art 5 such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, N hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, N 10 lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2 naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pirate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, 15 and N'(C4 alkyl) 4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate. 20
Biologically ActiveAgents
This disclosure contemplates that the LNPs provided herein and/or the various combination therapies provided herein may be used to deliver a variety of agents to a subject. Such agents typically will be biologically active agents. Biologically active agents are agents
25 that have an effect in vivo, and preferably a beneficial effect, such as desirable immune
modulation, immune stimulation, immune inhibition, cell killing, cell preservation, modified gene expression, protein replacement, and the like. Biologically active agents include but are not limited to prophylactic agents, therapeutic agents, and diagnostic agents. Biologically active agents include immunomodulatory agents such as immunostirnulatory or
30 immunoinhibitory agents, antigens, antibodies and antibody fragments such as antigen
binding antibody fragments, adjuvants, cytokines such as interleukins, anti-bacterial agents, anti-viral agents, anti-fungal agents, anti-parasitic agents, anti-cancer agents, anti
inflammatory agents, and the like.
Such agents may be, without limitation, nucleic acids, proteins or peptide, small organic compounds, carbohydrates and/or polysaccharides, and the like. They may be used to express nucleic acids and/or proteins in cells, particularly in cells that are deficient in such nucleic acids or proteins or have mutated versions of such nucleic acids or proteins. They 5 may be used to introduce and express nucleic acids or proteins that are not native to the cell or organism, as may be done for example in the context of an immunization or vaccination protocol. In this respect, the nucleic acid or protein may be foreign to the subject to whom it is administered (e.g., not naturally occurring in such subject, or not naturally occurring at all), N and it is administered to the subject to induce and/or boost an immune response to such N[0 nucleic acid or protein. The nucleic acids provided herein may be used for such a purpose. Other biologically active agents may be used alone or together with such nucleic acids or proteins, including formulated together with such nucleic acids or proteins, including formulated in the LNPs of this disclosure.
|5 Nucleic Acids As used herein, the term "nucleic acid" refers to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a W phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three 25 nucleotides). In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double-stranded DNA. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. Nucleic acids include any compound and/or substance that comprises a polymer of nucleotides. These polymers are referred to as polynucleotides. Nucleic acids may be or 30 may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P- D-ribo configuration, a-LNA having an a L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof Nucleic acids may be naturally occurring, for example, in the context of a genome, a 5 transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, N RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. N io Nucleic acids can be purified from natural sources, produced using recombinant expression
N systems and optionally purified, chemically synthesized, etc.
Furthermore, the terms "nucleic acid," "DNA," "RNA," and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic 15 acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2 aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5 zo methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5
propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7 deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 25 arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N
phosphoramidite linkages). A "nucleoside" refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase"). A "nucleotide"
30 refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides
may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphdioester linkages, in which case the polynucleotides would comprise regions of nucleotides. Modified nucleotide base pairing encompasses not only the standard adenosine thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed 5 between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base N pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or N i10 uracil. Any combination of base/sugar or linker may be incorporated into polynucleotides of N the present disclosure.
The skilled artisan will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite "T"s in a representative DNA sequence but where the sequence represents RNA, the "T"s would be substituted for "U"s. 15 Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) that are useful in the therapeutic agents described herein include, but are not
limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6 glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6 20 threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; I-methyladenosine; 2'-0 methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2'-0 methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; 25 N6,N6,2'-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6 hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2
methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; N I -methyl adenosine; N6, N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 30 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2 (halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2' deoxy-ATP; 2'-Deoxy-2'-a-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8
(alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8 (alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7 5 methyladenine; I -Deazaadenosine TP; 2'Fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2 Amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-Ethynyladenosine TP; 2 aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2'-a-Trifluoromethyladenosine TP; 2 Azidoadenosine TP; 2'-b-Ethynyladenosine TP; 2-Bromoadenosine TP; 2'-b N Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2',2'-difluoroadenosine TP; N 10 2'-Deoxy-2'-a-mercaptoadenosine TP; 2'-Deoxy-2'-a-thiomethoxyadenosine TP; 2'-Deoxy-2' NI b-aminoadenosine TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosineTP 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b-fluoroadenosine TP; 2'-Deoxy-2'-b iodoadenosine TP; 2-Deoxy-2'-b-mercaptoadenosine TP; 2'-Deoxy-2'-b thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-odoadenosine TP; 2 15 Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2 Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-tluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4' Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5'-Homo adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9 20 Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6 diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7 deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5 hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 2'-0 methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O 25 dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2' OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; ax-thio-cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP; 2'-Azido-2'-deoxy-CTP; 2' Deoxy-2'-a-aminocytidine TP; 2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2'-0 30 dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5 (propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5 propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4
(acetyl)cytosine; 1-methyl-I-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2 methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-I methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-I-methyl-I-deaza pseudoisocytidine; 4-thio-I -methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza 5 zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo
vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP; 2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP; 2'O-methyl-N4-Bz cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine N TP; 2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine TP; 2'-Deoxy-2'-a N 10 mercaptocytidine TP; 2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine
TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP; 2'-Deoxy-2'-b chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; 2' Deoxy-2'-b-mercaptocytidine TP; 2'-Deoxy-2'-b-thiomethoxycytidine TP; 2'-O-Methyl-5-(I propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'-Azidocytidine TP; 4'-Carbocyclic cytidine is TP; 4'-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2 thiocytidine'TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine'I'P; 5'-Homo-cytidine TP; 5 Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2'-O-dimethylguanosine; N2 20 methylguanosine; Wyosine; l,2'-O-dimethylguanosine; 1-methylguanosine; 2'-0
methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-0 ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-d imethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6 25 thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; NI-methyl-guanosine; a-thio
guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2'-Amino-2'-deoxy-GTP; 2'-Azido-2' deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP; 2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (nethyl)guanine; 7-(alkyl)guanine; 7-(deaza)guaninc; 7 30 (methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8 (halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine; -methyl-6-thio-guanosine; 6- methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7 methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6 thio-guanosine; N2-methyl-6-thio-guanosine; I -Me-GTP; 2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine TP; 2'-a-Ethynylguanosine TP; 2'-a 5 Trifluoromethylguanosine TP; 2'-b-Ethynylguanosine TP; 2'-b-Trifluoromethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'-Deoxy-2'-a-mercaptoguanosine TP; 2'-Deoxy-2'-a thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2-b-bromoguanosine TP; 2-Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b fluoroguanosine TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine TP; NI10 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'-Carbocyclic guanosine N TP; 4'-Ethynylguanosine TP; 5'-Homo-guanosine TP; 8-bromo-guanosine TP; 9 Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine; 1,2'-0 dimethylinosine; 2'-O-methylinosine; 7-methylinosine; 2-0-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; 15 deaza thymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3-methyluridine; 5 carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5 taurinomethyluridine; Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseduouridine; I-methyl pseudouridine; 2-0-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2' 20 0-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2'-O-dimethyluridine; 3-Methyl pseudo-Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5 (carboxyhydroxymethyl)uridine methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro-uridine; 5 aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine; 5 carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine 25 methyl ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5 carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5 carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5 Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5 methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methyluridine,), 30 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5 methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5 Oxyacetic acid- Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; NI-methyl-pseudo uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3- carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)- 2-thiouridine TP; 5-(iso Pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5 propynyl uracil; a-thio-uridine; I (aminoalkylamino-carbonyethylenyl)-2(thio)-pseudouraci1; I (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 5 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-pseudouracil; I (aminocarbonylethylenyl)-2(thio) pseudouracil; I (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; I (aminocarbonylethylenyl)-4 (thio)pseudouracil; I (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)-pseudouracil; I substituted 2,4-(dithio)pseudouracil; I substituted 4 N 10 (thio)pseudouracil; I substituted pseudouracil; l-(aminoalkylamino-carbonylethylenyl)-2 N (thio)-pseudouracil; I-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; I-Methyl-3 (3-amino-3-carboxypropyl)pseudo-UTP; I-Methyl-pseudo-UTP; 2 (thio)pseudouracil; 2' deoxy uridine; 2'fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl, 2'amino, 2'azido, 2'fluro-guanosine; 2'-Amino-2'-deoxy-UTP; 2'-Azido-2'-deoxy-UTP; 2'-Azido 15 deoxyuridine TP; 2'-O-methylpseudouridine; 2'deoxy uridine; 2' fluorouridine; 2'-Deoxy-2' a-aminouridine TP; 2'-Deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio )pseudouracil; 4-(thio)uracil; 4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2 20 (thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 24 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2 (thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil; 5 25 (alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5 (cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazole-I-alkyl)uracil; 5-(methoxy)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio )uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2 30 (thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5 (methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl) 2,4(dithio )uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5 (trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6
(azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; P seudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; I carboxymethyl-pseudouridine; 1-methyl-I-deaza-pseudouridine; I-propynyl-uridine; I taurinomethyl-I-methyl-uridine; I-taurinomethyl-4-thio-uridine; I-taurinomethyl 5 pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-I -methyl-I -deaza-pseudouridine; 2 thio-I-methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy pseudouridine; 4-thio-l -methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; (±)I-(2-Hydroxypropyl)pseudouridine TP; (2R)-1-(2 N 10 Hydroxypropyl)pseudouridine TP; (2S)-l-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2 N Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;I-(2,2,2-Trifluoroethyl)-pseudo-UTP; I (2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; I-(2,2-Diethoxyethyl)pseudouridine TP; 1 (2,4,6-Trimethylbenzyl)pseudouridine TP; I-(2,4,6-Trimethyl-benzyl)pseudo-UTP; 1-(2,4,6 15 Frimethyl-phenyl)pseudo-UTP; I-(2-Amino-2-carboxyethyl)pseudo-UTP;I-(2-Amino ethyl)pseudo-UTP; I-(2-H-ydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridineTP; 1-(3,4 Dimethoxybenzyl)pseudouridine'TP; 1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3 Amino-propyl)pseudo-UTP; I-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1-(4-Amino 20 4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP; I-(4-Amino-butyl)pseudo UTP; l-(4-Amino-phenyl)pseudo-UTP; 1-(/1-Azidobenzyl)pseudouridineTP; 1-(1 Brnmohenzyl)pseudouridine TP; 1-(4-Chinrnbenzyl)pseiidniiridine TP; 1-(4 Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine TP; 1-(4 Methanesulfonylbenzyl)pseudouridine TP; I-(4-Methoxybenzyl)pseudouridine TP; 1-(4 25 Methoxy-benzyl)pseudo-UTP; I-(4-Methoxy-phenyl)pseudo-UTP; 1-(4 Methylbenzyl)pseudouridine'TP; I-(4-Methyl-benzyl)pseudo-UTP; 1-(4 Nitrobenzyl)pseudouridine TP; I-(4-Nitro-benzyl)pseudo-UTP; I(4-Nitro-phenyl)pseudo UTP; 1-(4-Thiomethoxybenzyl)pseudouridine TP; 1-(4 Trifluoromethoxybenzyl)pseudouridine TP; I-(4-Trifluoromethylbenzyl)pseudouridine TP; 30 1-(5-Amino-pentyl)pseudo-UTP; I-(6-Amino-hexyl)pseudo-UTP; 1,6-Dimethyl-pseudo UTP; I-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP; I-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl } pseudouridine TP; I-Acetylpseudouridine TP; -Alkyl-6-(I-propynyl)-pseudo-UTP; I-Alkyl-6-(2-propynyl)-pseudo-UTP; I-Alkyl-6-allyl- cN pseudo-UTP; I-Alkyl-6-ethynyl-pseudo-UTP; I-Alkyl-6-homoally-pseudo-UTP; 1-Alkyl-6 vinyl-pseudo-UTP; 1-AllylpseudouridineTP; I-Aminomethyl-pseudo-UTP; I Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1 Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; I-Butyl-pseudo-UTP; 1 5 Cyanomethylpseudouridine TP; I-Cyclobutylmethyl-pseudo-UTP; I-Cyclobutyl-pseudo UTP; I-Cycloheptylmethyl-pseudo-UTP; I-Cycloheptyl-pseudo-UTP; I-Cyclohexylmethyl pseudo-UTP; I-Cyclohexyl-pseudo-UTP; I-Cyclooctylmethyl-pseudo-UTP; I-Cyclooctyl pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; I-Cyclopentyl-pseudo-UTP; 1 Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; I-Ethyl-pseudo-UTP; I N 10 Hexyl-pseudo-UTP; I-HomoallyipseudouridineTP; I-HydroxymethylpseudouridineTP; 1 N iso-propyl-pseudo-UTP; I-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-alpha thio-pseudo-UTP; I-MethanesulfonylmethylpseudouridineTP; I Methoxymethylpseudouridine TP; I-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP; 1-Methyl 6-(4-morpholino)-pseudo-UTP; 1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; I-Methyl-6 15 (substituted phenyl)pseudo-UTP; I-Methyl-6-amino-pseudo-UTP; I-Methyl-6-azido-pseudo UTP; 1-Methyl-6-bromo-pseudo-UTP; I-Methyl-6-butyl-pseudo-UTP; I-Methyl-6-chloro pseudo-UTP; I-Methyl-6-cyano-pseudo-UTP; I-Methyl-6-dimethylamino-pseudo-UTP; I Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP; I-Methyl-6-ethyl pseudo-UTP; I-Methyl-6-fluoro-pseudo-UTP; I-Methyl-6-formyl-pseudo-UTP; I-Methyl-6 20 hydroxyamino-pseudo-UTP; I-Methyl-6-hydroxy-pseudo-UTP; I-Methyl-6-iodo-pseudo UTP; I-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP; I-Methyl-6 methylamino-pseudo-UTP; I-Methyl-6-phenyl-pseudo-UTP; I-Methyl-6-propyl-pseudo UTP; I-Methyl-6-tert-butyl-pseudo-UTP; I-Methyl-6-trifluoronethoxy-pseudo-UTP; 1 Methyl-6-trifluoromethyl-pseudo-UTP; I-Morpholinomethylpseudouridine TP; I -Pentyl 25 pseudo-UTP; 1-Phenyl-pseudo-UTP; I-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; I-Propyl-pseudo-UTP; I-propynyl-pseudouridine; I-p-tolyl-pseudo-UTP; 1-tert-Butyl pseudo-UTP; 1-ThiomethoxymethylpseudouridineTP; I ThiomorpholinomethylpseudouridineTP; I-TrifluoroacetylpseudouridineTP; I Trifluorornethyl-pseudo-UTP; I-Vinylpseudouridine TP; 2,2'-anhydro-uridine TP; 2'-brorno 30 deoxyuridine TP; 2'-F-5-Methyl-2'-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP; 2'-a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP; 2'-b Trifluoromethyluridine'TP; 2'-Deoxy-2',2'-difluorouridine TP; 2-Deoxy-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b- azidouridine TP; 2'-Deoxy-2-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP; 2'-Deoxy 2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodourid ine TP; 2'-Deoxy-2'-b-mercaptouridine TP; 2' Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-0 Methyl-5-(I -propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic 5 uridine TP; 4'-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5 Cyanouridine TP; 5-Dimethylaminouridine TP; 5'-Homo-uridine TP; 5-iodo-2'-fluoro deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5 Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6 N (4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl) N 10 pseudo-UTP; 6-Anino-pseudo-UTP; 6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl N pseudo-UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP; 6-Fluoro pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo UTP; 6-lodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP; 6 15 Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP; 6 Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine 1-(4 methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy )-ethoxy] 20 ethoxy )-ethoxy}lpropionic acid; Pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy )-ethoxy} ethoxy]-ethoxy )-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy ]-ethoxy) ethoxy}}propionic acid; Pseudouridine TP l-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP I-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid; Pseudo-UTP-NI-4-butanoic acid; Pseudo 25 UTP-N1-5-pentanoic acid; Pseudo-UTP-NI-6-hexanoic acid; Pseudo-UTP-NI-7-heptanoic acid; Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N I -p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4 demethylwyosine;2,6-(diamino)purine;I-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: I,3-( diaza)-2-( oxo )-phenthiazin-1-yl;I,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6 30 (dioxa)-naphthalene;2 (amino)purine;2,4,5-(trimethyl)phenyl;2' methyl, 2'amino, 2'azido, 2'fluro-cytidine;2' methyl, 2'amino, 2'azido, 2'fluro-adenine;2'methyl, 2'amino, 2'azido, 2'fluro-uridine;2'-amino-2'-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2'-azido 2'-deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro-modified bases; 2'-O-methyl-ribose; 2-oxo-
7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-y; 2-pyridinone; 3 nitropyrrole; 3 (methyl)-7-(propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6 (methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(d imethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5-nitroindole; 6 5 (aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl pyrrolo-pyrimidin-2-on-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio )-3-(aza)-phenthiazin- yl; 7-(aminoalkylhydroxy)-I-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yI; 7-(aminoalkylhydroxy) 1,3-(diaza)-2-(oxo)-phenoxazin-1 -yl; 7-(aminoalkylhydroxy)-,3-( diaza)-2-( oxo) N phenthiazin-1-yi; 7-(aminoalkyihydroxy)-l,3-( diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; N 10 7-(guanidiniumalkylhydroxy)-I-(aza)-2-(thio )-3-(aza)-phenoxazinI-yl; 7 N (guanidiniumalkylhydroxy)-I-(aza)-2-(thio )-3-(aza)-phenthiazin-1-yl; 7 (guanidiniumalkylhydroxy)-I -(aza)-2-(thio)-3-(aza)-phenoxazin-I -yl; 7 (guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yI; 7-(guanidiniumalkyl hydroxy)-1,3-( diaza)-2-( oxo )-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-,3-(diaza)-2-( 15 oxo )-phenoxazin-1-yi; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl 7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted I -(aza)-2-(thio)-3-(aza)-phenoxazin- I -yl; 7 substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis
ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-y; Difluorotolyl; Hypoxanthine; 20 Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6 methyl-2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06 substituted purines; 0-alkylated derivative; ortho-(aminoalkylhydroxy)-6-pheny-pyrrolo pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin 25 TP; para-(aminoalkylhydiroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yi; para-substituted-6 phenyl-pyrrolo-pyrimidin-2-on-3-y; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7 (aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino pyridopyrimidin-3-y; pyrrolo-pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5'-TP; 30 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one
ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2' OH--ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'-O1--ara-guanosine
TP; 5-(2-carbomethoxyvinyl)uridine TP; and N6-(19-Amino-pentaoxanonadecyl)adenosine TP. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) includes a combination of at least two (e.g., 2, 3, 4 or more) of the 5 aforementioned modified nucleobases. In some embodiments, modified nucleobases in the polynucleotide (e.g., RNA
polynucleotide, such as mRNA polynucleotide) are selected from the group consisting of pseudouridine (y), NI-methylpseudouridine (mIV), NI-ethylpseudouridine, 2-thiouridine, N 4'-thiouridine, 5-methylcytosine, 2-thio-1 -methyl-I-deaza-pseudouridine, 2-thio-I -methyl N 1o pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2 N thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-I methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5 methyluridine,), 5-methoxyuridine and 2'-O-methyl uridine. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) includes a 15 combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified
nucleobases. Each possibility represents a separate embodiment of the present invention. In some embodiments, modified nucleobases in the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) are selected from the group consisting of I methyl-pseudouridine (ml ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), 20 pseudouridine (y), a-thio-guanosine and a-thio-adenosine. In some embodiments, the polynucleotide includes a combination of at least two (e.g., 2, 3, / or more) of the aforementioned modified nucleobases. Each possibility represents a separate embodiment of the present invention. Each possibility represents a separate embodiment ofthe present invention. 25 In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises pseudouridine (V) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (ml). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine 30 (mh I) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 5-methoxy-uridine (mo5J) and 5 methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA 5 polynucleotide, such as mRNA polynucleotide) comprises 2'-O-methyl uridine. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2'-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6 N methyl-adenosine (m6A). In some embodiments, the polynucleotide (e.g., RNA N i10 polynucleotide, such as mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and N 5-methyl-cytidine (m5C). Each possibility represents a separate embodiment of the present invention. In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) is uniformly modified (e.g., fully modified, modified throughout the entire 15 sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as any of those set forth above. Each possibility 20 1epieselnts a sepate cviibudiiient ofthe prcscnt invention. In some embodiments, the modified nucleobase is a modified cytosine. Examples of nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine. 25 In some embodiments, a modified nucleobase is a modified uridine. Example nucleobases and nucleosides having a modified uridine include 5-cyano uridine or 4'-thio uridine. Each possibility represents a separate embodiment of the present invention. In some embodiments, a modified nucleobase is a modified adenine. Example nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, I-methyl 30 adenosine (ml A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6 Diaminopurine. Each possibility represents a separate embodiment of the present invention. In some embodiments, a modified nucleobase is a modified guanine. Example nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine
(m11), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQl), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m I G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. Each possibility represents a separate embodiment of the present invention. 5 In some embodiments, polynucleotides function as messenger RNA (mRNA).
"Messenger RNA" (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex
N vivo. The basic components of an mRNA molecule typically include at least one coding Ni10 region, a 5'untranslated region (UTR), a 3'UTR, a 5'cap and a poly-A tail. Polynucleotides N may function as mRNA but can be distinguished from wild-type mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics. The mRNA, as provided herein, comprises at least one (one or more) ribonucleic acid 15 (RNA) polynucleotide having an open reading frame encoding at least one polypeptide of interest. In some embodiments, a RNA polynucleotide of an mRNA encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5 9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 polypeptides. In some embodiments, a RNA polynucleotide of an mRNA encodes at least 10, 20, 30, 40, 50 , 60, 70, 20 80, 90 or 100 polypeptides. In some embodiments, a RNA polynucleotide of an mRNA ciicodes at last 100 or at last 200 polypeptidc3.
In some embodiments, the nucleic acids are therapeutic mRNAs. As used herein, the term "therapeutic mRNA" refers to an mRNA that encodes a therapeutic protein. Therapeutic
proteins mediate a variety of effects in a host cell or a subject in order to treat a disease or
25 ameliorate the signs and symptoms of a disease. For example, a therapeutic protein can replace a protein that is deficient or abnormal, augment the function of an endogenous
protein, provide a novel function to a cell (e.g., inhibit or activate an endogenous cellular activity, or act as a delivery agent for another therapeutic compound (e.g., an antibody-drug conjugate). Therapeutic mRNA may be useful for the treatment of the following diseases and 30 conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune disorders.
Thus, the structures of the invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, the mRNA of the structures described herein can be administered to a subject, wherein the polynucleotides are translated in vivo to produce a therapeutic peptide. Provided are compositions, methods, kits, and reagents for diagnosis, treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agents of the invention include the structures, cells containing 5 structures or polypeptides translated from the polynucleotides contained in the structures. The structures may be induced for translation in a cell, tissue or organism. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell, tissue or organism is contacted with an effective amount of a composition containing a structure which contains the mRNA polynucleotides each of which has at least one translatable region encoding a 10 peptide. N An "effective amount" of the structures are provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the nucleic acids, and other determinants. In general, an effective amount of the nucleic acids
15 provides an induced or boosted peptide production in the cell. The mRNA ofthe present invention may be designed to encode polypeptides of interest selected from any of several target categories including, but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular 20 membrane bound proteins, nuclear proteins, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic
indication has been identified but which nonetheless have utility in areas of research and discovery. "Therapeutic protein" refers to a protein that, when administered to a cell has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or 25 pharmacological effect. The mRNA disclosed herein, may encode one or more biologics. As used herein, a
"biologic" is a polypeptide-based molecule produced by the methods provided herein and which may be used to treat, cure, mitigate, prevent, or diagnose a serious or life-threatening disease or medical condition. Biologics, according to the present invention include, but are 30 not limited to, allergenic extracts (e.g. for allergy shots and tests), blood components, gene therapy products, human tissue or cellular products used in transplantation, vaccines,
monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytics, and
immunomodulators, among others.
According to the present invention, one or more biologics currently being marketed or in development may be encoded by the mRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation of the encoding polynucleotides of a known biologic into the mRNA of the invention will result in improved therapeutic efficacy 5 due at least in part to the specificity, purity and/or selectivity of the construct designs. The mRNA disclosed herein, may encode one or more antibodies or fragments thereof. The term "antibody" includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fe region), antibody compositions with N polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and N i10 single-chain molecules), as well as antibody fragments. The term "immunoglobulin" (Ig) is N used interchangeably with "antibody" herein. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, 15 amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or 20 belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include, but are not limited to, "primatized" antibodies comprising variable 25 domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences. An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; 30 nanobodies; single-chain antibody molecules and multispecific antibodies formed from aitibudy fiagments. Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, may be encoded by the mRNA of the invention, including the heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. Also included are polynucleotide sequences encoding the subclasses, gamma and mu. Hence any of the subclasses of antibodies may be encoded in part or in whole and include the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA I and IgA2. According to the present invention, one or more antibodies or fragments currently 5 being marketed or in development may be encoded by the mRNA of the present invention. Antibodies encoded in the mRNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, blood, cardiovascular, CNS, poisoning (including antivenoms), dermatology, endocrinology, gastrointestinal, medical imaging, musculoskeletal, oncology, immunology, respiratory, sensory and anti-infective. N 10 In one embodiment, mRNA disclosed herein may encode monoclonal antibodies N and/or variants thereof. Variants of antibodies may also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives. In one embodiment, the mRNA disclosed herein may encode an immunoglobulin Fc region. In another embodiment, the mRNA may encode a 15 variant immunoglobulin Fe region. The mRNA disclosed herein, may encode one or more vaccine antigens. As used herein, a "vaccine antigen" is a biological preparation that improves immunity to a particular disease or infectious agent. According to the present invention, one or more vaccine antigens currently being marketed or in development may be encoded by the mRNA of the present 20 invention. Vaccine antigens encoded in the mRNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, cancer, allergy and infectious disease. The mRNA of the present invention may be designed to encode on or more antimicrobial peptides (AMP) or antiviral peptides (AVP). AMPs and AVPs have been 25 isolated and described from a wide range of animals such as, but not limited to, microorganisms, invertebrates, plants, amphibians, birds, fish, and mammals. The anti microbial polypeptides described herein may block cell fusion and/or viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example, the anti-microbial polypeptide can comprise or consist of a synthetic peptide corresponding to a region, e.g., a consecutive 30 sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the transmembrane subunit of a viral envelope protein, e.g., HIV-I gp120 or gp4I. The amino acid and nucleotide sequences of HIV- gp120 or gp41 are described in, e.g., Kuiken et al., (2008). "HIV Sequence Compendium," Los Alamos National Laboratory.
In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence. In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding viral protein 5 sequence. In other embodiments, the anti-microbial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a capsid N binding protein. In some embodiments, the anti-microbial polypeptide may have at least N 1o about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding sequence of the capsid binding protein. The anti-microbial polypeptides described herein may block protease dimerization and inhibit cleavage of viral proproteins (e.g., HIV Gag-pol processing) into functional proteins thereby preventing release of one or more enveloped viruses (e.g., HIV, HCV). In 15 some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence. In other embodiments, the anti-microbial polypeptide can comprise or consist ofa synthetic peptide corresponding to a region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a protease zo binding protein. In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding sequence of the protease binding protein. A non-limiting list of infectious diseases that the mRNA vaccine antigens or anti microbial peptides may treat is presented below: human immunodeficiency virus (HIV), [IV 25 resulting in mycobacterial infection, AIDS related Cacheixa, AIDS related Cytomegalovirus infection, HIV-associated nephropathy, Lipodystrophy, AID related cryptococcal meningitis, AIDS related neutropaenia, Pneumocysitisjiroveci (Pneumocystis carinii) infections, AID related toxoplasmosis, hepatitis A, B, C, D or E, herpes, herpes zoster (chicken pox), German measles (rubella virus), yellow fever, dengue fever etc. (flavi viruses), flu (influenza viruses), 30 haemorrhagic infectious diseases (Marburg or Ebola viruses), bacterial infectious diseases such as Legionnaires' disease (Legionella), gastric ulcer (Helicobacter), cholera (Vibrio), E. coli infections, staphylococcal infections, salmonella infections or streptococcal infections, tetanus (Clostridium tetani), protozoan infectious diseases (malaria, sleeping sickness, leishmaniasis, toxoplasmosis, i.e. infections caused by plasmodium, trypanosomes, leishmania and toxoplasma), diphtheria, leprosy, measles, pertussis, rabies, tetanus, tuberculosis, typhoid, varicella, diarrheal infections such as Amoebiasis, Clostridium difficile-associated diarrhea (CDAD), Cryptosporidiosis, Giardiasis, Cyclosporiasis and 5 Rotaviral gastroenteritis, encephalitis such as Japanese encephalitis, Wester equine encephalitis and Tick-borne encephalitis (TBE), fungal skin diseases such as candidiasis, onychomycosis, Tinea captis/scal ringworm, Tinea corporis/body ringworm, Tinea cruris/jock itch, sporotrichosis and Tinea pedis/Athlete's foot, Meningitis such as N Haemophilus influenza type b (Hib), Meningitis, viral, meningococcal infections and N i10 pneumococcal infection, neglected tropical diseases such as Argentine haemorrhagic fever, N Leishmaniasis, Nematode/roundworm infections, Ross river virus infection and West Nile virus (WNV) disease, Non-HIV STDs such as Trichomoniasis, Human papillomavirus (HPV) infections, sexually transmitted chlamydial diseases, Chancroid and Syphilis, Non-septic bacterial infections such as cellulitis, lyme disease, MRSA infection, pseudomonas, 15 staphylococcal infections, Boutonneuse fever, Leptospirosis, Rheumatic fever, Botulism, Rickettsial disease and Mastoiditis, parasitic infections such as Cysticercosis, Echinococcosis, Trematode/Fluke infections, Trichinellosis, Babesiosis, Hypodermyiasis, Diphyllobothriasis and Trypanosomiasis, respiratory infections such as adenovirus infection, aspergillosis infections, avian (H5N I) influenza, influenza, RSV infections, severe acute 20 respiratory syndrome (SARS), sinusitis, Legionellosis, Coccidioidomycosis and swine (HINI) influenza, sepsis such as bacteraemia, sepsis/septic shock, sepsis in premature infants, urinary tract infection such as vaginal infections (bacterial), vaginal infections (fungal) and gonococcal infection, viral skin diseases such as B19 parvovirus infections, warts, genital herpes, orofacial herpes, shingles, inner ear infections, fetal cytomegalovirus 25 syndrome, foodborn illnesses such as brucellosis (Brucella species), Clostridium perfringens (Epsilon toxin), E. Coli 0157:H7 (Escherichia coli), Salmonellosis (Salmonella species), Shingellosis (Shingella), Vibriosis and Listeriosis, bioterrorism and potential epidemic diseases such as Ebola haemorrhagic fever, Lassa fever, Marburg haemorrhagic fever, plague, Anthrax Nipah virus disease, Hanta virus, Smallpox, Glanders (Burkholderia mallei), 30 Melioidosis (Burkholderia pseudomallei), Psittacosis (Chlamydia psittaci), Q fever (Coxiella burnctii), Tularemia (Fancisella tularensis), rubella, mumps and polio. The mRNA disclosed herein, may encode one or more validated or "in testing" therapeutic proteins or peptides. According to the present invention, one or more therapeutic proteins or peptides currently being marketed or in development may be encoded by the mRNA of the present invention. Therapeutic proteins and peptides encoded in the mRNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, blood, cardiovascular, CNS, poisoning (including antivenoms), 5 dermatology, endocrinology, genetic, genitourinary, gastrointestinal, musculoskeletal, oncology, and immunology, respiratory, sensory and anti-infective.
The mRNA disclosed herein, may encode one or more cell-penetrating polypeptides. As used herein, "cell-penetrating polypeptide" or CPP refers to a polypeptide which may N facilitate the cellular uptake of molecules. A cell-penetrating polypeptide of the present N i10 invention may contain one or more detectable labels. The polypeptides may be partially
N labeled or completely labeled throughout. The mRNA may encode the detectable label completely, partially or not at all. The cell-penetrating peptide may also include a signal sequence. As used herein, a "signal sequence" refers to a sequence of amino acid residues bound at the amino terminus of a nascent protein during protein translation. The signal 15 sequence may be used to signal the secretion of the cell-penetrating polypeptide. In one embodiment, the mRNA may also encode a fusion protein. The fusion protein may be created by operably linking a charged protein to a therapeutic protein. As used herein, "operably linked" refers to the therapeutic protein and the charged protein being connected in such a way to permit the expression of the complex when introduced into the cell. As used 20 herein, "charged protein" refers to a protein that carries a positive, negative or overall neutral
electrical charge. Preferably, the therapeutic protein may be covalently linked to the charged protein in the formation of the fusion protein. The ratio of surface charge to total or surface amino acids may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9. The cell-penetrating polypeptide encoded by the mRNA may form a complex after 25 being translated. The complex may comprise a charged protein linked, e.g. covalently linked,
to the cell-penetrating polypeptide.
In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, "protein-binding partner"
30 includes, but is not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell where the mRNA may be introduced. The cell-penetrating polypeptide may also be capable of penetrating the first cell. In one embodiment, the mRNA may encode a cell-penetrating polypeptide which may comprise a protein-binding partner. The protein binding partner may include, but is not 5 limited to, an antibody, a supercharged antibody or afunctional fragment. The mRNA may be introduced into the cell where a cell-penetrating polypeptide comprising the protein binding partner is introduced. Some embodiments of the present disclosure provide a therapeutic mRNA that N includes at least one ribonucleic acid (RNA) polynucleotide having an open reading frame Ni10 encoding at least one antigenic polypeptide, in which the RNA polynucleotide of the RNA N includes at least one chemical modification. In some embodiments, the chemical modification is selected from pseudouridine, NI-methylpseudouridine, N] ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl--deaza pseudouridine, 2-thio-I-methyl-pseudouridine, 2-thio-5-aza-uridine , 2-thio 15 dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio pseudouridine, 4-methoxy-pseudouridine, 4-thio-I-methyl-pseudouridine, 4-thio pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine,), 5-methoxyuridine, and 2'-O-methyl uridine. Each possibility represents a separate embodiment of the present invention. 20 Any of the foregoing polynucleotides of the present disclosure, in some embodiments. are codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs 25 that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold 30 properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms. In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild 5 type mRNA sequence encoding a polypeptide.or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic N protein or polypeptide. In some embodiments, a codon optimized sequence shares less than Ni10 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally
N occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of 15 interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized
sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares between 65% and 85%
20 (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence
identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments, a codon optimized sequence shares between 65% and 75 or about 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a 25 naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide. In some embodiments a codon optimized RNA may, for instance, be one in which the levels of G/C are enhanced. The G/C-content of nucleic acid molecules may influence the stability of the RNA. RNA having an increased amount of guanine (G) and/or cytosine (C) 30 residues may be functionally more stable than nucleic acids containing a large amount of
adenine (A) and thymine (T) or uracil (U) nucleotides. W002/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA. As used herein, when referring to polypeptides the terms "site" as it pertains to amino acid based embodiments is used synonymously with "amino acid residue" and "amino acid 5 side chain." As used herein when referring to polynucleotides the terms "site" as it pertains to nucleotide based embodiments is used synonymously with "nucleotide." A site represents a position within a peptide or polypeptide or polynucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide or polynucleotide based NI molecules. N 10 As used herein the terms "termini" or "terminus" when referring to polypeptides or N polynucleotides refers to an extremity of a polypeptide or polynucleotide respectively. Such extremity is not limited only to the first or final site of the polypeptide or polynucleotide but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid 15 with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a f-ec carboxyl group (COOH)). Proteins are in some cases made up otfmultiple polypeptide chains brought together by disultide bonds or by non-covalent tbrces (multimers, oligomers). These proteins have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based 20 moicty such as an organic conjugate. As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but 25 otherwise identical) of a reference protein 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids which are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the disclosure. In some embodiments, a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 30 mutations as shown in any of the sequences provided or referenced herein. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that are greater than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than
80%, 75%, 70%, 65% or 60% identical to any of the sequences described herein can be utilized in accordance with the disclosure. Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree ofsequence similarity or identity with the reference molecules (e.g., reference 5 polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules). The term "identity" as known in
the art, refers to a relationship between the sequences of two or more polypeptides or
polynucleotides, as determined by comparing the sequences. In the art, identity also means N the degree of sequence relatedness between them as determined by the number of matches N 10 between strings of two or more amino acid residues or nucleic acid residues. Identity N measures the percent of identical matches between the smaller of two or more sequences with
gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., "algorithms"). Identity of related peptides can be readily calculated by known methods. "% identity" as it applies to polypeptide or polynucleotide sequences is defined as the 15 percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence
or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends 20 on a calculation of percent identity but may differ in value due to gaps and penalties
introduced in the calculation. Generally, variants of a particular polynucleotide or
polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment 25 programs and parameters described herein and known to those skilled in the art. Such tools
for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) "Identification of common 30 molecular subsequences." J. Mol. Biol. 147:195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. &
Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453.). More recently a Fast Optimal
Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, specifically in the definition of "identity" below. 5 As used herein, the term "homology" refers to the overall relatedness between
polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Polymeric molecules (e.g. nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or polypeptide molecules) that N share a threshold level of similarity or identity determined by alignment of matching residues N i10 are termed homologous. Homology is a qualitative term that describes a relationship between
N molecules and can be based upon the quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 15 65%,70%,75%,80%,85%, 90%,95%,or99% identical orsimilar. Theterm
"homologous" necessarily refers to a comparison between at least two sequences
(polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, 20 homologous polynucleotide sequences are characterized by the ability to encode a stretch of
at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4 5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least
25 20 amino acids. Homology implies that the compared sequences diverged in evolution from a common origin. The term "homolog" refers to a first amino acid sequence or nucleic acid sequence (e.g., gene (DNA or RNA) or protein sequence) that is related to a second amino
acid sequence or nucleic acid sequence by descent from a common ancestral sequence. The
30 term "homolog" may apply to the relationship between genes and/or proteins separated by the event of speciation or to the relationship between genes and/or proteins separated by the event of genetic duplication. "Orthologs" are genes (or proteins) in different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function in the course of evolution. "Paralogs" are genes (or proteins) related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one. The term "identity" refers to the overall relatedness between polymeric molecules, for 5 example, between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second N nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded N 10 for comparison purposes). In certain embodiments, the'length of a sequence aligned for
N comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second 15 sequence, then the molecules are identical at that position. The percent identity between the
two sequences is a function of the number of identical positions shared by the sequences,
taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment otthe two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a 20 mathematical algorithm. For example, the percent identity between two nucleic acid
sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; 25 Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been 30 incorporated into the ALIGN program (version 2.0) using a PAM]20 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences 5 include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al.,.J Molec. Biol., 215, 403 (1990)). The immunomodulatory agent may be an immunostimulatory agent or an NI immunoinhibitory agent. N 10 An immunostimulatory agent is an agent that stimulates an immune response
N (including enhancing a pre-existing immune response) in a subject to whom it is
administered, whether alone or in combination with another agent. Examples include antigens, adjuvants (e.g., TLR ligands such as imiquimod, imidazoquinoline, nucleic acids comprising an unmethylated CpG dinucleotide, monophosphoryl lipid A or other 15 lipopolysaccharide derivatives, single-stranded or double-stranded RNA, flagellin, muramyl dipeptide), cytokines including interleukins (e.g., [L-2, IL-7, IL-15 (or superagonist/mutant forms of these cytokines), IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand, etc.), immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain/antibody fragments of these molecules), and the like. 20 An immunoinhibitory agent is an agent that inhibits an immune response in a subject
to whom it is administered, whether alone or in combination with another agent. Examples include steroids, retinoic acid, dexamethasone, cyclophosphamide, anti-CD3 antibody or antibody fragment, and other immunosuppressants. Adjvants are agents that enhance an immune response. The adjuvant may be without
25 limitation alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from the
bark of the Q. saponaria tree such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), 30 ISCOMS (immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium), non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g., IMS 1312, water-based nanoparticles combined with a soluble immunostimulant, Seppic) Adjuvants may be TLR ligands. Adjuvants that act through TLR3 include without 5 limitation double-stranded RNA. Adjuvants that act through TLR4 include wihtout limitation derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related N to lipid A; OM Pharma SA, Meyrin, Switzerland). Adjuvants that act through TLR5 include N lo without limitation flagellin. Adjuvants that act through TLR7 and/or TLR8 include single N stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazoquinolinamines (e.g., imiquirnod, resiquimod). Adjuvants acting through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN. Another adjuvant class is phosphorothioate containing molecules such as 15 phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages. The antigen may be without limitation a cancer antigen, a self-antigen, a microbial antigen, an allergen, or an environmental antigen. The antigen may be peptide, lipid, or carbohydrate in nature, but it is not so limited. 20 A cancer antigen is an antigen that is expressed preferentially by cancer cells (i.e., it is expressed at higher levels in cancer cells than on non-cancer cells) and in some instances it is expressed solely by cancer cells. The cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell. The cancer antigen may be MART-I/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal associated 25 antigen (CRC)--C017-1 A/GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AMLI, prostate specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor/CD3-zeta chain, and CD20. The cancer antigen may be selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A 11, 30 MAGE-A 12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE B4), MAGE-Cl, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5). The cancer antigen may be selected from the group consisting of GAGE-L, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9. The cancer antigen may be selected from the group consisting of BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-I, CDK4, tyrosinase, p53, MUC family, HER2/neu, p2lras, RCASI, a-fetoprotein, E-cadherin, a-catenin, P-catenin, y catenin, p120ctn, gp1OOPmell 17, PRAME,NY-ESO-l, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 5 ganglioside, human papilloma virus proteins, Smad family of tumor antigens, Imp-, PI A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20, and c-erbB-2. Each possibility represents a separate embodiment of the present invention. N Microbial antigens are antigens derived from microbial species such as without N 10 limitation bacterial, viral, fungal, parasitic and mycobacterial species. As such, microbial N antigens include bacterial antigens, viral antigens, fungal antigens, parasitic antigens, and mycobacterial antigens. Examples of bacterial, viral, fungal, parasitic and mycobacterial species are provided herein. The microbial antigen may be part of a microbial species or it may be the entire microbe. 15 An anti-cancer agent is an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term. Several anti-cancer agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, 20 mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), anti microtubule agents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, 25 fludarabine, pentostatin, chlorodeoxyadenosine), anthracyclines, vinca alkaloids. or epipodophyllotoxins. Examples of anti-cancer agents include without limitation Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; 30 Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Bortezomib (VELCADE); Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin (a platinum-containing regimen);
Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolernycin; Cisplatin (a platinum-containing regimen); Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquone; Docetaxel (TAXOTERE); Doxorubicin; 5 Droloxifene; Dromostanolone; Duazomycin; Edatrexate; Eflornithine; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin; Erbulozole; Erlotinib (TARCEVA), Esorubicin; Estramustine; Etanidazole; Etoposide; Etoprine; Fadrozole; Fazarabine; Fenretinide; Floxuridine; Fludarabine; 5-Fluorouracil; Flurocitabine; Fosquidone; Fostriecin; N Gefitinib (IRESSA), Gemcitabine; Hydroxyurea; Idarubicin; Ifosfamide; Ilmofosine; N i10 Imatinib mesylate (GLEEVAC); Interferon alpha-2a; Interferon alpha-2b; Interferon alpha Sn I; Interferon alpha-n3; Interferon beta-I a; Interferon gamma-I b; Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLIMID, REVIMID); Letrozole; Leuprolide; Liarozole; Lometrexol; Lomustine; Losoxantrone; Masoprocol; Maytansine; Mechlorethamine; Megestrol; Melengestrol; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Metoprine; 15 Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed (ALIMTA), Pegaspargase; Peliomycin; Pentamustine; Pentomone; Peplomycin; Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate; Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin; Prednimustine; 20 Procarbazine; Puromycin; Pyrazofurin; Riboprine; Rogletimide; Safingol; Semustine; Simtrazene; Sitogluside; Sparfosate; Sparsomycin; Spirogermanium; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tamsulosin; Taxol; Taxotere; Tecogalan; Tegafur; Teloxantrone; Temoporfin; Temozolomide (TEMODAR); Teniposide; Teroxirone; Testolactone; Thalidomide (THALOMID) and derivatives thereof; 25 Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan; Toremifene; Trestolone; Triciribine; Trimetrexate; Triptorelin; Tubulozole; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine; Vincristine; Vindesine; Vinepidine; Vinglycinate; Vinleurosine; Vinorelbine; Vinrosidine; Vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin. 30 The anti-cancer agent may be an enzyme inhibitor including without limitation tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor. The tyrosine kinase inhibitor may be without limitation Genistein (4',5,7 trihydroxyisoflavone), Tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]-propanedinitrile,
Herbimycin A, Daidzein (4',7-dihydroxyisoflavone), AG-126, trans-i-(3'-carboxy-4' hydroxyphenyl)-2-(2",5"-dihydroxy-phenyl)ethane, or HDBA (2-Hydroxy5-(2,5 Dihydroxybenzylamino)-2-hydroxybenzoic acid. The CDK inhibitor may be without limitation p21, p 2 7 , p57, p15,p16, p18, orpl9. The MAP kinase inhibitor maybe without 5 limitation KY12420 (C23 1-1 2 4 0 ),8 CNI-1493,PD98059,or4-(4-Fluorophenyl)-2-(4 inethylsulfinyl phenyl)-5-(4-pyridyl) IH-imidazole. The EGFR inhibitor may be without limitation erlotinib (TARCEVA), gefitinib (IRESSA), WHI-P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 N (ZACTIMA), AEE788, and AG1458. N 10 The anti-cancer agent may be a VEGF inhibitor including without limitation N bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin. The anti-cancer agent may be an antibody or an antibody fragment including without 15 limitation an antibody or an antibody fragment including but not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that 20 binds simultaneously to HER-2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory conditions such as RSV infection), ibritumomab tiuxetan (ZEVALIN, indicated for Non-Hodgkin's lymphoma), cetuximab (ERBITUX), MDX-447, 25 MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CD1), IOR EGF/R3, celogovab (ONCOSCINT OV103), epratuzumab (LYMPHOCIDE), pemtumomab (THERAGYN), and Gliomab-- (indicated for brain cancer, melanoma). A diagnostic agent, which may be referred to herein as an imaging agent, is an agent that emits signal directly or indirectly thereby allowing its detection in vivo. Diagnostic 30 agents such as contrast agents and radioactive agents that can be detected using medical imaging techniques such as nuclear medicine scans and magnetic resonance imaging (MRI). Imaging agents for magnetic resonance imaging (MRI) include Gd(DOTA), iron oxide or gold nanoparticles; imaging agents for nuclear medicine include 2T1, gamma-emitting radionuclide 99 mTc; imaging agents for positron-emission tomography (PET) include positron-emitting isotopes, (I8)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64, gadoamide, and radioisotopes of Pb(II) such as 203 Pb, and I In; imaging agents for in vivo fluorescence imaging such as fluorescent dyes or dye-conjugated nanoparticles. In other 5 embodiments, the agent to be delivered is conjugated, or fused to, or mixed or combined with an diagnostic agent.
The compounds and compositions may be administered to virtually any subject type that is likely to benefit from delivery of agents as contemplated herein. Human subjects are N preferred subjects in some embodiments of the invention. Subjects also include animals such N 10 as household pets (e.g., dogs, cats, rabbits, ferrets, etc.), livestock or farm animals (e.g., cows,
N pigs, sheep, chickens and other poultry), horses such as thoroughbred horses, laboratory
animals (e.g., mice, rats, rabbits, etc.), and the like. Subjects also include fish and other aquatic species.
The subjects to whom the agents are delivered may be normal subjects. Alternatively 15 they may have or may be at risk of developing a condition that can be diagnosed or that can benefit from localized delivery of one or more particular agents. Such conditions include cancer (e.g., solid tumor cancers), infections (particularly infections localized to particular regions or tissues in the body), autoimmune disorders, allergies or allergic conditions, asthma, transplant rejection, and the like. In some embodiments, the subjects have been 20 diagnosed with a genetic defect and are being administered a nucleic acid based therapeutic. Agents may be administered systemically or locally. Agents may be administered in effective amounts. An effective amount is a dosage of the agent sufficient to provide a medically desirable result. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the 25 condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. The invention provides pharmaceutical compositions. Pharmaceutical compositions 30 are sterile compositions that comprise agents and may comprise delivery vehicles, nanoparticles and the like, preferably in a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other subject contemplated by the invention. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the cells, nanoparticles and agent(s) are combined to facilitate administration. The components of the pharmaceutical compositions are commingled in a manner that precludes interaction that would substantially impair their 5 desired pharmaceutical efficiency. The compounds and compositions, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Pharmaceutical parenteral formulations include N 10 aqueous solutions of the ingredients. Aqueous injection suspensions may contain substances
N which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively, suspensions of ingredients may be prepared as oil-based suspensions such as are known in the art or that will be readily apparent to those of ordinary skill in the art based on this disclosure. is This disclosure further contemplates use of LNPs together with one or more secondary agents, including agents that would normally be indicated for the subject. In some instances, the LNPs may be administered substantially simultaneously with the secondary agents. By substantially simultaneously, it is meant that a LNP is administered to a subject close in time with the administration of the secondary agent, including for 20 example with I hour, within 30 minutes, within 10 minutes, or within 5 minutes.
In some instances, the secondary agent(s) may be administered prior to the LNP. For
example, the secondary agent(s) may be administered prior to and within 24 hours, or within
18 hours, or within 12 hours, or within 6 hours, or within 3 hours, or within 2 hours of the LNP administration. The secondary agent(s) may be administered 18-24 hours prior to LNP 25 administration, or 12-18 hours prior to LNP administration, or 6-12 hours prior to LNP administration, or 2-6 hours prior to LNP administration.
Subjects who have been administered one or more secondary agents 2 or more hours prior to LNP administration may be referred to as having been pre-medicated with such agent(s). Subjects who have been administered one or more secondary agents within I hour 30 prior to LNP administration may be referred to as having been co-mediated with such agent(s). In some instances, the secondary agent(s) may be administered continuously to the subject, on an as needed basis or on a regular schedule (e.g., every day, every two days, etc.).
In other instances, the secondary agent may be administered before or after the
administration of the LNP.
Such secondary agents may include but are not limited to anti-histamines, anti-platelet agents, and non-steroidal anti-inflammatory drugs. In certain embodiments, the LNPs are not 5 formulated with and subjects are not pre- or co-medicated with a corticosteroid, such as but
not limited to dexamethasone.
In certain embodiments, single secondary agents having anti-inflammatory and anti platelet effects are used. An example of such an agent is aspirin. N In certain embodiments, a combination of aspirin, clopidrogrel (Plavix@), and an anti Ni10 histamine such as but not limited to diphenhydramine (Benadryl), fexofenadine (Allegra), N loratadine (Claritin), or cetirizine is used. One or more of the secondary agents may be
administered once per LNP administration while others may be administered more frequently. For example, clopidrogrel (Plavix@) may be administered once per LNP administration while aspirin and/or the anti-histamine may be administered daily.
15 Anti-histamines include 1-I receptor antagonists and Hl receptor inverse agonists. Examples of H I receptor antagonists include but are not limited to acrivastine,
alimemazine, alimemazine tartrate, antazoline, astemizole, azatadine, azatadine maleate,
azelastine, bamipine, benzquinamide, bepotastine, bepotastine besilate, bilastine bromazine, bromopheniramine, buclizine, carbinoxamine, chlorphenoxamine, chlorcyclizine, 20 cinnopentazone histapyrrodine, chlorodipheynhydramine, chloropyramine, chlorophenamine, Chlorpromazine, cinnarizine, clemastine, clemizole, clocinizine, cyclizine, cyproheptadine, desloratadine, deptropine, dexchlorpheniramine, dexbrompheniraine, dimenhydrinate,
dimetindene, dimetotiazine, diphenhydramine (Benadryl), piphenylpyraline, doxepin, doxylamine, ebastine, efletirizine, embramine, emedastine, epinastine, fexofenadine 25 (Allegra), flunarizine, homochlorcyclizine, hydroxyzine, isothipendyl, ketotifen, levocabastine (2nd genereation), loratadine (Claritin), mebhydroline, meclozine, mepyramine, mequitazine, methdilazine, mirtazapine, mizolastine, niaprazine, olopatadine, orphenadrine, oxatomide, oxomemazine, pemirolast, phenindamine, pheniramine, phenyltoloxamine, pimethixene, piprinhydrinate, promethazine, propiomazine,
30 pyrrobutamine, quetiapine, quifenadine, rupatadine, setastine, terfenadine, thenyldiamine,
thiethylperazine, thonzylamine, tolpropamine, trimethobenzamine, tripelennamine,
triprolidine and tritoqualine.
Examples of Hl receptor inverse agonists include but are not limited to pyrilamine, cetirizine, levocetirizine, and desoratadine. Anti-platelet agents include but are not limited to activation inhibitors, aggregation inhibitors, adhesion antagonists, anti-coagulation drugs (that do not target platelets directly), 5 and agents that reduce platelet count or numbers.
Examples of activation inhibitors include but are not limited to (1) thrombin receptor
PAR-I inhibitors such as SCH 530348 (vorapaxar), E-5555 (atopaxar), SCH79797, FR 171113, RWJ 56110, BMS-200661, RWJ-58259, SCH205831, Pipal-7 pepducin, Plpal-12 N pepducin; (2) thrombin receptor PAR-4 inhibitors such as ML 354, tcY-NH2, P4pal-10 N 10 pepducin, P4pal-i l pepducin; (3) FSLLRY-NH2 (PAR-2 peptide antagonist); (4) TxA2 N receptor antagonists such as AH 23,848, SQ 29,548, or R 68,070, S-1452, iosartan, seratrodast; (5) thromboxane receptor antagonists such as terutroban; (6) ADP P2Y12 receptor inhibitors such as ticlopidine, clopidogrel, prasugrel, ticagrelor, cangrelor, elinogrel., AZD6140, AR-C69931, CoA; (7) ADP P2Y Ireceptor inhibitors such as A2P5P, A3P5P, 15 MRS2179, MRS2279, MRS2500, palmitoyl-CoA (also acts on P2Y12), and other compounds from SAR study by Thalji et al. 2010; (8) 5-HT2A antagonists such as R-1012444, naftidrofuryl, sarpogrelate, AT-1015; (9) thromboxane syntahase inhibitors such as dazoxiben, CS-518 (TXA2 synthase inhibitor), SB 203580, U63557A, imidazo (1,5-2) pyridine-5-hexanoic acid; (10) COX-l inhibitors such as aspirin, NCX-4016, ridogrel, 20 S18886, picotamide, ramatroban (also TXA2 receptor antagonist), SC-560, FR122047, mofezolac, P6, TFAP, ibuprofen and naproxen (also Cox-2 inhibitors); (11) COX-2 inhibitors such as triflusal (also COX-1 and PDE inhibitor), Etoricoxib, rofecoxib, celecoxib, mcloxicam; and (12) P13K inhibitors such as AZD6482. Examples of aggregation inhibitors include but are not limited to (1) GPa/IIa 25 Inhibitors such as EMS16; (2) GPVI inhibitors such as monoclonal antibodies and Fab fragments of mAb 12A5; (3) GPIIb/IIIa inhibitors such as abciximab, eptifibatide, tirofiban; (4) PDE inhibitors such as dipyridamole (also adenosine reuptake inhibitor), cilostazol (PDE3 inhibitor that results in increased cAMP, and activated PKA), and (5) ADP receptor antagonists. Other platelet aggregation inhibitors include aspirin, clopidrogrel (Plavix@), 30 aspirin/pravastatin, cilostazol, prasugrel, aspirin/dipyridamole, ticagrelor, cangrelor, elinogrel, dipyridamole, and ticlupidine. Examples of adhesion antagonists (to fibrinogen) include but are not limited to CIqTNF-related protein-1, DZ-697b, RG12986.
Examples of non-platelet anti-coagulation agents include but are not limited to warfarin; Xa inhibitors such as rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, otamixaban; thrombin inhibitors such as bivalirudin, hirudin, dabigatran, lepirudin, desirudin, argatroban, melagatran, dabigatran, CDSO3, FDSO3, SDSO3, and additional sulphated 5 benzofurans allorsteric inhibitors reported by Sidhu et al. paper.
Examples of agents that reduce platelet count or number include but are not limited to (1) cAMP phosphodiesterase inhibitors (e.g., anagrelide), 6,7-dichloro-,5-dihydroimidazo
[2,1-b]quinazolin-2(3H)-one or 6,7-dichloro-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2 N one (U.S. Patents 3,932,407; 4,146,718; RE31,617, Haematologica 1992 77:40-3), (2) N i10 antibodies to cell surface receptors specifically expressed by platelets ormegakaryocytes
N such as glycoprotein Jib/IIla receptor antibodies, (3) most chemotherapeutic anti-cancer drugs such as busulphan (Br. J. Haematol. 1986 62:229-37), hydroxyurea (N EngI J Med 1995 332:1132-6), hepsulfan, phosphorus-32 (Br J Radiol 1997 70:1169-73), pipobroman (Scand J. Haematol 1986 37:306-9), cyclophosphamide (J Cell Physiol 1982 112:222-8), 15 certain alkylating agents and certain antimetabolites, (4) cytokines, growth factors and
interleukins such as alpha-interferon (Cancer Immunol Immunother 1987 25:266-73), gamma-interteron, transtorming growth tactor-beta, neutrophil activating peptide-2 and its
analogs (U.S Patent 5,472,944), macrophage inflammatory protein and its analogs (U.S. Patent 5,306,709), (5) compounds secreted by either platelets or megakaryocytes such as 20 platelet-factor 4 (U.S. Patent 5,185,323), transforming growth factor-beta, the 12-17 kD glycoprotein produced by megakaryocytes, thrombin and thrombospondin and its amino (1 174 amino acid) terminal fragment (J Lab Clin Med 1997 129:231-8), and (6) other agents including anti-cheloid agents such as Tranilast (Rizaben) (J Dermatol 1998 25:706-9); forskolin and spleen anti-maturation factor (U.S. Patent 4,088,753). 25 Anti-platelet agents may also be characterized as anti-thrombotic agents, fibrinolytic
agents, direct thrombin inhibitors, glycoprotein Ilb/Ila receptor inhibitors, agents that bind to cellular adhesion molecules and inhibit the ability of white blood cells to attach to such molecules, calcium channel blockers, beta-adrenergic receptor blockers, cyclooxygenase-2 inhibitors, and angiotensin system inhibitors. 30 Anti-thrombotic agents are defined as agents which prevent the formation of a blood
thrombus via a number of potential mechanisms and they include fibrinolytic agents, anti
coagulant agents, and inhibitors of platelet function.
Fibrinolytic agents are defined as agents that lyse a thrombus (e.g., a blood clot), usually through the dissolution of fibrin by enzymatic action. Examples of thrombolytic agents include but are not limited to ancrod, anistreplase, bisobrin lactate, brinolase, Hageman factor (i.e. factor XII) fragments, molsidomine, plasminogen activators such as 5 streptokinase, tissue plasminogen activators (TPA) and urokinase, and plasmin and plasminogen. Anti-coagulant agents also include inhibitors of factor Xa, factor TFPI, factor VIla, factor IXc, factor Va, factor Villa as well as inhibitors of other coagulation factors. Anti-coagulant agents are agents which inhibit the coagulation pathway by impacting N negatively upon the production, deposition, cleavage and/or activation of factors essential in N 10 the formation of a blood clot. Anti-coagulant agents include but are not limited to vitamin K antagonists such as coumarin and coumarin derivatives (e.g., warfarin sodium); glycosoaminoglycans such as heparins both in unfractionated form and in low molecular weight form; ardeparin sodium, bivalirudin, bromindione, coumarin dalteparin sodium, desirudin, dicumarol, lyapolate sodium, nafamostat mesylate, phenprocoumon, sulfatide, and 15 tinzaparin sodium. Other "anti-coagulant" and/or "fibrinolytic" agents include Plasminogen; Streptokinase; Urokinase: Anisoylated Plasminogen-Streptokinase Activator Complex; Pro Urokinase; (Pro-UK); rTPA (alteplase or activase; r denotes recombinant); rPro-UK; Abbokinase; Eminase; Streptase; Anagrelide Hydrochloride; Bivalirudin; Dalteparin 20 Sodium; Danaparoid Sodium; Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin Sodium; Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; retaplase; Trifenagrel; Warfarin; Dextrans. Still other anti-coagulant agents include, but are not limited to, Ancrod; Anticoagulant Citrate Dextrose Solution; Anticoagulant Citrate Phosphate Dextrose Adenine Solution; 25 Anticoagulant Citrate Phosphate Dextrose Solution; Anticoagulant Heparin Solution; Anticoagulant Sodium Citrate Solution; Ardeparin Sodium; Bromindione; Desirudin; Dicumarol; Heparin Calcium; Heparin Sodium; Lyapolate Sodium; Nafamostat Mesylate; Phenprocoumon. Clot lysing agents include, but are not limited to, tissue plasminogen activator, 30 streptokinase, and nimodipine, Inhibitors of platelet function are agents that impair the ability of mature platelets to perform their normal physiological roles (i.e., their normal function). Platelets are normally involved in a number of physiological processes such as adhesion, for example, to cellular and non-cellular entities, aggregation, for example, for the purpose of forming a blood clot, and release of factors such as growth factors (e.g., platelet-derived growth factor (PDGF)) and platelet granular components. One subcategory of platelet function inhibitors are inhibitors of platelet aggregation which are compounds which reduce or halt the ability of 5 platelets to associate physically with themselves or with other cellular and non-cellular components, thereby precluding the ability of a platelet to form a thrombus. Examples of useful inhibitors of platelet function include but are not limited to acadesine, anagrelide, anipamil, argatroban, aspirin, clopidogrel, cyclooxygenase inhibitors N such as nonsteroidal anti-inflammatory drugs and the synthetic compound FR-122047, N i10 danaparoid sodium, dazoxiben hydrochloride, diadenosine 5',5"'-P,P4-tetraphosphate N (Ap4A) analogs, difibrotide, dilazep dihydrochloride, 1,2- and 1,3-glyceryl dinitrate, dipyridamole, dopamine and 3-methoxytyramine, efegatran sulfate, enoxaparin sodium, glucagon, glycoprotein Ib/Ila antagonists such as Ro-43-8857 and L-700,462, ifetroban, ifetroban sodium, iloprost, isocarbacyclin methyl ester, isosorbide-5-mononitrate, itazigrel, 15 ketanserin and BM-13.177, lamifiban, lifarizine, molsidomine, nifedipine, oxagrelate, PGE, platelet activating factor antagonists such as lexipafant, prostacyclin (PGI2), pyrazines, pyridinol carbamate, ReoPro (i.e., abciximab), sultinpyrazone, synthetic compounds BN 50727, BN-52021, CV-415 1, E-5510, FK-409, GU-7, KB-2796, KBT-3022, KC-404, KF 4939, OP-41483, TRK-100, TA-3090, TFC-612 and ZK-36374, 2,4,5,7-tetrathiaoctane, 20 2,4,5,7-tetrathiaoctane 2,2-dioxide, 2,4,5-trithiahexane, theophyllin pentoxifyllin, thromboxane and thromboxane synthetase inhibitors such as picotamide and sulotroban, ticlopidine, tirofiban, trapidil and ticlopidine, trifenagrel, trilinolein, 3-substituted 5,6-bis(4 methoxyphenyl)-1,2,4-triazines, and antibodies to glycoprotein JIb/IIIa as well as those disclosed in U.S. Patent 5,440,020, and anti-serotonin drugs, Clopridogrel; Sulfinpyrazone; 25 Aspirin; Dipyridamole; Clofibrate; Pyridinol Carbamate; PGE; Glucagon; Antiscrotonin drugs; Caffeine; Theophyllin Pentoxifyllin; Ticlopidine. "Direct thrombin inhibitors" include hirudin, hirugen, hirulog, agatroban, PPACK, thrombin aptamers. "Glycoprotein IIb/ilIa receptor inhibitors" are both antibodies and non-antibodies, and 30 include but are not limited to ReoPro (abcixamab), lanifiban, tirofiban. "Calcium channel blockers" are a chemically diverse class of compounds having important therapeutic value in the control of a variety of diseases (Fleckenstein, Cir. Res. v. 52, (suppl. 1), p. 13 - 1 6 (1983); Fleckenstein, Experimental Facts and Therapeutic Prospects,
John Wiley, New York (1983); McCall, D., Curr Pract Cardiol, v. 10, p. 1-11 (1985)). Calcium channel blockers are a heterogeneous group of drugs that prevent or slow the entry of calcium into cells by regulating cellular calcium channels. (Remington, The Science and Practice of Pharmacy, Nineteenth Edition, Mack Publishing Company, Eaton, PA, p.963 5 (1995)). Most of the currently available calcium channel blockers, and useful according to the present invention, belong to one of three major chemical groups of drugs, the dihydropyridines, such as nifedipine, the phenyl alkyl amines, such as verapamil, and the benzothiazepines, such as diltiazem. Other calcium channel blockers useful according to the invention, include, but are not limited to, amrinone, amlodipine, bencyclane, felodipine, 10 fendiline, flunarizine, isradipine, nicardipine, nimodipine, perhexilene, gallopamil, tiapamil N and tiapamil analogues (such as 1993RO-11-2933), phenytoin, barbiturates, and the peptides dynorphin, omega-conotoxin, and omega-agatoxin, and the like and/or pharmaceutically acceptable salts thereof. "Beta-adrenergic receptor blocking agents" are a class of drugs that antagonize the 15 cardiovascular effects of catecholamines in angina pectoris, hypertension, and cardiac arrhythmias. Beta-adrenergic receptor blockers include, but are not limited to, atenolol, acebutolol, alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol, hedroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol, metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol, practolol, practolol, sotalonadolol, 20 tiprenolol, tomalolol, timolol, bupranolol, penbutolol, trimepranol, 2-(3-(1,1-dimethylethyl) amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHClI, 1-butylamino-3-(2,5-dichlorophenoxy) 2-propanol, I -isopropylam ino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol, 3 isopropylamino-I -(7-methylindan-4-yloxy)-2-butanol, 2-(3-t-butylamino-2-hydroxy propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy)phthalide. 25 The above-identified compounds can be used as isomeric mixtures, or in their respective levorotating or dextrorotating form. Cyclooxygenase-2 (COX-2) is a recently identified form of a cyclooxygenase. "Cyclooxygenase" is an enzyme complex present in most tissues that produces various prostaglandins and thromboxanes from arachidonic acid. Non-steroidal, anti-inflammatory 30 drugs exert most of their anti-inflammatory, analgesic and antipyretic activity and inhibit hormone-induced uterine contractions and certain types of cancer growth through inhibition of the cyclooxygenase (also known as prostaglandin G/H synthase and/or prostaglandin endoperoxide synthase). Initially, only one form of cyclooxygenase was known, the
"constitutive enzyme" or cyclooxygenase- (COX-1). It and was originally identified in bovine seminal vesicles. Cyclooxygenase-2 (COX-2) has been cloned, sequenced and characterized initially from chicken, murine and human sources (See, e.g., U.S. Patent 5,543,297, issued August 6, 5 1996 to Cromlish , et al., and assigned to Merck Frosst Canada, Inc., Kirkland, CA, entitled: "Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity"). A number of selective "COX-2 inhibitors" are known in the art. These include, but are not limited to, COX-2 inhibitors described in U.S. Patent 5,474,995 "Phenyl heterocycles N as cox-2 inhibitors"; U.S. Patent 5,521,213 "Diaryl bicyclic heterocycles as inhibitors of Ni10 cyclooxygenase-2"; U.S. Patent 5,536,752 "Phenyl heterocycles as COX-2 inhibitors"; U.S. N Patent 5,550,142 "Phenyl heterocycles as COX-2 inhibitors"; U.S. Patent 5,552,422 "Aryl substituted 5,5 fused aromatic nitrogen compounds as anti-inflammatory agents"; U.S. Patent 5,604,253 "N-benzylindol-3-yl propanoic acid derivatives as cyclooxygenase inhibitors"; U.S. Patent 5,604,260 "5-methanesulfonamido-1-indanones as an inhibitor of 15 cyclooxygenase-2"; U.S. Patent 5,639,780 N-benzyl indol-3-yl butanoic acid derivatives as cyclooxygenase inhibitors"; U.S. Patent 5,677,318 Diphenyl-1,2-3-thiadiazoles as anti inflammatory agents"; U.S. Patent 5,691,374 "Diaryl-5-oxygenated-2-(5H) -furanones as COX-2 inhibitors"; U.S. Patent 5,698,584 "3,4-diaryl-2-hydroxy-2,5-dihydrofurans as prodrugs to COX-2 inhibitors"; U.S. Patent 5,710,140 "Phenyl heterocycles as COX-2 20 inhibitors"; U.S. Patent 5,733,909 "Diphenyl stilbenes as prodrugs to COX-2 inhibitors"; U.S. Patent 5,789,413 "Alkylated styrenes as prodrugs to COX-2 inhibitors"; U.S. Patent 5,817,700 "Bisaryl cyclobutenes derivatives as cyclooxygenase inhibitors"; U.S. Patent 5,849,943 "Stilbene derivatives useful as cyclooxygenase-2 inhibitors"; U.S. Patent 5,861,419 "Substituted pyridines as selective cyclooxygenase-2 inhibitors"; U.S. Patent 25 5,922,742 "Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2 inhibitors"; U.S. Patent 5,925,631 "Alkylated styrenes as prodrugs to COX-2 inhibitors"; all of which are commonly assigned to Merck Frosst Canada, Inc. (Kirkland, CA). Additional COX-2 inhibitors are also described in U.S. Patent 5,643,933, assigned to G. D. Searle & Co. (Skokie, IL), entitled: "Substituted sulfonylphenylheterocycles as cyclooxygenase-2 and 5 3n lipoxygenase inhibitors." Aspirin is an example of a COX-2 inhibitor. A number of the above-identified COX-2 inhibitors arc prodrugs of selective COX-2 inhibitors, and exert their action by conversion in vivo to the active and selective COX-2 inhibitors. The active and selective COX-2 inhibitors formed from the above-identified
COX-2 inhibitor prodrugs are described in detail in WO 95/00501, published January 5, 1995, WO 95/18799, published July 13, 1995 and U.S. Patent 5,474,995, issued December 12, 1995. Given the teachings of U.S. Patent 5,543,297, entitled: "Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity," a person of ordinary skill in the 5 art would be able to determine whether an agent is a selective COX-2 inhibitor or a precursor of a COX-2 inhibitor, and therefore part of the present invention. Non-steroidal anti-inflammatory drugs include but are not limited to naproxen sodium, diclofenac, sulindac, oxaprozin, diflunisal, aspirin, piroxicam, indomethocin, N etodolac, ibuprofen, fenoprofen, ketoprofen, mefenamic acid, nabumetone, tolmetin sodium, N 10 and ketorolac tromethamine. N In some embodiments, the secondary agent may be an agent that inhibits the production of natural IgM, IgG, and/or activation of Bla and/or Bib cells by LNPs. Such agents may be antagonists of a surface receptor of Bl a cells (e.g., CD36 and C5a) or BI b cells, for examples, antibodies or small molecule inhibitors that bind the surface receptor and 15 interfere with its binding to its cognate ligands (e.g., lipid component such as phosphatidylcholine in certain LNPs). In other embodiments, the secondary agent may be an agent that inhibits the activation of platelets and/or complement system (classical pathway or alternative pathway) by LNPs. Such agents may be CD36 antagonists, TLR antagonists, or antagonists of any 20 component in the complement cascade. Such antagonists may be antagonistic antibodies specific to one of the targets. In some examples, the antagonists may be a protease inhibitor that targets one or more of the serine protease component in the complement system. Other CD36 antagonists include, but are not limited to, salvianolic acid or metabolites thereof(e.g., RA and DSS), 3- cinnamoyl indole, 13-pentyl berberine, hexarelin, or certain fatty acids such 25 as DHA. It is to be understood that the disclosure contemplates use of one or more of the foregoing secondary agents with any of the LNP provided herein, including for example those that comprise a cationic lipid such as MC3, a helper lipid such as DSPC or DOPE, a structural lipid such as cholesterol, and a methoxy-PEGylated lipid such as DMG-PEG, 30 including when such methoxy-PEGylated lipid is used at a molar percentage of greater than 0.5% including 1.5%. Thus, the disclosure contemplates that LNPs that would otherwise trigger a platelet response may be used together with secondary agents that include one or more anti-platelet secondary agents. Such combinations are intended to reduce frequency and/or severity of ABC and toxicity related to LNP use in vivo. Also provided herein are methods for reducing drug responses, including ABC and dose-limiting toxicity, associated with LNPs encapsulating mRNAs. 5 ABC is a threshold phenomenon, which means that the dose of an agent such as LNPs must reach a threshold to induce clinically signicant ABC (substantial). Accordingly, it is contemplated that using a dose lower than the threshold could reduce ABC or prevent its occurrence. Alternatively, the LNPs described herein can lower BIa and/or Bib and/or N natural IgM stimulating activity and thus increase the dosing threshold. N 10 In some embodiments, a method for reducing ABC of lipid LNPs encapsulating an N mRNA can be performed by at least (i) administering to a subject in need thereof a first dose of the LNPs, and (ii) administering to the subject a second dose of the LNPs; wherein the first dose, the second dose, or both are equal to or less than about 0.3 mg/kg. For example, the first dose, the second dose, or both can be equal to or less than 0.2 mg/kg or 0.1 mg/kg. In 15 some examples, the first dose, the second dose, or both, can range from about 0.1-0.3 mg/kg.
The interval between the first dose and the second dose can be less than 2 weeks, e.g, less than 10 days, less than I week, less than 4 days, or less than 2 days. When subsequent doses are required, the same low doses described herein may be used. The interval between two consecutive doses may be less than 2 weeks, for example, less than 10 days, less than I week, 20 less than 4 days, or less than 2 days. Dose-limiting toxicity, such as CARPA, refers to side effects of a drug or other treatment that are serious enough to prevent an increase in dose or level of treatment. It is
contemplated that using treatment regimens that could maintain the serum level of LNPs below the threshold for triggering clinically significant dose-limiting toxicity would reduce 25 such toxicity or prevent its occurrence. Accordingly, provided herein is a method for delivering lipid nanoparticles (LNPs) encapsulating an mRNA to a subject without promoting LNP-related toxicity. Such a method comprises administering an amount of the LNPs to a subject during a period, wherein the serum level of the LNPs in the subject during the administration period is not sufficient to 30 induce LNP-related toxicity. The LNP-related toxicity may be coagulopathy, disseminated intravascular coagulation (DIC), vascular thrombosis, activation-related pseudoallergy (CARPA), acute phase response (APR), or a combination thereof.
It is within the knowledge of those skilled in the art to select suitable doses of the mRNA-encapsulating LNPs and the duration of the administration (e.g., infusion) so as to maintain the serum level of the LNPs below the threshold. For example, when a large dose is needed to reach the intended therapeutic effects, a longer administration period can be used. 5 Occurrence of any of the dose-limiting toxicity can be monitored via conventional
approaches in medical practice. The dose and administration period can be adjusted upon
showing of any symptom associated with the toxicity. In some examples, the dose of the LNPs may be lower than 1 mg/kg, e.g., 0.5 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg. In N other examples, the LNP dose may range from 0.5 to I mg/kg (e.g., 0.3 to 0.5 mg/kg). The N 10 administration period may range from 30 minutes to 3 hours, for example 1-2 hours. In some
N instances, the administration period is no less than I hour, for example, no less than 1.5 hours, no less than 2 hours, no less than 2.5 hours, or no less than 3 hours. In any of the methods described herein, the mRNA encapsulated in LNPs can be a therapeutic mRNA, which may code for a therapeutic protein. The mRNA encapsulated in 15 LNPs may also be a mRNA encoding a vaccine antigen. In some instances, the mRNA
encapsulated in LNPs may encode multiple proteins. In some embodiments, the LNPs used
in this method can be any of the LNPs described herein, Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific 20 embodiments are, therefore, to be construed as merely illustrative, and not limitative ofthe
remainder of the disclosure in any way whatsoever. All publications cited herein are
incorporated by reference for the purposes or subject matter referenced herein.
25 Exemplary Assay Methods:
1. Bead assays by How cytometry:
Streptavidin CML latex beads (Polysciences Inc) were coupled with biotinylated DSPC (6mm beads) or biotinylated PEG (10 mm beads) following manufacturer's recommendations. Coupled Beads (DSPC coupled and PEG coupled) were incubated with 30 diluted serum from mice injected with different LNPs for 30 minutes at room temperature.
After washing, beads were then incubated with a rat anti-mouse IgM IgG (BD biosciences) for 15 minutes at room temperature. After washing, cells were resuspended in PBS + 2% BSA and analyzed by flow cytometry with a BD Fortesssa (BD Biosciences). Titers of anti
LNP IgM were calculated based on standard curve obtained with an anti-PEG IgM
monoclonal antibody. Analysis was performed with FlowJo and Prism Software.
2. In vitro platelet activation assay with LNPs or LNPs components
5 Blood samples were collected in 6mL BD Vacutainer containing ImL anticoagulant
citrate dextrose (BD biosciences) and centrifuged with no acceleration and no brake at 200 x g, 22°C, for 20 minutes. The top, transparent layer of platelet rich plasma (PRP) was transferred into a I5mL conical tube and washed in PBS+2% fetal calf serum. After counting, N 10 cells were incubated at room temperature for different time points with different LNPs or
N 10 LPS or different LNP components and stained with anti-CD41, CD31 and CD62P fluorescently labeled for 20min on ice. After washing cells were fixed and analyzed by flow cytometry with a BD Fortessa (BD Biosciences). Analysis was performed with FlowJo and Prism Software.
15 3. In vitro platelet aggregation with macrophages, B cells
Blood sample were collected in 6mL BD Vacutainer containing 1mL anticoagulant citrate
dextrose (BD biosciences). 10-25ml of blood were incubated at room temperature for different time points at room temperature with different LNPs or LPS and stained with anti
CD41, CD] lb, CD19 and F4/80 fluorescently labeled for 20min on ice. After washing cells 20 were fixed and analyzed by flow cytometry with a BD Fortessa (BD Biosciences). Analysis was performed with FlowJo and Prism Software.
4. In vivo platelet activation assay
Mice were injected intravenously with different LNPs. After different time points, Blood
25 sample were collected in 6mL BD Vacutainer containing lmL anticoagulant citrate dextrose
(BD biosciences) and centrifuged with no acceleration and no brake at 200 x g, 22°C, for 20 minutes. The top, transparent layer of platelet rich plasma (PRP) was transferred into a l5mL 5 conical tube and washed in PBS+2% fetal calf serum. After counting, 10 cells were stained with anti-CD41, CD31 and CD62P fluorescently labeled for 20min on ice. After washing 30 cells were fixed and analyzed by flow cytometry with a BD Fortessa (BD Biosciences). Analysis was performed with FlowJo and Prism Software.
5. In vivo platelet aggregation with macrophages, B cells
Mice were injected intravenously with different LNPs. After different time points, Blood sample were collected in 6mL BD Vacutainer containing ImL anticoagulant citrate dextrose (BD biosciences).I0-25ml of blood were the stained with anti-CD41, CDllb, CD19 and 5 F4/80 fluorescently labeled for 20min on ice. After washing cells were fixed and analyzed by flow cytometry with a BD Fortessa (BD Biosciences). Analysis was performed with FlowJo and Prism Software.
N 6. In vivo splenic B cell activation assay:
10 Spleen of injected animals with fluorescent LNPs were collected in saline buffer. Splenocytes cell suspension were prepared by gently pressing the spleen through a 70-M mesh cell strainer (Fisher Scientific). After washing, red blood cells was lysed and cells were resuspended in PBS+2% fetal calf serum. After washing and counting, 105 cells were stained with anti-CD19, CD86 and CD69 fluorescently labeled for 20min on ice. After washing cells 15 were fixed and analyzed by flow cytometry with a BD Fortessa (BD Biosciences). Analysis was performed with FlowJo and Prism Software.
7. In vivo LNP interaction with B cells:
Spleen of injected animals with fluorescent LNPs were collected in saline buffer. 20 Splenocytes cell suspension were prepared by gently pressing the spleen through a 70-gM mesh cell strainer (Fisher Scientific). After washing, red blood cells was lysed and cells were resuspended in PBS+2% fetal calf serum. After washing and counting, 103 cells were stained with anti-CD19 and CD5 fluorescently labeled for 20min on ice. After washing cells were fixed and analyzed by flow cytometry with a BD Fortessa (BD Biosciences). Analysis was 25 performed with FlowJo and Prism Software.
8. In vitro splenic B cell activation assay:
Spleen of injected animals with fluorescent LNPs were collected in saline buffer. Splenocytes cell suspension were prepared by gently pressing the spleen through a 70-ltM 30 mesh cell strainer (Fisher Scientific). After washing, red blood cells was lysed and cells were resuspended in PBS+2% Fetal calf serum. After counting, 105 cells were incubated at 37C for the indicated time points with different LNPs or medium. After incubation, cells were stained anti-CD19, CD86 and CD69 fluorescently labeled for 20min on ice. After washing cells were
Claims (15)
1. A method for delivering a therapeutic level of a protein of interest to a subject, wherein the method comprises administering multiple doses of a lipid nanoparticle (LNP) to 2023203042
the subject; wherein the LNP encapsulates an mRNA coding for the protein of interest, wherein the LNP comprises an ionizable lipid, a helper lipid, a structural lipid, and a PEG lipid, wherein the PEG lipid is a compound of Formula (V-OH):
(V-OH), or a pharmaceutically acceptable salt thereof, wherein: r is an integer between 1 and 100, inclusive; and R5 is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and wherein the LNP does not induce a drug response associated with an LNP.
2. Use of a lipid nanoparticle (LNP) in the manufacture of a medicament for delivering a therapeutic level of a protein of interest to a subject; wherein the LNP encapsulates an mRNA coding for the protein of interest, wherein the LNP comprises an ionizable lipid, a helper lipid, a structural lipid, and a PEG lipid, wherein the PEG lipid is a compound of Formula (V-OH):
(V-OH), or a pharmaceutically acceptable salt thereof, wherein: r is an integer between 1 and 100, inclusive; and
R5 is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; wherein the LNP does not induce a drug response associated with an LNP; and wherein multiple doses of the LNP are administered to the subject.
3. The method of claim 1 or the use of claim 2, wherein said drug response is a CD19(+) 2023203042
mediated immune response.
4. The method of claims 1 or 3, or the use of claims 2 or 3, wherein said drug response is selected from induction of IgM, induction of IgG, induction of memory B cells, an ABC response, and an anti-drug antibody (ADA) response.
5. The method of any one of claims 1, 3 or 4, or the use of any one of claims 2-4 wherein the PEG-lipid comprises a PEG molecule of an average molecular weight of: (i) 2,000 Da; or (ii) less than 2,000 Da, optionally around 1,500 Da, around 1,000 Da, or around 500 Da.
6. The method of any one of claims 1 or 3-5, or the use of any one of claims 2-5, wherein the compound of Formula (V-OH) is Cmpd452:
(Cmpd452) or a pharmaceutically acceptable salt thereof.
7. The method of any one of claims 1 or 3-5, or the use of any one of claims 2-5, wherein the PEG-lipid is HO-PEG2000-ester-C18.
8. The method of any one of claims 1 or 3-5, or the use of any one of claims 2-5, wherein the compound of Formula (V-OH) is Cmpd403:
(Cmpd403). 2023203042
9. The method of any one of claims 1 or 3-8, or the use of any one of claims 2-8, wherein the helper lipid is: (i) a non-cationic helper lipid comprising at least one fatty acid chain of at least 8C and at least one polar head group moiety, wherein the structural lipid is a sterol; (ii) a zwitterionic non-cationic helper lipid, or oleic acid; (iii) a lipid that is not a phosphatidyl choline (PC); or (iv) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
10. The method of any one of claims 1 or 3-9, or the use of any one of claims 2-9, wherein the helper lipid is a non-cationic helper lipid and the structural lipid is cholesterol, and wherein the LNP has a molar ratio of about 45-65% cationic lipid, about 0.15-15% PEG lipid, about 15-45% cholesterol and about 5-25% non-cationic helper lipid.
11. The method of any one of claims 1 or 3-10, or the use of any one of claims 2-10, wherein the LNP comprises less than 0.5 % (w/w) of PEG lipid.
12. The method of any one of claims 1 or 3-11, or the use of any one of claims 2-11, wherein the LNP is characterized as having reduced binding to B1a cells and/or reduced B1a cell activation activity.
13. The method of any one of claims 1 or 3-11, or the use of any one of claims 2-11, wherein administration of the LNP does not activate CD36, optionally wherein: (i) the helper lipid competitively inhibits phosphatidylcholine from binding to CD36; or (ii) the helper lipid does not bind or has low binding activity to CD36.
14. The method of any one of claims 1 or 3-13, or the use of any one of claims 2-13, wherein the LNP encapsulating the mRNA encoding the protein of interest is administered two or more times, three or more times, or four or more times; and/or wherein administration is repeated once, twice, 3, 4, 5, 6, 7, 8, 9, 10, or more times. 2023203042
15. The method of any one of claims 1 or 3-14, or the use of any one of claims 2-14, wherein the interval between a first dose and a second dose is about 21 days or less; and/or wherein administration further comprises administering to the subject an additional agent that inhibits immune responses.
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