(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(l•J w;~:s~:!~:::.:opcrty ~ llllllllllll~~~~~~~~;~~~~~~~~~~~~~~~~.~~~~~~~~~~llllllllll
`WO 2018/232357 Al
`
`(43) International Publication Date ~
`20 December 2018 (20.12.2018) WI P 0 I PC T
`
`(51) International Patent Classification:
`A61K 9/51 (2006.01)
`A61K 47128 (2006.01)
`A61K 3117105 (2006.01)
`A61K 47154 (2017.01)
`A61K 3117088 (2006.01)
`A61K 48100 (2006.01)
`A61K 47114 (2017.01)
`C12N 15188 (2006.01)
`A61K 47124 (2006.01)
`
`(21) International Application Number:
`PCT/US2018/037922
`
`(22) International Filing Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`15 June 2018 (15.06.2018)
`
`English
`
`English
`
`(30) Priority Data:
`62/520,530
`62/590,200
`
`US
`15 June 2017 (15.06.2017)
`22 November 2017 (22.11.2017) US
`
`(71) Applicant: MODERNATX, INC. [US/US]; 200 Technol(cid:173)
`ogy Square, Cambridge, MA 02139 (US).
`
`(72) Inventors: HOGE, Stephen; 320 Bent Street, Cam(cid:173)
`bridge, MA 02141 (US). SCHARITER, Joseph; 207
`Hammond Street, Waltham, MA 02451 (US). BOWER(cid:173)
`MAN, Charles; 745 South St, Waltham, MA 02453 (US).
`
`SMITH, Michael, H.; 25 Whiting Way, Needham, MA
`02492 (US). XIA, Yan; 140 Lyman St., Apt. 5, Waltham,
`MA 02452 (US).
`
`(74) Agent: LOCKHART, Helen, C. et al.; Wolf, Greenfield &
`Sacks, P.C., 600 Atlantic Avenue, Boston, MA 02210-2206
`(US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
`HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP,
`KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, VA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`
`(54) Title: RNA FORMULATIONS
`
`" Day 36 (2wl<s post-boost)
`Day 21 p wl<.s post-prime)
`
`6~-------r------~--------r-------~-------,
`****
`$
`@
`®®
`~
`
`~
`@
`
`®
`
`®
`
`1% PEGT·
`1% PEGT·
`1.5%PEGT-
`mtxlng (standard mi.xing/0.5% PEG mlKing/0.5% PEG
`formu!a:ti.o.n}
`pO·'>t··insettlm'!
`fin;ll <tdditlon
`
`FIG.15
`
`(57) 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.
`
`[Continued on next page]
`
`---;;;;;;;;;;;;;;;
`;;;;;;;;;;;;;;; -------
`---
`--
`
`--;;;;;;;;;;;;;;; -;;;;;;;;;;;;;;; -
`
`PROTIVA - EXHIBIT 2052
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc.
`IPR2018-00739
`
`

`

`W 0 2018/23 23 57 Al 11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR, NE, SN, TD, TG).
`
`Published:
`-
`with international search report (Art. 21(3))
`-
`with sequence listing part of description (Rule 5.2(a))
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`RNA FORMULATIONS
`
`RELATED APPLICATIONS
`
`This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional
`
`5
`
`application number 62/520,530, filed June 15, 2017 and U.S. provisional application number
`
`62/590,200, filed November 22, 2017, which are both incorporated by reference herein in
`
`their entirety.
`
`10
`
`The present embodiments relate generally to lipid nanoparticles, and more
`
`specifically, to lipid nanoparticles having a certain distribution of one or more components.
`
`FIELD OF INVENTION
`
`BACKGROUND
`
`It is of great interest in the fields of therapeutics, diagnostics, reagents, and for
`
`15
`
`biological assays to be able to control protein expression. Most methods rely upon
`
`regulation at the transcriptional level (e.g., from DNA to mRNA), but not at the translational
`
`level (e.g., from mRNA to protein). Although attempts have been made to control protein
`
`expression on the translational level, the low levels of translation, the immunogenicity, and
`
`other delivery issues have hampered the development of mRNA as a therapeutic.
`
`20
`
`There remains a need in the art to be able to design, synthesize, and deliver a nucleic
`
`acid, e.g., a ribonucleic acid (RNA) such as a messenger RNA (mRNA) encoding a peptide
`
`or polypeptide of interest inside a cell, whether in vitro, in vivo, in-situ, or ex vivo, so as to
`
`effect physiologic outcomes which are beneficial to the cell, tissue or organ and ultimately to
`
`an orgamsm.
`
`25
`
`SUMMARY
`
`Lipid nanoparticles having a certain distribution of one or more components, related
`
`compositions, and methods associated therewith are provided. The present disclosure is
`
`based, in part, on the discovery that the distribution of certain components within the lipid
`
`30
`
`nanoparticles can influence and/or dictate physical (e.g., stability) and/or biological (e.g.,
`
`efficacy, intracellular delivery, immunogenicity) properties of the lipid nanoparticles.
`
`Inventive lipid nanoparticles having a certain distribution of one or more components may
`
`1
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`not suffer from one or more limitations of conventional particulate carriers, even though the
`
`inventive lipid nanoparticles may contain the same or similar molecules (e.g., at the molar
`
`ratios, at the same weight percentages) as the conventional particulate carrier. Compositions
`
`comprising inventive lipid nanoparticles may have advantageous biological and physical
`
`5
`
`properties.
`
`Methods for controlling the distribution of components capable of imparting
`
`beneficial properties to the lipid nanoparticle have also been discovered. In some cases,
`
`these methods may be readily applied to the formulation process using relatively simple
`
`techniques.
`
`10
`
`In one set of embodiments, compositions are provided. In one embodiment, a
`
`composition comprises lipid nanoparticles (LNPs) that comprise an ionizable lipid, a PEG
`
`lipid, and inaccessible mRNA, and a relatively small amount of accessible mRNA. In such
`
`cases, no more than about 50% (e.g., no more than about 45%, 40%, 35%, 30%, 25%, 20%,
`
`15%, 10%, 5%, 3%, or 1%) of mRNA in the composition is accessible mRNA and the half-
`
`15
`
`life time of the PEG lipid in serum is relatively short, e.g., less than or equal to about 3.0
`
`hours (e.g, less than or equal to about 2.75, 2.5, 2.25, 2.0, 175, 1.5, 1.25, 1.0, 0.75, 0.5, or
`
`0.25 hours). In some cases, no more than 30% of mRNA in the composition is accessible
`
`mRNA. In certain cases, no more than 5% of mRNA in the composition is accessible
`
`mRNA. In some cases, the quantitative value of the amount of accessible mRNA is
`
`20
`
`generated using an ion-exchange chromatography (lEX) assay and/or is not generated using
`
`a Ribogreen assay. In some embodiments, the lipid nanoparticles may also comprise a
`
`structural lipid and/or a neutral lipid. In some cases, the ionizable lipid is an ionizable
`
`amino lipid.
`
`In another embodiment, a composition comprises lipid nanoparticles (LNPs)
`
`25
`
`comprising an ionizable lipid, a PEG lipid, and mRNA and having an exterior region and
`
`one or more interior regions. The majority of the mRNA is positioned in the one or more
`
`interior regions and the majority of the PEG lipid is positioned within the exterior region.
`
`For instance, at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%,
`
`or 100%) of the mRN A is positioned within the one or more interior regions and at least
`
`30
`
`about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the PEG
`
`lipid is positioned within the exterior region. In some embodiments, the lipid nanoparticles
`
`may also comprise a structural lipid and/or a neutral lipid. In some cases, the ionizable lipid
`
`2
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`is an ionizable amino lipid. In some embodiments, the composition further comprises a
`
`continuous phase. In some such cases, the exterior region is in direct contact with the
`
`continuous phase.
`
`In one embodiment, a composition comprises lipid nanoparticles (LNPs) comprising
`
`5
`
`an ionizable lipid, a PEG lipid, and mRNA. The majority of the PEG lipid is surface
`
`accessible and the majority of the mRNA in the composition is inaccessible. For instance, at
`
`least about 50% (e.g., at least about 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
`
`100%) of the PEG lipid in the lipid nanoparticles is surface accessible and no more than
`
`about 50% of mRNA (e.g., no more than about 45%, 40%, 35%, 30%, 25%, 20%, 15%,
`
`10
`
`10%, 5%, 3%, 1%, or 0%) in the composition is accessible mRNA. In some embodiments,
`
`the lipid nanoparticles may also comprise a structural lipid and/or a neutral lipid. In some
`
`cases, the ionizable lipid is an ionizable amino lipid.
`
`In another embodiment, a composition comprises lipid nanoparticles (LNPs)
`
`comprising an ionizable lipid, a PEG lipid, and mRNA. The surface polarity of the lipid
`
`15
`
`nanoparticles is relatively low (e.g., lower than a threshold) and the half-life time of the PEG
`
`lipid is relatively short. For instance, the half-life time of the PEG lipid in serum is less than
`
`or equal to about 3.0 hours (e.g., less than or equal to about 2.75, 2.5, 2.25, 2.0, 175, 1.5,
`
`1.25, 1.0, 0.75, 0.5, or 0.25 hours) and the normalized general polarization of laurdan in the
`
`lipid nanoparticles is greater than or equal to about 0.5 (e.g., greater than or equal to about
`
`20
`
`0.55, 0.6, 0.65, 0.7, 0.75, 0.8, or 0.85). In some cases, the normalized general polarization of
`
`laurdan in the lipid nanoparticles is greater than or equal to about 0.5 and less than or equal
`
`to about 0.9. In some embodiments, the lipid nanoparticles may also comprise a structural
`
`lipid and/or a neutral lipid. In some cases, the ionizable lipid is an ionizable amino lipid.
`
`In one embodiment, a composition comprises lipid nanoparticles (LNPs) comprising
`
`25
`
`an ionizable lipid, a PEG lipid, and mRNA. The surface polarity of the lipid nanoparticles is
`
`less than a threshold and the half-life time of the PEG lipid is relatively short. For instance,
`
`the half-life time of the PEG lipid in serum is less than or equal to about 3.0 hours (e.g., less
`
`than or equal to about 2.75, 2.5, 2.25, 2.0, 175, 1.5, 1.25, 1.0, 0.75, 0.5, or 0.25 hours). In
`
`some cases, the surface polarity of the lipid nanoparticles is less than that of a comparative
`
`30
`
`lipid nanoparticle. In some cases, the comparative lipid nanoparticles formed via a
`
`nanoprecipitation reaction, wherein the comparative lipid nanoparticles comprise the same
`
`ionizable lipid, PEG lipid, and mRNA as the lipid nanoparticles, and wherein greater than
`
`3
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`about 30% (e.g., greater than about 35%, greater than about 40%, greater than about 45%,
`
`greater than about 50%, greater than about 55%, greater than about 60%, greater than about
`
`65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than
`
`about 85%, greater than about 90%, greater than about 95%, 100%) of the PEG lipid
`
`5
`
`nanoparticles in the comparative lipid nanoparticles originated from the nanoprecipitation
`
`reaction. In some embodiments, the lipid nanoparticles may also comprise a structural lipid
`
`and/or a neutral lipid. In some cases, the ionizable lipid is an ionizable amino lipid.
`
`In another embodiment, a composition comprises lipid nanoparticles (LNPs)
`
`comprising an ionizable lipid, a PEG lipid, and mRNA. The majority of the PEG lipid is
`
`10
`
`surface accessible and the surface polarity is relatively low. For instance, greater than about
`
`50% (e.g., at least about 55%,60% 65%,70%,75%, 80%, 85%,90%,95%, or 100%) of the
`
`PEG lipid in the lipid nanoparticles is surface accessible and the normalized general
`
`polarization of laurdan in the lipid nanoparticles is greater than or equal to about 0.5 (e.g.,
`
`greater than or equal to about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, or 0.85). In some cases, the
`
`15
`
`normalized general polarization of laurdan in the lipid nanoparticles is greater than or equal
`
`to about 0.5 and less than or equal to about 0.9. In some embodiments, the lipid
`
`nanoparticles may also comprise a structural lipid and/or a neutral lipid. In some cases, the
`
`ionizable lipid is an ionizable amino lipid.
`
`In one embodiment, a composition comprises lipid nanoparticles (LNPs) comprising
`
`20
`
`an ionizable lipid, a PEG lipid, and mRNA. The majority of the PEG lipid is surface
`
`accessible and the surface polarity is lower than a threshold. For instance, greater than about
`
`50% (e.g., at least about 55%,60% 65%,70%,75%, 80%, 85%,90%,95%, or 100%) of the
`
`PEG lipid in the lipid nanoparticles is surface accessible. In some cases, the surface polarity
`
`of the lipid nanoparticles is less than that of a comparative lipid nanoparticle. In some cases,
`
`25
`
`the comparative lipid nanoparticles formed via a nanoprecipitation reaction, wherein the
`
`comparative lipid nanoparticles comprise the same ionizable lipid, PEG lipid, and mRNA as
`
`the lipid nanoparticles, and wherein greater than about 30% (e.g., greater than about 35%,
`
`greater than about 40%, greater than about 45%, greater than about 50%, greater than about
`
`55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than
`
`30
`
`about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater
`
`than about 95%, 100%) of the PEG lipid nanoparticles in the comparative lipid nanoparticles
`
`originated from the nanoprecipitation reaction. In some embodiments, the lipid
`
`4
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`nanoparticles may also comprise a structural lipid and/or a neutral lipid. In some cases, the
`
`ionizable lipid is an ionizable amino lipid.
`
`In another embodiment, a composition comprises lipid nanoparticles (LNPs)
`
`comprising an ionizable lipid, a PEG lipid, and mRNA and having an exterior region and
`
`5
`
`one or more interior regions. The majority of the PEG lipid is positioned within the exterior
`
`region and the surface polarity of the lipid nanoparticles is relatively low. For instance, at
`
`least about 60% (e.g., at least about 65%,70%,75%, 80%, 85%,90%,95%, or 100%) of the
`
`PEG lipid is positioned within the exterior region and the normalized general polarization of
`
`laurdan in the lipid nanoparticles is greater than or equal to about 0.5 (e.g., greater than or
`
`10
`
`equal to about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, or 0.85). In some cases, the normalized general
`
`polarization of laurdan in the lipid nanoparticles is greater than or equal to about 0.5 and less
`
`than or equal to about 0.9. In some embodiments, the lipid nanoparticles may also comprise
`
`a structural lipid and/or a neutral lipid. In some cases, the ionizable lipid is an ionizable
`
`amino lipid. In some embodiments, the composition further comprises a continuous phase.
`
`15
`
`In some such cases, the exterior region is in direct contact with the continuous phase.
`
`In one embodiment, a composition comprises lipid nanoparticles (LNPs) comprising
`
`an ionizable lipid, a PEG lipid, and mRNA and having an exterior region and one or more
`
`interior regions. The majority of the PEG lipid is positioned within the exterior region and
`
`the surface polarity of the lipid nanoparticles is lower than a threshold. For instance, at least
`
`20
`
`about 60% (e.g., at least about 65%,70%,75%, 80%, 85%,90%,95%, or 100%) of the PEG
`
`lipid is positioned within the exterior region. In some cases, the surface polarity of the lipid
`
`nanoparticles is less than that of a comparative lipid nanoparticle. In some cases, the
`
`comparative lipid nanoparticles formed via a nanoprecipitation reaction, wherein the
`
`comparative lipid nanoparticles comprise the same ionizable lipid, PEG lipid, and mRNA as
`
`25
`
`the lipid nanoparticles, and wherein greater than about 30% (e.g., greater than about 35%,
`
`greater than about 40%, greater than about 45%, greater than about 50%, greater than about
`
`55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than
`
`about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater
`
`than about 95%, 100%) of the PEG lipid nanoparticles in the comparative lipid nanoparticles
`
`30
`
`originated from the nanoprecipitation reaction. In some embodiments, the lipid
`
`nanoparticles may also comprise a structural lipid and/or a neutral lipid. In some cases, the
`
`ionizable lipid is an ionizable amino lipid. In some embodiments, the composition further
`
`5
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`comprises a continuous phase. In some such cases, the exterior region is in direct contact
`
`with the continuous phase.
`
`In another embodiment, a composition comprises lipid nanoparticles (LNPs)
`
`comprising an ionizable lipid and inaccessible mRNA, and a relatively small amount of
`
`5
`
`accessible mRNA. In such cases, no more than about 50% (e.g., no more than about 45%,
`
`40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, or 1%) of mRNA in the composition is
`
`accessible mRN A and the normalized general polarization of laurdan in the lipid
`
`nanoparticles is greater than or equal to about 0.5 (e.g., greater than or equal to about 0.55,
`
`0.6, 0.65, 0.7, 0.75, 0.8, or 0.85). In some cases, the normalized general polarization of
`
`10
`
`laurdan in the lipid nanoparticles is greater than or equal to about 0.5 and less than or equal
`
`to about 0.9. In some embodiments, the lipid nanoparticles may also comprise a PEG lipid,
`
`structural lipid, and/or a neutral lipid. In some cases, the ionizable lipid is an ionizable
`
`amino lipid.
`
`In one embodiment, a composition comprises lipid nanoparticles (LNPs) comprising
`
`15
`
`an ionizable lipid and inaccessible mRNA, and a relatively small amount of accessible
`
`mRNA. In such cases, no more than about 50% (e.g., no more than about 45%, 40%, 35%,
`
`30%,25%,20%, 15%, 10%,5%, 3%, or 1 %) ofmRNA in the composition is accessible
`
`mRNA and the surface polarity is lower than a threshold. In some cases, the surface polarity
`
`of the lipid nanoparticles is less than that of a comparative lipid nanoparticle. In some cases,
`
`20
`
`the comparative lipid nanoparticles formed via a nanoprecipitation reaction, wherein the
`
`comparative lipid nanoparticles comprise the same ionizable lipid and mRNA as the lipid
`
`nanoparticles, and wherein greater than about 30% (e.g., greater than about 35%, greater
`
`than about 40%, greater than about 45%, greater than about 50%, greater than about 55%,
`
`greater than about 60%, greater than about 65%, greater than about 70%, greater than about
`
`25
`
`75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than
`
`about 95%, 100%) of the PEG lipid nanoparticles in the comparative lipid nanoparticles
`
`originated from the nanoprecipitation reaction. In some embodiments, the lipid
`
`nanoparticles may also comprise a structural lipid, a PEG lipid, and/or a neutral lipid. In
`
`some cases, the ionizable lipid is an ionizable amino lipid.
`
`30
`
`In another embodiment, a composition comprises lipid nanoparticles (LNPs)
`
`comprising an ionizable lipid and mRNA and having an exterior region and one or more
`
`interior regions. The majority of the mRNA is positioned in the one or more interior regions
`
`6
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`and the surface polarity of the lipid nanoparticles is relatively low. For instance, at least
`
`about 60% (e.g., at least about 65%,70%,75%, 80%, 85%,90%,95%, or 100%) of the
`
`mRNA is positioned within the one or more interior regions and the normalized general
`
`polarization of laurdan in the lipid nanoparticles is greater than or equal to about 0.5 (e.g.,
`
`5
`
`greater than or equal to about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, or 0.85). In some cases, the
`
`normalized general polarization of laurdan in the lipid nanoparticles is greater than or equal
`
`to about 0.5 and less than or equal to about 0.9. In some embodiments, the lipid
`
`nanoparticles may also comprise a structural lipid, a PEG lipid, and/or a neutral lipid. In
`
`some cases, the ionizable lipid is an ionizable amino lipid. In some embodiments, the
`
`10
`
`composition further comprises a continuous phase. In some such cases, the exterior region
`
`is in direct contact with the continuous phase.
`
`In one embodiment, a composition comprises lipid nanoparticles (LNPs) comprising
`
`an ionizable lipid and mRNA and having an exterior region and one or more interior regions.
`
`The majority of the mRNA is positioned in the one or more interior regions and the surface
`
`15
`
`polarity is lower than a threshold. For instance, at least about 60% (e.g., at least about 65%,
`
`70%,75%, 80%, 85%, 90%, 95%, or 100%) of the mRNA is positioned within the one or
`
`more interior regions. In some cases, the surface polarity of the lipid nanoparticles is less
`
`than that of a comparative lipid nanoparticle. In some cases, the comparative lipid
`
`nanoparticles formed via a nanoprecipitation reaction, wherein the comparative lipid
`
`20
`
`nanoparticles comprise the same ionizable lipid and mRNA as the lipid nanoparticles, and
`
`wherein greater than about 30% (e.g., greater than about 35%, greater than about 40%,
`
`greater than about 45%, greater than about 50%, greater than about 55%, greater than about
`
`60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than
`
`about 80%, greater than about 85%, greater than about 90%, greater than about 95%, 100%)
`
`25
`
`of the PEG lipid nanoparticles in the comparative lipid nanoparticles originated from the
`
`nanoprecipitation reaction. In some embodiments, the composition further comprises a
`
`continuous phase. In some such cases, the exterior region is in direct contact with the
`
`continuous phase.
`
`In one embodiment, a composition comprises lipid nanoparticles (LNPs) comprising
`
`30
`
`an ionizable lipid and inaccessible mRNA, and very little accessible mRNA, e.g., no more
`
`than about 30% (e.g., no more than about 25%, 20%, 15%, 10%, 5%, 3%, or 1%) of mRNA
`
`in the composition is accessible mRNA. In some embodiments, the lipid nanoparticles may
`
`7
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`also comprise a structural lipid, a PEG lipid, and/or a neutral lipid. In some cases, the
`
`ionizable lipid is an ionizable amino lipid.
`
`In another embodiment, a composition comprises lipid nanoparticles (LNPs)
`
`comprising an ionizable lipid, a PEG lipid, and mRNA. A substantial amount of the lipid
`
`5
`
`nanoparticles in the composition are enhanced lipid nanoparticles. The enhanced lipid
`
`nanoparticles have more inaccessible mRNA than the accessible mRNA. For instance, at
`
`least about 50% (e.g., at least about 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
`
`100%) of the lipid nanoparticles in the composition are enhanced lipid nanoparticles. In
`
`some embodiments, the lipid nanoparticles may also comprise a structural lipid and/or a
`
`10
`
`neutral lipid. In some cases, the ionizable lipid is an ionizable amino lipid.
`
`In certain embodiments, at least about 95% of the PEG lipid in the composition is
`
`surface accessible. In some embodiments, at least about 95% of the PEG lipid is surface
`
`accessible in at least about 95% of the LNPs in the composition. In certain embodiments, at
`
`least about 95% of the mRNA in the composition is inaccessible. In some embodiments, at
`
`15
`
`least about 95% of the mRNA is inaccessible in at least about 60% (e.g., at least about 65%,
`
`at least about 70%, at least about 75%, at least about 80% at least about 85%, at least about
`
`90%, at least about 95%) of the LNPs in the composition. In some embodiments, at least
`
`about 95% of the mRNA is inaccessible in at least about 95%.
`
`In some embodiments, the PEG lipid comprising two or more aliphatic groups that
`
`20
`
`are indirectly attached. In certain cases, the PEG lipid is not a hydroxyl-PEG lipid. In some
`
`cases, the PEG lipid is a methoxy-PEGylated lipid. In certain cases, the PEG lipid does not
`
`have the following structure:
`
`0
`
`HOVo~Rs
`
`wherein r is 45. In other embodiments, the PEG lipid does have the above structure. In
`
`25
`
`some cases, the PEG-lipid is not Compounds 419, 420, 421, 422, 423, 424, 425, 426, 427, or
`
`428. In other cases, the PEG-lipid is Compounds 419, 420, 421, 422, 423, 424, 425, 426,
`
`427, or 428. In certain cases, the LNPs have a molar ratio of ionizable amino lipid:
`
`structural lipid: neutral lipid: PEG-lipid other than 50:38.5:10:1.5. In certain cases, the
`
`LNPs have a molar ratio of ionizable amino lipid: structural lipid: neutral lipid: PEG-lipid of
`
`30
`
`50:38.5:10:1.5. In some cases, the PEG lipid is less than 1.5 in the molar ratio of ionizable
`
`amino lipid: structural lipid: neutral lipid: PEG-lipid. In other cases, the PEG lipid is not
`
`8
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`less than 1.5 in the molar ratio of ionizable amino lipid: structural lipid: neutral lipid: PEG(cid:173)
`
`lipid.
`
`In some embodiments, at least about 50%, at least about 55%, at least about 60%, at
`
`least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about
`
`5
`
`85%, at least about 90%, or at least about 95% of the mRNA in the composition is fully
`
`encapsulated. In some cases, the quantitative value of the amount of accessible mRNA
`
`and/or fully encapsulated mRNA is generated using an ion-exchange chromatography (lEX)
`
`assay and/or is not generated using a Ribogreen assay.
`
`In some embodiments, surface polarity is determined using one or more fluorescent
`
`10
`
`probes. The one or more fluorescent probes may comprise prodan. The one or more
`
`fluorescent probes may comprise laurdan.
`
`In general, the compositions may have relatively high in vitro and/or in vivo
`
`expression. The composition may have an in vitro expression of mRNA that is greater than
`
`a threshold value. The threshold value may be the value from a comparative lipid
`
`15
`
`nanoparticle. In some cases, an in vitro expression of the mRNA in the composition is
`
`greater than comparative lipid nanoparticles formed via a nanoprecipitation reaction,
`
`wherein the comparative lipid nanoparticles comprise the same ionizable lipid, PEG lipid,
`
`and mRNA as the lipid nanoparticles, and wherein greater than 30% of the PEG lipid
`
`nanoparticles in the comparative lipid nanoparticles originated from the nanoprecipitation
`
`20
`
`reaction. The composition may have an in vivo expression of mRNA that is greater than a
`
`threshold value. The threshold value may be the value from a comparative lipid
`
`nanoparticle. In some cases, an in vivo expression of the mRNA is greater than comparative
`
`lipid nanoparticles formed via a nanoprecipitation reaction, wherein the comparative lipid
`
`nanoparticles comprise the same ionizable lipid, PEG lipid, and mRNA as the lipid
`
`25
`
`nanoparticles, and wherein greater than 30% of the PEG lipid nanoparticles in the
`
`comparative lipid nanoparticles originated from the nanoprecipitation reaction.
`
`In one set of embodiments, compositions comprising precursor lipid nanoparticles
`
`are provided. In one embodiment, a composition comprises lipid nanoparticles (LNPs)
`
`comprising an ionizable lipid, a PEG lipid, and mRNA, wherein at least about 50% of the
`
`30
`
`lipid nanoparticles in the composition are precursor lipid nanoparticles, the precursor lipid
`
`nanoparticles have more mRNA associated with the ionizable lipid than the PEG lipid, and
`
`the precursor lipid nanoparticles comprise at least about 0.01 mol% and less than or equal to
`
`9
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`about 1.0 mol% of the PEG lipid. In some cases, at least about 50% (e.g., at least about
`
`60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the mRNA in the precursor lipid
`
`nanoparticles is associated with the ionizable lipid. In some instances, less than about 50%
`
`(e.g., less than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the mRNA in the
`
`5
`
`precursor lipid nanoparticles is associated with the PEG lipid. In certain cases, the ratio of
`
`mRNA associated with the ionizable lipid to mRNA associated with the PEG lipid in the
`
`precursor lipid nanoparticles is at least about 2:1 (e.g., at least about 3:1,4:1, or 5:1). In
`
`some cases, the composition further comprises an organic solvent (e.g., ethanol). In some
`
`instances, the precursor lipid nanoparticles comprise at least about 0.05 mol% (e.g., at least
`
`10
`
`about 0.1 mol%, 0.2 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, or 0.8
`
`mol%) of the PEG lipid.
`
`In one aspect, the present disclosure is based, at least in part, on the discoveries that
`
`lipid nanoparticles (LNPs) may be designed in order to provide stealth delivery of
`
`therapeutic payload without inducing a damaging innate immune response. Components of
`
`15
`
`prior art LNPs, such as phosphatidylcholine, induce the production of natural IgM and/or
`
`IgG molecules, which may be mediated by activation of B 1 cells, such as B 1a and/or B 1b
`
`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 (CARP A). In some
`
`20
`
`embodiments, the LNPs of the invention are designed as a composition having optimal
`
`surface properties that avoid immune cell recognition. Highly effective compositions having
`
`enriched populations of LNPs that avoid immune activation are provided in aspects of the
`
`invention.
`
`In some aspects, the invention is a composition comprising an enriched population of
`
`25
`
`LNPs, wherein the LNPs have an outer shell and an inner core and comprise an ionizable
`
`lipid, a phospholipid, a PEG lipid, and optionally a structural lipid, wherein at least about
`
`50% (e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 100%) of the LNPs comprise RNA
`
`encapsulated within the inner core and wherein the outer shell comprises at least about 80,
`
`85, 90, 95 or 100% of the PEG lipid. In some aspects, between about 90 and 100% of the
`
`30
`
`LNPs comprise RNA encapsulated within the inner core and the outer shell comprises at
`
`least about 95% of the total PEG lipid. In some aspects, between about 95 and 100% of the
`
`10
`
`

`

`wo 2018/232357
`
`PCT/US2018/037922
`
`LNPs comprise RNA encapsulated within the inner core and about 95% of the total PEG
`
`lipid in the outer shell.
`
`In some aspects, the invention is a composition comprising an enriched population of
`
`lipid nanoparticles (LNPs), wherein the LNPs have an outer shell and an inner core and
`
`5
`
`comprise an ionizable lipid, a phospholipid, a PEG lipid, and optionally a structural lipid,
`
`wherein at least 95% of the LNPs comprise RNA encapsulated within the inner coreand
`
`wherein the outer shell comprises greater than 95% of the total PEG lipid.
`
`In other aspects, the invention is a composition comprising an enriched population
`
`of lipid nanoparticles (LNPs) comprising RNA, wherein the LNPs have an outer shell and an
`
`10
`
`inner core and comprise an ionizable lipid, a phospholipid, a PEG lipid, and optionally a
`
`structural lipid, wherein at least 95% of the RNA in the composition is encapsulated within
`
`the LNPs and wherein the outer shell comprises greater than 95% of the total PEG lipid.
`
`According to other aspects the invention is a composition comprising an enriched
`
`population of lipid nanoparticles (LNPs), wherein the LNPs have an outer shell and an inner
`
`15
`
`core and comprise an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid,
`
`wherein at least 50% (e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%) of the LNPs have an
`
`outer shell fluidity value of greater than a threshold polarization level and wherein RNA is
`
`encapsulated within the and wherein the outer shell comprises greater than 95% of the total PEG
`
`lipid.
`
`20
`
`In yet other aspects the invention is a composition comprising an enriched
`
`population of lipid nanoparticles (LNPs), wherein the LNPs have an outer shell and an inner
`
`core and comprise an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid,
`
`wherein at lea