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`EXHIBIT 4
`EXHIBIT 4
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`Molecular Therapy
`Nucleic Acids
`Original Article
`
`NO) GENE«CELL
`
`td THERAPY
`
`Optimization of Lipid Nanoparticles
`for Intramuscular Administration
`of MRNA Vaccines
`
`Kimberly J. Hassett,! Kerry E. Benenato,' Eric Jacquinet,' Aisha Lee,'! Angela Woods,' Olga Yuzhakov,!
`Sunny Himansu,! Jessica Deterling,' Benjamin M. Geilich,! Tatiana Ketova,' Cosmin Mihai,! Andy Lynn,'
`Iain McFadyen,' Melissa J. Moore,' Joseph J. Senn,' Matthew G.Stanton,'? Orn Almarsson,' Giuseppe Ciaramella,'»*
`and Luis A. Brito!
`
`'Moderna Therapeutics, 200 Technology Square, Cambridge, MA 02139, USA
`
`vectored vaccines do. Lastly, proteins produced by nucleic acid based
`vaccines can provide a morenatural presentation to the immune sys
`tem,yielding better T cell responses.’ Even so, morethan two decades
`after thefirst proof of conceptreport,’ no nucleic acid based vaccine
`has been approved for use in humans.
`
`mRNAvaccines have the potential to tackle many unmet med-
`ical needs that are unable to be addressed with conventional
`vaccine technologies. A potent and well-tolerated delivery tech-
`nology is integral to fully realizing the potential of mRNA vac-
`cines. Pre-clinical and dinical studies have demonstrated that
`mRNA delivered intramuscularly (IM) with first-generation
`lipid nanoparticles (LNPs) generates robust
`immune re-
`A key factor hampering both DNA and mRNAvaccine development
`is the lack of a potent, well tolerated delivery system. Because DNA
`sponses. Despite progress made over the past several years,
`there remains significant opportunity for improvement, as
`requires delivery to the nucleus, an inherently inefficient process,
`the most advanced LNPs were designed for intravenous (IV)
`high doses (1 2 mg) and an electroporation device are required to
`delivery of siRNAto the liver. Here, we screened a panel ofpro-
`generate robust immune responses. Although recent advances in
`prietary biodegradable ionizable lipids for both expression and
`DNAelectroporation have shown promise, the broad adoption of
`the technology will likely be limited dueto the necessity of a special
`immunogenicity in a rodent model when administered IM. A
`ized device and the pain associated with electroporation.” An
`subset of compoundswasselected and further evaluated for
`tolerability, immunogenicity, and expression in rodents and
`advantage ofmRNAover DNAis that mRNAonlyrequires cytosolic
`non-human primates (NHPs). A lead formulation was identi-
`delivery. In rodents, early studies showed that intramuscular admin
`istration of buffer formulated mRNAcan lead to measurablelevels of
`fied that yielded a robust immune response with improved
`immunogenicity.’ However, a recentphaseItrial of a rabies mRNA
`tolerability. More importantly for vaccines, increased innate
`vaccine administered in Ringer’s buffer yielded no immunogenicity
`immune stimulation driven by LNPs does not equate to
`unless delivered with a high pressure intra dermal injection device.'°
`increased immunogenicity,
`illustrating that mRNA vaccine
`tolerability can be improved without affecting potency.
`
`INTRODUCTION
`Sincethe first active immunization, vaccines have provided increased
`life expectancy and improved public health, saving countless lives."*
`Today, a variety of technologies exist for vaccine development,
`includinglive and attenuatedviruses, recombinantproteins, synthetic
`peptides, glycoconjugates, and nucleic acids.’ Nucleic acid (DNA and
`mRNA)basedvaccines offer several advantages over other technolo
`gies. They can be rapidly produced with reduced development time
`and costs by using a common manufacturing platform and purifica
`tion methods regardless of the antigen. Unlike manufacturing for
`other vaccines, these methods would not include propagation of
`viruses or purification of a recombinantprotein. The antigen would
`be expressedin situ, allowing for transmembrane domains to be pre
`sent, if needed, and multimeric complexes to be formed.’ Addition
`ally, nucleic acids do not suffer from anti vector immunity like viral
`
`the need for more
`these results highlight
`Although promising,
`potent intracellular delivery technologies for mRNAvaccines. One
`such technology is lipid nanoparticles (LNPs). LNPsare typically
`composed of an ionizablelipid, cholesterol, PEGylated lipid, and a
`helper lipid such as distearoylphosphatidylcholine (DSPC). Early
`work with small interfering RNA (siRNA) identified the ionizable
`lipid as the primary driver of potency.’ '’ The most clinically
`
`Received 28 September 2018; accepted 26 January 2019;
`https://doi.org/10.1016/j.omtn.2019.01.013.
`Present address: GenerationBio, 215First Street, Suite 150, Cambridge, MA 02142,
`USA
`
`>Present address: Beam Therapeutics, 325 Vassar Street, Cambridge, MA 02139,
`USA
`
`Correspondence: Luis A. Brito, Moderna Therapeutics, 200 Technology Square,
`Cambridge, MA 02139, USA.
`E-mail: luis. brito@modernatx.com
`
`(2)
`
`This is an open access article underthe otBY NC NDlicense (http://creativecommons.org/licenses/by nc nd/4.0/).
`
`lecular Therapy: Nucleic Acids Vol. 15 April 2019 © 2019 The Authors.
`
`1
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`Document 193-4 Filed 01/16/24 Page 3 of 19 PagelD #: 12561
`Case 1:22-cv-00252-MSG
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`Molecular Therapy: Nucleic Acids
`
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`Figure 1. Pharmacokinetics of LNPs containing MC3 after IM administration in mice
`Lipid concentration (nanograms per gram)after IM administration of modified MRNA encoding luciferase formulated in LNPs containing MCS(graytriangles) in muscle,liver,
`and spleen up to 24 h post injection (n = 3 per group per time point).
`
`advanced LNPcontainsthe ionizablelipid MC3 and has been shown
`to be safe in humans after intravenous (IV) administration of
`siRNA.'* Our own vaccinetrials with MC3 based LNPsforinfluenza
`gave 100% seroconversion with a 100 ug dose of modified mRNA.
`However, consistent with other vaccines,'*'® we did observe mild
`to moderate local and systemic adverse events.'’ As healthy individ
`uals ranging from day old newbornsto the elderly receive vaccines,
`critical features for broad vaccine adoption are minimal injection
`site reactivity and high tolerability. To date, the only LNPsevaluated
`for intramuscular (IM) mRNAvaccinedelivery were originally opti
`mized for IV delivery of siRNAto theliver.'®*’ Although there are
`preclinical reports of novel LNPs being evaluated for vaccines, no
`rationale has been provided regarding formulation composition or
`selection.””-
`
`Here, we describe rational evolution and selection of an improved
`formulation for IM administration ofmRNA,focusing on the impact
`of the ionizable lipid componentas the primary driver of expression
`andtolerability. Our previous experience with IV administration of
`the proprietary ionizable lipids showed rapid clearance compared.
`to MC3,”° resulting in improved systemic tolerability. Our work
`here illustrates that the ideal formulation for IV expression is not
`necessarily ideal for IM expression. Additionally, we also show that
`increased innate immunestimulation driven by the LNP is not neces
`sary for increased immunogenicity,
`illustrating that we have an
`opportunity to improve vaccine tolerability without affecting vaccine
`potency.
`
`RESULTS
`Observations of mild to moderate adverse events in our clinical work
`with MC3”’ and data showing slow MC3clearanceafter IV adminis
`tration”’ fueled a hypothesis that the adverse events mightbe related
`to the extended presence ofMC3at theinjection site. Mass spectrom
`
`etry analysis ofmuscle tissue revealed that, 24 h after IM injection,the
`MC3concentration only decreased by 50% compared to Cmax (Fig
`ure 1A). Further, MC3 wasalso detectable in liver and spleen 24 h
`post IM injection (Figures 1B and 1C). Thus, IM administration of
`MC3 formulated mRNA LNPsresulted in extended local and sys
`temic lipid exposure.
`
`The goal of the work described here was to identify a new ionizable
`lipid with improvedtolerability and a potency equal or better than
`that of MC3. To do so, we screened 30 novel LNPs,each containing
`a different ionizable lipid in place of MC3. Each LNP formulation
`maintained the same lipid nitrogen to phosphate ratio (N:P) and
`molar composition oflipid components (ionizable lipid, cholesterol,
`phospholipid, and polytheylene glycol [PEG]lipid). Co formulation
`of mRNAsencodingfirefly luciferase and the H10N8influenza hem
`agglutinin (HA) antigen allowed both protein expression and immu
`nogenicity to be evaluated in the samestudy. Luciferase activity was
`measured by whole body imaging 6 h post IM injection ofthe first
`dose. Immunogenicity was evaluated by quantifying « H10 immuno
`globulin (Ig)G titers 2 weeks after the second dose, which was admin
`istered 3 weeks after the first. The ionizable lipids screened hereall
`contain a tertiary amine with ester containing lipid tails to enable
`rapid in vivo metabolism.”° In addition, wealso tested the quaternary
`ammonium containing lipid N [1 (2,3 Dioleoyloxy)propyl] N,N,N
`trimethylammonium (DOTAP).
`
`Consistent with our previous publications, MC3 formulated mRNA
`yielded robust titers and protein expression at a low dose (0.001 mg
`per kg).'””“ In contrast, we observed nodetectable protein expression
`or immunogenicity for DOTAP containing LNPs (Figure 2A). Many
`of our novel biodegradable lipids proved superior to MC3 for
`both protein expression and immunogenicity upon IM administra
`tion. However, there was no strong relationship between protein
`
`2
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`Molecular Therapy: Nucleic Acids Vol. 15 April 2019
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`

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`Case 1:22-cv-00252-MSG Document 193-4 Filed 01/16/24 Page 4 of 19 PageID #: 12562
`Filed 01/16/24 Page 4 of 19 PagelD #: 12562
`Case 1:22-cv-00252-MSG Document 193-4
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`www.moleculartherapy.org
`
`A
`IM (0.001 mpk) immunogenicity
`
`IM (0.001mpk) expression
`IM (0.01mpk) expression
`IV (0.5mpk) expression
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`
`expression (log)
`
`Lipid pK,
`
`Figure 2. Expression and Immunogenicity from LNPs Containing Novellonizable Lipids in Mice
`(A) Thirty novellipid LNPs, A through E’ were compared to a D (MC) LNP control for expression and immunogenicity. Lipids are arrangedleft to right in order of pKa from low
`(A) to high (DOTAP). Expression measured by luminescencein flux (photons per second)6 h after administration of modified MRNA encoding luciferase delivered at 0.5 mg/kg
`lVinCD 1 mice, 0.01 mg/kg IM or 0.001 mg/kg IM in BALB/c mice(n = 5 per group). Immunogenicity measured by H10 specific IgG titers measured 2 weeksafter two doses
`administered 3 weeks apart delivered IM at 0.001 mg/kg IM in BALB/c mice (n = 5 per group). Data are represented as logs fold change compared to MC3. Squares
`containing an X indicate >4 fold change (logs) lower than for MC3.(B) Logs fold increase in expression was comparedto the logs fold change in immunogenicity at the low
`doselevel administered IM (0.001 mg/kg}. Thefive lead novel lipids and MC3 LNPsare labeled accordingly: MC3 (gray triangles), lipid H (green circles), lipid M (orange
`squares), lipid P (purple diamonds),lipid Q (tan inverted triangles), andlipid N (yellow hexagons). (C) Lipid pK, versus fold increasein immunogenicity at 0.001 mg/kg IM for
`lipids A through E’. (D) Circulating IgG antibody (microgramspermilliliter of serum) 6 h after administration of 0.2 mg/kg modified MRNAs encoding the heavy chain and light
`chain of an influenza monoclonal antibody formulated at a 2:1 massratio in LNPs containing MC3 or novellipids (n = 5 per group). *p < 0.05; “p < 0.01; *“p < 0.001; “p<
`0.0001, ordinary one way ANOVAwith Dunnett’s multiple comparisons test of each novellipid versus MC3.
`
`expression and immunogenicity (r = 0.54). Of the 14 lipids yielding
`higher @ H10 IgG titers than MC3, four lipids yielded significantly
`less luciferase expression relative to MC3, whereas four lipids yielded
`significantly greater luciferase activity (Figure 2B). The twolipids
`with the highest «& H10 IgG titers were only 1.3 fold better than
`MC3 with regard to protein expression,
`illustrating that protein
`expression upon IM administration was a poor predictor of
`immunogenicity.
`
`Wealso foundlittle correspondence in rank between the LNPs with
`regard to IM versus IV expression (Figure 2A),illustrating that for
`mulations can behave differently when administered locally versus
`systemically. A possible explanation for the lack ofcorrelation be
`tween IM and IV performance could be that the optimal physical
`or chemical properties differ between the two routes. One strong
`determinant of immunogenicity was the lipid pKa, with a range of
`6.6 6.9 being optimal for IM immunogenicity (Figure 2C). This dif
`fers from the optimal pKa range for IV delivery of siRNAs and
`mRNAs,which has been reported as 6.2 6.6.''”? mRNA encapsula
`tion efficiencies and LNP sizes ranged from 69% to 100% and from
`50 to 142 nm, respectively. While there was norelationship between
`encapsulation efficiency and either IM protein expression or immu
`
`nogenicity, there was a relationship between both readouts and
`LNPsize, with the best performing formulations being 75 95 nm
`(Figures $1A and S1B).
`
`For further study, we picked the five ionizable lipids exhibiting the
`greatest increase in & H10 IgG titers compared to MC3 (colored
`symbols in Figure 2; structures in Figure 3A). Notably, the pKa
`for all five lipids was very close to 6.75 (Figure 2C). As an additional
`measure of potency, we compared the ability of each lead LNP to
`drive the expression ofa secreted IgG antibody after IM administra
`tion in mice (Figure 2D). With the exception oflipid Q, the other
`four lipids yielded higher IgG serum concentrations than MC3
`(p < 0.05).
`
`To understand the biodegradability of these lipids, we measuredlipid
`levels after IM administration. As expected, IM delivery ofthese LNPs
`in CD 1 mice wasfollowed by rapid clearance (Figures 3B 3D). All
`lead lipids degraded faster than MC3 in muscle (Figure 3B), spleen
`(Figure 3C), andliver (Figure 3D). 24 h post injection, the amount
`of lipid present in muscle dropped considerably from peak levels
`for all formulations tested, though lipids H and Q did not return to
`baselinelevels by 48 h. Liver and spleenlipid levels closely followed
`
`Molecular Therapy: Nucleic Acids Vol. 15 April2019
`
`3
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`

`

`Case 1:22-cv-00252-MSG Document 193-4 Filed 01/16/24 Page 5 of 19 PageID #: 12563
`Filed 01/16/24 Page 5 of 19 PagelD #: 12563
`Case 1:22-cv-00252-MSG Document 193-4
`
`Molecular Therapy: Nucleic Acids
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`Figure 3. Chemical Structure and Pharmacokinetics of Lead Lipids
`(A) Chemical structures and pK, of MC3 and novellipids. (B D) Lipid concentration (nanograms per gram) after IM administration of modified mRNA encodingluciferase
`formulated in LNPs containinglipid H @reencircles), lipid M (orange squares), lipid P (purple diamonds), lipid Q (tan invertedtriangles), andlipid N (yellow hexagons)in (B}
`muscle, (C)liver, and (D) spleen up to 48 h post injection (n = 3 per group pertime point).
`
`IM lipidlevels, though lipid H showed a peak at 6 h that dropped by
`24 h in the spleen andliver.
`
`Immunogenicity in non human primates (NHPs) wasevaluated after
`IM injections ofH1ON8 mRNAformulated with thefive lead lipids as
`LNPs. ELISA antibodytiters (Figure 4A) and HAItiters (Figure 4B)
`were notstatistically different for any group (one way ANOVA,
`p > 0.05), except lipid P was significantly lower than MC3after the
`first dose (one way ANOVA,p < 0.01) by ELISA andafter the second
`dose (one way ANOVA,p < 0.001) by HAItiter. Immune responses
`were measurable after a single dose by ELISA. After a second dose,
`both HAI and ELISAtiters boosted considerably, indicating strong
`immunepriming.
`
`Wealso tested protein expression of the five lead lipids in NHPs.
`500 wg IgG mRNAformulated in LNPs was injected IM, and serum
`antibody expression levels were monitored for 2 weeks. While three
`out ofthe five selected lipids yielded expression comparableto that
`of MC3 based LNPs, lipid H (p < 0.001) and lipid M (p = 0.05)
`showedsignificantly more expression over time then MC3 (Fig
`ure 4C). Forlipid H, the maximum antibody concentration measured
`24 h post injection was three times the antibody concentration
`measured with MC3 formulated material.
`
`To assesstolerability in NHPs,thesite of injection was monitored for
`edema (Figure 4D) and erythema(Figure 4E) 1 and 3 days after injec
`tion and was rated based on severity. Despite enhanced protein
`
`4
`
`Molecular Therapy: Nucleic Acids Vol. 15 April 2019
`
`

`

`Case 1:22-cv-00252-MSG Document 193-4 Filed 01/16/24 Page 6 of 19 PageID #: 12564
`Case 1:22-cv-00252-MSG Document 193-4 Filed 01/16/24 Page 6 of 19 PagelD #: 12564
`
`www.moleculartherapy.org
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`Figure 4. Expression and Immunogenicity in Non-human Primates
`(A and B) Immunogenicity measured by H10 specific (A) ELISA or (B) HAI at days 0, 21 (8 weeks after the first dose), and 42 (8 weeks after the second dose). Each dosein
`cynomolgus monkeys contained 5 yg modified mRNA encoding H10N8 formulated in LNPs containing either MC3 (graytriangles),lipid H (greencircles), lipid M (orange
`squares), lipid P (purple diamonds), lipid Q (tan inverted triangles), orlipid N (yellow hexagons) (n = 3 per group). (C) Circulating IgG levels (in microgramspermilliliter) after a
`500 yg IM administration in cynomolgus monkeys of modified MRNA encoding heavy andlight chain antibodiesin a 2:1 weight ratio formulated in LNPs containing MC3 or
`novellipids (n = 3 per group). (D and E) Site of injection was monitored for (D) edema and (E) erythema 1 and 3 days afterinjection.(F) Circulating IL 6 levels (in picograms per
`milliliter) 6 h after administration. *p > 0.05; **p > 0.001, two way ANOVAwith Dunnett’s multiple comparisontest of eachlipid versus MC3at each time point. *““p > 0.01;
`*"*") > 0.0001, z test of areas under the curve (AUCs) for each novellipid versus MC3.
`
`expression, NHPs injected with lipid H based LNPs exhibited no
`signs of swelling or redness 1 or 3 days post injection, with all
`NHPsreceiving a score of 0 for both edema and erythema.All other
`novellipids evaluatedelicited mild to moderate scores for edema and
`erythemain at least 1 animal dosed. The MC3 group had one NHP
`receive a score of 3 for edema on day 1 post injection, resolving to
`a score of 1 on day 3 post injection. All lipids tested, except for lipid
`H,elicited an erythemascore of 1 in at least one NHP. Serum inter
`leukin (IL) 6 levels were comparable for all lipids based on one way
`ANOVA(Figure 4F). The NHPs in the MC3 group with the highest
`
`level of IL 6 also showed the highest level of edema, indicating a
`strong innate immuneresponsein that individual animal.
`
`To assess and compare thelocal tolerability of the different ioniz
`able lipid LNPs, we administered 0.01 mg or 0.1 mg mRNA ex
`pressing prM E from the Zika virus formulated in either MC3,
`lipid H, lipid M, lipid P, lipid Q, or lipid N in Sprague Dawley
`rats IM. Serum cytokines in rats receiving both the high and low
`doses were measured 6 h after administration, using a 22 plex
`Luminex panel. Changes were observed in eotaxin, GRO alpha,
`
`Molecular Therapy: Nucleic Acids Vol. 15 April2019
`
`5
`
`€
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`Case 1:22-cv-00252-MSG Document 193-4 Filed 01/16/24 Page 7 of 19 PageID #: 12565
`Case 1:22-cv-00252-MSG Document 193-4 Filed 01/16/24 Page 7 of 19 PagelD #: 12565
`
`Molecular Therapy: Nucleic Acids
`
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`Figure 5. Tolerability in Rats
`Serum concentrations (in picograms permilliliter) of cytokines (A) eotaxin, (B) GRO alpha, (C) IP 10, (D) RANTES, and (©) MCP 1 were measured 6 h after a single IM
`administration of 0.01 mg or 0.1 mg modified MRNA encoding prMEfrom Zika virus formulated in LNPs containing MC3 (gray),lipid H (green),lipid M (orange), lipid P (purple),
`lipid Q {tan), orlipid N (yellow) (n = 3 per group). (F |) Representative histology sections stained with H&E 2 days after a single IM administration of 0.1 mg of modified MRNA
`encoding prMEfrom Zika virus formulated in LNPs containing MCSorlipid H in the (F and H) muscle and (G and1) skin. (F) MC3 muscle; (G) MC3 skin; (H)lipid H muscle;
`() lipid H skin.
`
`IP 10, RANTES, and MCP 1 (Figures 5A 5E). With the exception
`of IP 10 at the 0.01 mg dose, lipid H induced the lowest systemic
`cytokine production.
`
`Forty eight hours after administration, animals were sacrificed, and
`the injection sites were collected, paraffin embedded, sectioned,
`H&Estained, and blindly reviewed by a pathologist (Table 1). To
`evaluate, compare, and rank thelocal tolerability ofeach LNP,various
`endpoints were evaluated and graded, including mixed cell inflam
`mation at the injection site and in the dermis, myofiber necrosis,
`and relative number of degenerated neutrophils. MC3 formulated
`mRNAwasthe worst tolerated lipid tested, whereas lipid H was the
`best tolerated lipid tested (Figures 5F 51).
`
`Rats dosed with MC3formulationsat both the high and low dosesdis
`played a dose dependent mixed cell inflammation characterized by
`edema; numerous intact and degenerate neutrophils; macrophages;
`and a few lymphocytes distending endomysium, epimysium, and
`
`adjacent connective tissue of the muscle and compressing myofibers
`at the injection site (Figures 5F and S3A). A dose dependent multi
`focal degeneration and/or necrosis of individual myofibers,infiltrated
`by inflammatory cells at times, was also observed. The mixed inflam
`mation observed in the muscle extended into the subcutaneous
`portion of the skin (Figures 5G and S3B). The subcutaneoustissue
`was expanded by edema and numerousintact and degenerate neutro
`phils, macrophages, and a few lymphocytes.
`
`The dose related mixed cell inflammation observed in rats adminis
`tered lipid H was lower in magnitude and severity when compared to
`the rats given MC3 (Figure 5H). The relative amount of degenerate
`neutrophils was also lower, and it is worth noticing that there was
`less degeneration and/or regeneration and/ornecrosis in the myofib
`ers. The extension and spillage of the inflammation from the
`muscular injection site into the subcutaneous tissue was also less
`severe and with much less edema than in animals given MC3
`(Figure 51).
`
`6
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`
`Table 1. Pathology Summary
`
`Degenerate Neutrophils
`Mixed-Cell Inflammation
`Degenerate Neutrophils
`Mixed-Cell Inflammation
`Muscle Fiber Necrosis
`Formulation and Dose
`MO
`
`0.01 mg
`0.1 mg
`
`Lipid H
`0.01 mg
`
`0.1 mg
`Lipid M
`0.01 mg
`
`0.1 mg
`Lipid P
`0.01 mg
`
`0.1 mg
`Lipid Q
`
`0.01 mg
`0.1 mg
`Lipid N
`
`0.01 mg
`0.1 mg
`
`2.3
`2.3
`
`1
`
`1.3
`
`2
`
`1.7
`
`23
`
`2.3
`
`2.3
`2
`
`0.7
`13
`
`2.4
`2.7
`
`1.8
`
`2.9
`
`2
`
`2.7
`
`2.2
`
`2.8
`
`2.2
`2.9
`
`14
`2
`
`17
`33
`
`1
`
`2.3
`
`13
`
`2
`
`17
`
`2.3
`
`2
`3
`
`2
`2.3
`
`2
`2
`
`0
`
`13
`
`17
`
`2
`
`13
`
`2.3
`
`0.7
`2.5
`
`0
`0
`
`0
`1
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0.7
`
`0
`1
`
`0
`0
`
`Rats (n = 3 per group) were injected IM with 0.01 or 0.1 mg modified mRNA encoding prM-E from the Zika virus formulated in LNPs containing MC3orlipid H. Average histo-
`pathology scores on a 0-4 scale were recorded for events occurring in the muscle and skin.
`
`DISCUSSION
`mRNAvaccines delivered with LNPs have the potential to address
`numerous unmet medical needs not accessible with current vaccine
`technologies. Multiple reports from the siRNA field have shown
`that the ionizable lipid is the primary driver of LNP potency.’”*
`In this work, we observed the impact of ionizable lipid identity on
`expression,
`immunogenicity, and tolerability when delivered IM.
`Our working hypothesis was that the inclusion of a biodegradable
`lipid within an LNP would lead to vaccines with improved tolera
`bility, as the lipid would be cleared quickly from thesite of injection
`following mRNAdelivery, and other tissues would also have minimal
`exposure to the lipid due to metabolic breakdown and clearance.
`Interestingly, throughoutour initial screening, we noticedlittle corre
`lation between expression and vaccine immunogenicity, indicating
`that expression aloneis insufficient to identify improved mRNA vac
`cine formulations. Wealso observed a divergence in the best express
`ing formulations between the IV and IM routes of administration.
`
`Ionizable lipid pK,is thoughtto affect the protein opsonization ofthe
`particles, cellular uptake, and endosomal escape efficiency. The
`optimal lipid pK, for siRNA mediated knockdown in the liver has
`been reported to be between 6.2 and6.5, in line with our finding of
`the optimal pK, for mRNAdelivery to and expression in the liver
`as between 6.2 and 6.8.'''*?? However, the best lipids with respect
`to protein expression after IV administration generally had lower
`pK,s than the best lipids for protein expression after IM administra
`tion. Lipids such as V (pK, = 6.87) and AC (pK, = 7.09) showlittle to
`
`no expression after IV administration yet were someofthe highest ex
`pressing lipids after IM administration, indicating a yet to be eluci
`dated difference between these two routes of administration.
`Differentcell types have shown variations in endosomeacidification,
`demonstrating the need for additional work to better understand the
`performance of LNPs in the context of mRNA delivery across multi
`ple tissues.”*”° We also foundthat optimal lipid pK, for immunoge
`nicity was between 6.6 and 6.8. Independent of cytosolic mRNA
`delivery, lipid pK, may also play a role in formulation interactions
`with the immunesystem. Although this research area has not been
`thoroughly explored, a recent report illustrates how ionizable lipids
`can drive uptake andtransfection in immunecells, demonstrating po
`tential areas of research for LNP mediated delivery of mRNA vac
`cines.”’ Although lipid pK, was found to be an important factor for
`driving immunogenicity, it was not the only factor, as manylipids
`fell within that pK, range and were no better than the MC3 control.
`In addition to differences in pK,, lipid H also showed an improve
`ment in endosomal escape efficiency, consistent with our previously
`published report on this class oflipids (Figure $4).”°
`
`Multiple previous reports speak to the need for a balance between
`expression and immunestimulation for optimal mRNA vaccine po
`tency.”*”° Pollardet al. documented the negative impact ofinterferon
`signaling on the magnitude of mRNA expression.”” The mRNAs we
`used all contained a base modification on uridine to minimize innate
`immuneactivation.”**° As the mRNA is immunesilent compared
`with canonical uridine containing mRNA, both antigen selection
`
`Molecular Therapy: Nucleic Acids Vol. 15 April2019
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`Molecular Therapy: Nucleic Acids
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`MATERIALS AND METHODS
`and delivery system are important to generate potent immune re
`sponses. LNPs have been shown to beeffective adjuvants for protein
`mRNASynthesis and Formulation
`subunit vaccines, butit is unclear how importantthat adjuvant mech
`UTRsequences and mRNAproduction processes were performed as
`previously described.’° Briefly, mRNA was synthesized in vitro by T7
`anism is for inducing immuneresponses from an mRNAvaccine. We
`previously showed that MC3 based LNPsgenerated innate immune
`RNA polymerase mediated transcription from a linearized DNA
`activation and a potentcellular infiltrate.*' The histopathology pre
`template, which incorporates the 5’ and 3’ UTRs andapoly(A)tail.
`sented here for lipid H, compared to that for MC3, is consistent
`The final mRNAutilizes Cap1 and full replacement of uridine with
`with improvedtolerability and reduced innate immunestimulation.
`N1 methyl pseudouridine. mRNA encoding influenza HA genes
`originated from the H10N8 strain®*, and the mRNA encoding
`The reduction in inflammatory cell infiltrate, myofiber damage, and
`systemic cytokines support the hypothesis that mRNA vaccines
`prM E from Zika utilized the signal sequences from human IgE
`may not require a strong adjuvant response for potent immune
`(MDWTWILFELVAAATRVHS)and the prM and E genes from an
`responses.
`Asian ZIKV strain (Micronesia 2007; GenBank: EU545988), which
`is >99% identical to circulating American strains.*° All coding se
`quences were generated using a proprietary algorithm.
`
`The improved tolerability and safety mediated by the inclusion of
`biodegradable lipids within LNPs correlate well with lipid halflife
`after IV delivery.”*** The lead ionizablelipids in this study showed
`improved biodegradability while maintaining immune
`titers
`compared. to MC3. Thetolerability data suggest that this increased
`biodegradability leads to a reduction in injection site inflammation.
`Our data also showthat extendedresidencetime of the ionizable lipid
`post transfection is not required for a robust immune response.
`Indeed, clearance is preferred to extended residence, which results
`in undesirable inflammation at the site of injection beyond when
`the protein antigen is cleared. Interestingly, the data also indicate
`that biodegradability is not the only factor in tolerability lipid H
`wasthebest tolerated lipid yet showed a biodegradability similar to
`that of the other lead lipids tested. Degradation andtolerability of
`the lipid metabolites likely contribute to the tolerability of any
`formulation.
`
`Other components, such as PEG, may play a role in vaccine po
`tency due to the impact of anti PEG responses that have been
`well described for IV administered liposomal
`therapeutics. To
`date, there is no published information on the impact of anti
`PEG responses across other routes of administration. The field
`of viral vector delivery has described how anti vector immunity
`can substantially reduce
`immune
`response and can even
`completely prevent vaccine boosting when a homologous vector
`is used for both priming and boosting.** Given that we see a sub
`stantial increase in immunetiters after a second dose, we do not
`believe that a neutralizing anti PEG response affects the LNP
`based vaccines we describe here.
`
`Thetolerability of any new vaccine is a key performancecriterion,
`as vaccines are given to healthy individuals throughout different
`stages of life, from 1 day old neonates to the elderly. Here, we
`have described the identification, performance, and tolerability
`assessment of novel ionizable lipids for inclusion in mRNA vaccine
`formulations. We focused on the ionizable lipid component of the
`LNP, as it has been previously demonstrated to be the primary
`driver of LNP potency andtolerability. Given their improved toler
`ability and increased antigen expression, the formulations we iden
`tified have the potential for both active and passive immunizatio