Methods and compositions for RNA delivery to the central nervous system for prolonged protein expression

Abstract

Provided herein is a method of treating or preventing or diagnosing a central nervous system central nervous system disease, disorder, trauma or injury. The method comprises use of a lipid nanoparticle comprising at least one nucleic acid encoding an antibody or antigen-binding fragment and the lysosomal enzyme glucocerebrosidase. Also provided is a lipid nanoparticle that can be used with the method disclosed herein.

Description

[0001]METHODS^AND^COMPOSITIONS^FOR^RNA^DELIVERY^TO^THE^CENTRAL^NERVOUS^SYSTEM^FOR^ PROLONGED^PROTEIN^EXPRESSION^ TECHNICAL^FIELD^ The^present^disclosure^relates^to^compositions^and^methods^for^treating^preventing^or^diagnosing^a^ central^nervous^system^diseases^or^disorders.^^In^particular,^the^compositions^include^lipid^ nanoparticles^(LNPs)^for^the^delivery^of^nucleic^acid,^such^as^mRNA^(LNP‐mRNA)^encoding^for^ polypeptides^such^as^glucocerebrosidase^and / or^other^lysosomal^enzymes,^or^antibodies^or^antigen‐ binding^fragments.^ BACKGROUND^ Neurological^disorders^are^often^chronic^conditions^that^bring^a^heavy^burden^to^patients,^their^ families^and^the^health^care^system.^Extensive^investigations^on^potential^treatments^have^been^ ongoing^for^decades,^but^the^clinical^success^rate^of^therapy^targeted^to^the^central^nervous^system^ (CNS)^is^low^when^compared^to^other^organs.^^It^is^widely^accepted^that^beyond^the^complex^nature^of^ neural^communication,^a^major^hurdle^for^brain^therapy^is^delivery^of^a^therapeutic^to^the^target^site^or^ sites.^^The^brain^is^particularly^difficult^to^target,^largely^due^to^the^presence^of^the^blood‐brain^barrier^ (BBB),^shielding^the^brain^parenchyma^from^the^periphery^and^preventing^the^passage^of^most^small^ molecules^and^nearly^all^macromolecules.^^ In^virtually^all^diseases^in^the^CNS^and^periphery,^the^absence^or^downregulation^of^one^or^many^ endogenous^proteins^is^known^to^participate^in^disease^pathology.^This^has^given^rise^to^a^ considerable^number^of^protein^biologics‐based^supplementation^or^replacement^therapies^in^the^ periphery.^^ One^such^example^is^in^Lysosomal^storage^disorders^(LSDs),^a^family^of^over^70^rare^inherited^ metabolic^disorders,^caused^by^genetic^point^mutations^or^haploinsufficiency^leading^to^enzyme^ dysfunctions^that^engender^disease.^The^standard^of^care^for^several^LSDs^is^enzyme^replacement^ therapy^(ERT),^where^recombinant^enzymes^are^infused^intravenously^(IV)^typically^every^two^weeks.^ While^many^ERTs^effectively^address^peripheral^symptoms,^for^example^Imiglucerase™^in^Gaucher^ Disease^(GD)^type^1,^they^do^not^cross^the^BBB^and^therefore^offer^little^to^no^improvement^of^ neurological^symptoms.^For^direct^brain^ERT,^cerliponase^alfa^is^a^recombinant^enzyme^that^is^ administered^directly^into^the^CNS^via^intracerebroventricular^(ICV)^injection^as^an^ERT^for^neuronal^ ceroid^lipofuscinosis^type^II^but^requires^biweekly^infusions.^Therefore,^despite^significant^advances^in^ recombinant^protein^biologics,^the^inability^of^these^proteins^to^penetrate^the^BBB,^as^well^as^short^ protein^half‐lives,^limit^the^utility^of^protein^supplementation^in^the^CNS.^ As^an^example^of^lysosomal^storage^disease,^Gaucher^Disease^(GD)^is^an^LSD^caused^by^monogenic^ mutations^in^the^GBA^gene^coding^for^the^lysosomal^enzyme^Glucocerebrosidase^(GCase),^resulting^in^a^ deficiency^in^GCase^function.^GCase^is^a^vital^enzyme^in^the^lysosome^responsible^for^healthy^ metabolic^breakdown^of^its^substrate^glucosylceramide.^GD^is^subdivided^into^types^1 / 2 / 3;^type^1^is^ ‘non‐neuronopathic’,^while^types^2^(Acute)^and^3^(Chronic)^lead^to^a^spectrum^of^neurological^disease.^ Type^2^manifests^as^rapid^neurodegeneration^and^death^in^infancy^or^early^childhood,^whereas^type^3^ causes^a^range^of^symptoms^of^varying^severity,^from^saccade^eye^movements^to^epilepsies,^to^ dementia.^GD^and^GCase^deficiency^have^also^been^shown^to^be^strongly^correlated^with^Parkinson’s^ disease^and^related^neurological^symptoms.^Given^that^many^patients^develop^intermediate^ phenotypes^across^GD^1‐3,^the^contemporary^view^is^that^GD^is^most^accurately^considered^as^a^ continuum^of^phenotypes.^An^LNP‐RNA^strategy^for^long‐term,^in‐brain^GCase^supplementation^could^ usher^in^efficacious,^re‐dosable,^and^titratable^treatments^for^neuronopathic^GD^and^Parkinson’s^ disease.^ The^therapeutic^or^diagnostic^use^of^protein^biologics^such^as^recombinant^enzymes,^monoclonal^ antibodies,^etc.^has^also^significantly^increased^in^recent^years,^however,^several^drawbacks^related^to^ delivery^reduce^their^potential^in^CNS^therapeutic^applications.^^The^main^challenges^remain^the^ inability^of^proteins^to^cross^the^BBB^and^access^the^brain^parenchyma,^as^well^as^the^short^half‐life^of^ proteins,^which^restricts^the^time^course^of^therapeutic^effects.^^^ An^alternative^to^direct^protein^delivery^is^therefore^needed^to^address^the^biodistribution^challenges^ of^the^brain.^One^approach^could^consist^of^delivering^the^genetic^material^(nucleic^acids)^that^encodes^ the^desired^protein^instead^of^the^protein^itself.^^However,^nucleic^acids^are^readily^broken^down^in^ extracellular^milieu,^are^immunogenic,^and^do^not^readily^enter^the^cell^compartment^they^need^to^ reach.^Non‐viral^delivery^vectors^such^as^lipid^nanoparticles^have^been^developed^to^address^these^ issues^and^deliver^nucleic^acids^to^the^cell^cytoplasm.^When^the^LNP‐encapsulated^cargo^is^messenger^ RNA,^the^RNA^can^readily^be^translated^into^the^desired^protein.^However,^delivery^of^LNP‐RNA^ complexes^to^brain^cells^remains^a^challenge.^There^remains^a^need^for^methods^to^deliver^antibodies^ to^the^CNS^at^therapeutic^concentrations^and / or^for^prolonged^duration.^^ In^various^non‐CNS^applications,^LNP‐messengerRNA^has^been^delivered^to^cells^and^leads^to^active^ protein^synthesis.^Similarly,^mRNA^encapsulated^in^SS‐cleavable^proton‐activated^lipid‐like^material^ (ssPalm)^nanoparticles^was^shown^to^deliver^exogenous^mRNA,^encoding^proteins,^into^neuronal^cells^ and^astrocytes^via^intracerebroventricular^(ICV)^administration8.^^Intrathecal^lumbar^injection^of^LNP‐ mRNA^was^found^to^mostly^target^the^dorsal^root^ganglia9,^measured^by^intracellular^protein^ production.^^Similarly,^transfecting^neurons,^astrocytes,^and^microglia / macrophages^with^an^LNP‐ mRNA^construct^encoding^human^IL‐10^in^the^intralesional^area^of^a^spinal^cord^lesion^promoted^ neuroprotection^and^functional^recovery^in^a^spinal^cord^injury^model^(Gál,^L.^et^al.^Restoration^of^ Motor^Function^through^Delayed^Intraspinal^Delivery^of^Human^IL‐10‐Encoding^Nucleoside‐Modified^ mRNA^after^Spinal^Cord^Injury.^Research^(Wash^D^C)^6,^0056^(2023);^and^PARDI,^N.^et^al.^WO^ 2023 / 086830,^but^there^was^no^demonstration^of^secreted^IL‐10^in^the^CNS^nor^of^prolonged^ expression).^^Intracerebral^LNPs^were^also^used^to^produce^intracellular^Cre^proteins^(inducing^Cre‐ dependent^gene^recombination)^in^neurons,^astrocytes^and^microglia,^but^with^a^limited^spatial^ distribution^in^mouse^brains^(Tuma,^J.^et^al.^Lipid^Nanoparticles^Deliver^mRNA^to^the^Brain^after^an^ Intracerebral^Injection.^Biochemistry^62,^3533–3547^(2023)).^ Delivery^of^LNP‐RNA^complexes^to^brain^cells^remains^a^challenge.^There^remains^a^need^for^improved^ LNP^delivery^systems^that^can^offer^prolonged^expression^and / or^wide^distribution^of^the^RNA^ expressed^protein^in^the^CNS.^^Also,^the^BBB^largely^prevents^the^entry^of^proteins^such^as^enzymes^or^ antibodies^to^the^brain^compartment,^hindering^their^use^for^neurological^applications.^Furthermore,^ diffusion^of^enzymes^or^antibodies^within^the^brain^parenchyma^is^limited^due^to^their^size.^ ^ SUMMARY^ According^to^an^embodiment,^there^is^provided^a^lipid^nanoparticle^including^at^least^one^nucleic^acid^ encoding^a^polypeptide^or^an^antibody^or^antigen‐binding^fragment,^wherein^the^polypeptide,^ antibody^or^antigen‐binding^fragment^is^capable^of^secretion^from^the^cell^into^an^interstitial^and / or^ cerebrospinal^fluid^of^the^subject.^In^embodiments,^the^polypeptide,^antibody^or^antigen‐binding^ fragment^further^comprises^a^secretion^signal.^In^embodiments^the^polypeptide^is^glucocerebrosidase^ (GCase)^or^functional^fragment^thereof.^In^embodiments^of^the^invention,^the^secreted^polypeptide,^ antibody^or^antigen‐binding^fragment^is^present^in^the^CSF^of^the^subject^at^a^first^concentration^at^a^ first^time^point^and^at^a^second^concentration^at^a^later^second^time^point.^ According to an embodiment, the^second^concentration^of^secreted^polypeptide,^antibody^or^antigen‐ binding^fragment^is^at^least^30%,^at^least^40%,^at^least^50%,^at^least^60%,^at^least^70%,^at^least^80%,^at^ least^90%,^or^at^least^95%^of^the^first^concentration.^In^embodiments,^the^first^time^point^is^about^3^ hours^after^contacting^the^LNP^with^the^cell.^In^embodiments,^the^second^time^point^is^about^48^hours^ after^contacting^of^the^LNP^with^the^cell.^ In^embodiments,^the^lipid^nanoparticle,^secreted^polypeptide,^antibody^or^antigen‐binding^fragment^is^ present^in^the^CSF^of^the^subject^for^a^period^of^at^least^1^week,^at^least^2^weeks,^at^least^3^weeks,^at^ least^4^weeks,^at^least^6^weeks,^or^at^least^8^weeks.^ In^embodiments,^the^polypeptide^is^glucocerebrosidase^or^a^functional^fragment^thereof^is^present^in^ the^CSF^of^the^subject^for^a^period^of^at^least^24^hours,^at^least^36^hours,^or^at^least^48^hours.^In^ embodiments,^the^lipid^nanoparticle^comprises^an^ionizable^lipid,^which^may^be^an^aminolipid^cationic^ at^neutral^pH.^ In^embodiments,^at^least^one^nucleic^acid^comprises^a^nucleic^acid^encoding^a^light^chain^and^a^nucleic^ acid^encoding^a^heavy^chain.^In^embodiments,^the^nucleic^acid^encoding^the^light^chain^and^the^nucleic^ acid^encoding^the^heavy^chain^are^at^a^ratio^of^about^1:1^to^1:4^(w / w).^ In^embodiments,^the^ionizable^lipid^has^a^pKa^between^5.0^and^7.0.^ In^some^embodiments,^the^lipid^nanoparticle^further^comprises^a^non‐cationic^helper^lipid.^^In^some^ embodiments,^the^non‐cationic^helper^lipid^is^a^generally^cylindrical‐shaped^lipid.^In^some^ embodiments,^non‐cationic^helper^lipid^is^distearoylphosphatidylcholine^(DSPC)^or^ dioleoylphosphatidylglycerol^(DOPG)^or^sphingomyelin.^ In^embodiments,^the^non‐cationic^helper^lipid^has^a^phosphatidylethanolamine^content^that^is^less^ than^2^mol%.^In^some^embodiments,^the^lipid^nanoparticle^includes^a^sterol.^In^further^embodiments,^ the^lipid^nanoparticle^includes^a^polymer^lipid^conjugate.^^ According^to^embodiments,^there^is^provided^^a^lipid^nanoparticle^wherein^the^lipid^nanoparticle^ comprises^(a)^an^ionizable,^amino,^cationic^lipid;^(b)^a^non‐cationic^helper^lipid;^(c)^a^sterol;^and^(d)^a^ polymer^lipid^conjugate,^and^wherein^the^molar^ratio^of^ionizable^lipid / non‐cationic^helper^ lipid / sterol / polymer^lipid^conjugate^is^20‐50 / 10‐40 / 38‐39.5 / 0.5‐2.0.^ In^embodiments,^the^molar^ratio^of^ionizable^lipid / non‐cationic^helper^lipid / sterol / polymer^lipid^ conjugate^is^20 / 40 / 38‐39.5 / 0.5‐2.0.^In^embodiments,^the^molar^ratio^of^ionizable^lipid / non‐cationic^ helper^lipid / sterol / polymer^lipid^conjugate^is^50 / 10 / 38‐39.5 / 0.5‐2.0.^ In^some^embodiments,^the^polypeptide^is^glucocerebrosidase^which^comprises^an^amino^acid^ sequence^of^at^least^80%,^at^least^85%,^at^least^90%,^at^least^95%,^or^at^least^98%^sequence^identity^to^ SEQ^ID^NO:^1.^In^some^embodiments,^the^secretion^signal^comprises^an^amino^acid^sequence^of^at^least^ 80%,^at^least^85%,^at^least^90%,^at^least^95%,^or^at^least^98%^sequence^identity^to^SEQ^ID^NO:^5.^In^ some^embodiments,^the^nucleic^acid^comprises^a^nucleotide^sequence^of^at^least^60%,^at^least^70%,^at^ least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ID^NO:^2.^ In^some^embodiments,^the^nucleic^acid^further^comprises^a^nucleotide^sequence^of^at^least^60%,^at^ least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^ SEQ^ID^NO:^3^or^4.^In^embodiments,^the^nucleic^acid^further^comprises^a^nucleotide^sequence^of^at^ least^60%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^ sequence^identity^to^SEQ^ID^NO:^6.^In^embodiments,^the^nucleic^acid^further^comprises^a^nucleotide^ sequence^of^at^least^60%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^ least^95%^sequence^identity^to^SEQ^ID^NO:^7.^In^embodiments,^the^nucleic^acid^comprises^a^nucleotide^ sequence^of^at^least^60%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^ least^95%^sequence^identity^to^SEQ^ID^NO:^8.^^ In^embodiments,^the^nucleic^acid^is^RNA,^which^may^be^mRNA,^circular^RNA^(circRNA),^and / or^self‐ amplifying^RNA^(saRNA).^^In^embodiments,^the^cell^of^the^central^nervous^system^is^a^glial^cell^selected^ from^an^astrocyte^or^oligodendrocyte^or^an^ependymal^cell^of^the^cerebral^ventricles^or^the^choroid^ plexus.^In^embodiments,^the^lipid^nanoparticle^is^part^of^a^pharmaceutical^formulation.^^ According^to^embodiments,^there^is^provided^a^lipid^nanoparticle^and^method^for^use^in^treating^or^ preventing^a^central^nervous^system^disease,^disorder,^trauma^or^injury^in^the^subject.^ In^some^embodiments,^the^central^nervous^system^disease,^disorder,^trauma^or^injury^is^Gaucher^ Disease^Type^1,^Gaucher^Disease^Type^2,^Gaucher^Disease^Type^3,^Parkinson’s^Disease^and^related^ synucleinopathies,^Alzheimer’s^Disease,^Lewy^Body^Dementia,^Multiple^Sclerosis,^or^any^combination^ thereof.^ In^embodiments,^there^is^provided^the^use^of^the^lipid^nanoparticle^for^the^manufacture^of^a^ medicament^for^a^central^nervous^system^disease,^wherein^the^central^nervous^system^disease,^ disorder,^trauma^or^injury,^in^some^embodiments,^is^Gaucher^Disease^Type^1,^Gaucher^Disease^Type^2,^ Gaucher^Disease^Type^3,^Parkinson’s^Disease^and^related^synucleinopathies,^Alzheimer’s^Disease,^ Lewy^Body^Dementia,^Multiple^Sclerosis,^or^any^combination^thereof.^ According^to^an^embodiment,^there^is^provided^a^method^of^treating,^preventing^or^diagnosing^a^ central^nervous^system^disease,^disorder,^trauma^or^injury^in^a^subject,^the^method^including:^^ contacting^a^lipid^nanoparticle^(LNP)^with^a^cell^of^the^central^nervous^system^of^the^subject,^the^lipid^ nanoparticle^including^at^least^one^nucleic^acid^encoding^a^polypeptide^capable^of^secretion^from^the^ cell^into^an^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^In^embodiments,^the^polypeptide^is^an^ antibody^or^antigen‐binding^fragment,^glucocerebrosidase^(GCase)^or^a^functional^fragment^thereof.^ In^embodiments^the^nucleic^acid^is^an^mRNA,^a^circular^RNA^(“circRNA”)^or^a^self‐amplifying^RNA^ (“saRNA”).^ In^embodiments,^the^contacting^of^the^LNP^with^the^cell^results^in^the^least^one^nucleic^acid^entering^ the^cell^and^production^of^the^polypeptide^in^the^cell.^In^embodiments,^the^production^of^the^ polypeptide,^the^polypeptide^is^secreted^from^the^cell^to^the^interstitial^and / or^cerebrospinal^fluid^ (CSF)^of^the^subject.^In^embodiments,^the^secreted^polypeptide^is^present^in^the^CSF^of^the^subject^for^ a^period^of^at^least^24^hours,^at^least^36^hours,^or^at^least^48^hours.^In^embodiments,^it^is^present^for^a^ period^of^at^least^1^week,^at^least^2^weeks,^at^least^3^weeks,^at^least^4^weeks,^at^least^6^weeks,^or^at^ least^8^weeks.^^In^some^embodiments,^the^secreted^polypeptide^is^present^in^the^CSF^of^the^subject^at^a^ first^concentration^at^a^first^time^point^and^at^a^second^concentration^at^a^later^second^time^point.^In^ embodiments,^the^first^time^point^is^about^3^hours^after^contacting^the^LNP^with^the^cell.^In^ embodiments,^the^second^time^point^is^about^48^hours^after^contacting^the^LNP^with^the^cell.^ In^embodiments,^the^second^concentration^is^at^least^30%,^at^least^40%,^at^least^50%,^at^least^60%,^at^ least^70%,^at^least^80%,^at^least^90%,^or^at^least^95%^of^the^first^concentration.^ In^embodiments^there^is^provided^^a^method^of^treating,^preventing^or^diagnosing^a^central^nervous^ system^disease,^disorder,^trauma^or^injury^in^a^subject,^the^method^including:^^contacting^a^lipid^ nanoparticle^(LNP)^with^a^cell^of^the^central^nervous^system^the^cell^of^the^central^nervous^system^is^ selected^from^one^or^more^of:^^(a)^a^glial^cell;^and^^(b)^an^ependymal^cell.^ In^further^embodiments,^the^glial^cell^is^selected^from^an^astrocyte,^an^oligodendrocyte,^or^an^ oligodendrocyte^precursor^cell,^and / or^the^ependymal^cell^is^from^the^cerebral^ventricles^or^the^ choroid^plexus.^In^embodiments,^the^polypeptide^is^diffusible^within^the^interstitial^and / or^ cerebrospinal^fluid^of^the^subject.^ In^embodiments,^the^method^of^any^one^of^the^preceding^claims,^wherein^contacting^of^the^LNP^with^ the^cell^comprises^administering^the^LNP^via^injection^via^an^intracerebroventricular^(ICV)^or^lumbar^ intrathecal^route,^cisterna^magna^route^or^via^a^catheter^to^the^central^nervous^system^of^the^subject.^ In^embodiments,^the^polypeptide^comprises^trastuzumab.^ In^embodiments,^there^is^provided^the^methods^described^above^wherein^the^cell^of^the^CNS^ comprises^neurons;^interneurons;^glia;^astrocytes;^oligodendrocytes;^microglia;^ependymal^cells;^ radial^glia;^neurovascular^cells^including^endothelial^cells,^pericytes,^and^smooth^muscle^cells;^ progenitor^cells;^and / or^meningeal^cells.^^ In^embodiments,^the^contacting^is^in^vitro^or^in^vivo.^ In^embodiments,^there^is^provided^a^method^of^treating,^preventing^or^diagnosing^a^central^nervous^ system^disease,^disorder,^trauma^or^injury^in^a^subject,^the^method^including:^^contacting^a^lipid^ nanoparticle^(LNP)^according^to^the^embodiments^with^a^cell^of^the^central^nervous^system^of^the^ subject,^the^lipid^nanoparticle^including^at^least^one^nucleic^acid^encoding^a^polypeptide^capable^of^ secretion^from^the^cell^into^an^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ ^ BRIEF^DESCRIPTION^OF^THE^DRAWINGS^ FIGURE^1^depicts^a^proposed^mechanism^for^delivery^of^a^secretory^protein,^such^as^the^enzyme^ glucocerebrosidase,^to^the^central^nervous^system^using^an^LNP‐mRNA^as^described^herein.^ FIGURE^2^shows^an^embodiment^of^the^therapeutic^Bioreactor^strategy^to^produce^exogenous^human^ GCase.^^ FIGURE^2A^depicts^the^deficit^of^GCase^enzymatic^function^in^neural^cells,^leading^to^accumulation^of^ un‐metabolized^glucosylceramide^that^engenders^disease,^such^as^Gaucher^Disease^Type^1,^Gaucher^ Disease^Type^2,^Gaucher^Disease^Type^3,^Parkinson’s^Disease^and^related^synucleinopathies,^ Alzheimer’s^Disease,^Lewy^Body^Dementia,^and^Multiple^Sclerosis.^^ FIGURE^2B^shows^an^embodiment^of^the^therapeutic^treatment^strategy^to^harness^glial^cells^(such^as^ astrocytes^and^oligodendrocytes)^via^LNP‐mRNA^transfection^to^produce^and^secrete^GCase^that^can^ then^diffuse^to^brain‐wide^sites^of^action.^Secreted^GCase^protein^can^enter^un‐transfected^cells,^such^ as^neurons^but^also^other^glial^or^immune^or^vascular^cells,^to^restore^GCase^enzyme^activity^broadly^ in^brain^cells.^ FIGURE^3A^shows^a^schematic^of^an^intracerebroventricular^(ICV)^injection^of^LNP‐mRNA^to^the^brain^ of^a^mouse,^where^the^LNP‐mRNA^encodes^for^the^fluorescent^reporter^protein^mCherry,^bilaterally^ injected^in^mice.^^ FIGURE^3B^shows^mCherry^expression^in^brain^slices^of^the^white^matter^tract^of^the^corpus^callosum^ at^2^days^post‐injection,^where^mCherry^was^used^as^a^reporter^to^visualize^cells^translating^the^mRNA^ cargo^into^mCherry^protein^(note^that^mCherry^is^not^secreted).^The^cells^instructed^to^express^ mCherry^have^a^widespread^distribution^in^the^brain,^but^most^pronounced^mCherry^protein^is^found^ in^the^corpus^callosum,^hippocampus,^ventricles,^and^septum.^ FIGURE^4A^shows^brain^images^of^LNP‐mRNA^uptake^in^oligodendrocytes^(olig2+),^following^ICV^ injection,^uptake^of^LNP‐mRNA^encoding^for^mCherry^induced^expression^of^mCherry^that^was^ visualized^within^immunostained^oligodendrocytes^in^white^matter^tracts^in^mice.^^The^broken^lined^ box^in^the^upper^images^(100^µm^scale)^are^the^areas^enlarged^in^the^corresponding^lower^images^(20^ µm^scale).^ FIGURE^4B^shows^brain^images^of^LNP‐mRNA^uptake^in^astrocytes^(GFAP+),^following^ICV^injection,^ uptake^of^LNP‐mRNA^encoding^for^mCherry^induced^expression^of^mCherry^that^was^visualized^within^ immunostained^astrocytes^in^white^matter^tracts^in^mice.^^The^broken^lined^box^in^the^upper^images^ (100^µm^scale)^are^the^areas^enlarged^in^the^corresponding^lower^images^(20^µm^scale).^ FIGURES^5A‐B^shows^brain^images^following^intracerebroventricular^(ICV)^injection^of^the^LNP‐ mRNA^encoding^for^mCherry^in^mice.^^The^images^show^immunostaining^of^cells^expressing^mCherry^ in^the^choroid^plexus^and^ventricle^walls^in^the^mice^at^various^magnifications.^^^ FIGURE^5C^shows^mCherry^expression^in^astrocytes^in^the^CA3^of^the^hippocampus^that^co‐localize^ with^GFAP^staining,^but^not^neurons^(NeuN).^Zoom^inset^panel^shows^the^dense^neuronal^cell^body^ layer^with^mCherry^negative^neurons^(NeuN)^but^expression^in^a^GFAP+^astrocyte.^ FIGURE^6^shows^spinal^cord^images^of^LNP‐mRNA^uptake.^^LNPs^with^encapsulated^mRNAs^encoding^ for^mCherry^were^injected^via^the^intrathecal^lumbar^route^in^a^mouse,^and^the^animal^was^perfused^ and^fixed^tissue^was^collected^two^days^after^LNP‐mRNA^injection.^^The^images^show^immunostaining^ of^cells^in^the^spinal^cord^expressing^mCherry.^ FIGURE^7^shows^mCherry^expression^in^coronal^brain^slices^of^the^white^matter^tract^of^the^corpus^ callosum^at^2^days^post‐injection,^where^mCherry^was^used^to^visualize^LNP‐mRNA^transfection^of^ five^different^LNP^formulations^(LNP^“D”,^LNP^“E”,^LNP^“F”,^LNP^“G”,^and^LNP^“H”)^from^TABLE^1.^ FIGURE^8^shows^a^schematic^of^the^protocol^used^to^demonstrate^that^LNP‐mRNA^can^induce^ production^and^secretion^of^a^desired^protein^to^the^CSF.^^The^concentration^of^FGF21^protein^in^ cerebrospinal^fluid^(CSF)^was^measured^after^bilateral^ICV^injection^in^mice^of^two^different^ formulations^of^LNP‐mRNA^(i.e.^LNP(1)‐FGF‐21^(LNP‐G^FGF‐21)^and^LNP(2)‐FGF‐21^(LNP‐H^FGF‐21))^ coding^for^FGF‐21^(graph^at^the^right^side^of^the^figure)^as^compared^to^CSF^control^and^LNP‐mRNA^ EPO^negative^control^(i.e.^mRNA^encoding^EPO^does^not^yield^increases^in^FGF‐21).^All^formulations^ were^injected^at^1^mg / mL^(1^µg^mRNA / hemisphere).^^This^demonstrates^the^specificity^of^the^assay^ but^importantly^that^protein^production^and^secretion^is^specific^to^mRNA^encoded^protein.^^After^ LNP‐mRNA^FGF‐21^injection^CSF^was^collected^and^FGF‐21^was^measured^using^a^U‐PLEX^FGF21^ assay.^^^^ FIGURE^9^shows^changes^in^CSF^FGF‐21^concentrations^at^3^hours^and^48^hours^post^injection^in^ response^to^varying^doses^of^LNP‐mRNA^(total^of^1^µL / hemisphere^injected^bilaterally)^encoding^for^ FGF‐21^(LNP(2)‐FGF‐21)^injected^ICV.^^CSF^FGF‐21^protein^was^measured^using^a^quantitative^U‐PLEX^ assay.^^Dashed^line^corresponds^to^CSF^FGF‐21^concentration^at^3^hours^post‐injection^at^a^dose^of^1^ mg / mL^mRNA.^ FIGURE^10^shows^the^concentration^of^EPO^protein^in^CSF^after^ICV^injection^of^LNP‐mRNA^(1^µg^ mRNA / hemisphere)^encoding^the^secreted^factor^EPO^(LNP‐mRNA‐EPO)^(LNP^“M”)^in^mice^(graph^at^ the^right^side^of^the^figure),^where^after^injection,^CSF^was^collected^and^EPO^was^measured^using^a^U‐ PLEX^EPO^assay^as^described^herein^(depicted^in^the^left^side^of^the^figure).^^Control^CSF^is^a^negative^ control^from^un‐injected^animals,^and^LNP‐FGF‐21^is^a^negative^injection^control^showing^LNP‐mRNA^ FGF‐21^production^does^not^alter^CSF^EPO^concentrations.^ FIGURE^11^shows^changes^in^CSF^EPO^concentrations^at^3^hours^and^48^hours^post^injection^in^ response^to^varying^doses^of^LNP‐mRNA^encoding^for^EPO^(LNP‐mRNA‐EPO)^(LNP^“M”)^injected^ICV.^^ CSF^EPO^protein^was^measured^using^a^quantitative^U‐PLEX^assay.^^Dashed^line^corresponds^to^CSF^ [EPO]^3^hours^post‐injection^at^a^dose^of^1^mg / mL^mRNA.^ FIGURE^12^shows^coronal^and^sagittal^views^of^the^mouse^brain^atlas^with^the^ intracerebroventricular^(ICV)^injection^site^(grey^triangle^“ICV^inject”^‐^coronal^and^sagittal),^with^the^ lateral^ventricle^(LV)^injections^sites^in^black^and^cerebral^spinal^fluid^(CSF)^collections^site^(grey^ triangle^“CSF^collect”^–^sagittal^only)^and^the^approximate^distance^between^the^injection^and^ collection^sites^(i.e.^8^mm^–^sagittal^only).^ FIGURE^13^shows^an^example^LNP‐mRNA‐mCherry^expression^in^a^human^iPSC^neurosphere^culture^ model^comprised^of^neurons^and^astrocytes.^iPSC^neurospheres^were^transfected^with^LNP‐mRNA‐ mCherry^(LNP^“F)^at^300nM,^30nM,^3nM,^and^0.3nM^mRNA^at^43^days^in^vitro^and^imaged^initially^ from^4^hours^to^24^hours^post^transfection,^leading^to^increased^mCherry^expression^as^measured^by^ fluorescence.^Cultures^were^subsequently^imaged^regularly^for^31^days^post^transfection,^revealing^ sustained^mCherry^expression^over^the^course^of^the^imaging^period^and^with^a^calculated^half‐life^of^ 11.8^days^(calculated^from^the^30nM^treatment^group).^ FIGURE^14^shows^an^example^LNP‐circRNA‐NeonGreen^expression^in^a^human^iPSC^neurosphere^ culture^model^comprised^of^neurons^and^astrocytes.^iPSC^neurospheres^were^transfected^with^LNP‐ circRNA‐NeonGreen^(LNP^“T”)^at^30nM^mRNA^at^43^days^in^vitro^and^imaged^regularly^for^15^days^ post^transfection,^revealing^sustained^NeonGreen^expression^over^the^course^of^the^imaging^period^ and^with^a^calculated^half‐life^of^3.3^days.^ FIGURE^15^shows^example^luminescence^data^from^human^iPSC^neurosphere^cultures^transfected^ with^150nM^or^15nM^LNP‐mRNA‐Gluc^(LNP^“U”)^exhibit^luminescent^signals^in^the^supernatant^in^a^ dose‐dependent^manner.^ FIGURE^16^shows^a^schematic^of^an^embodiment^of^the^therapeutic^human^GCase^mRNA^construct^ leading^to^expression^of^functional^GCase^protein.^Numbered^segments^are^modular^components^of^ the^final^mRNA^sequence^that^can^be^individually^tuneable.^ FIGURE^17A^shows^an^in^vitro^western^blot^example^of^exogenous^GCase^(AKA^GBA1)^secretion.^ measured^from^culture^supernatant.^Exogenous^GCase^DNA^was^transfected^HEK293T^cells^and^ compared^to^mCherry^transfection^control.^ FIGURE^17B^shows^an^in^vitro^western^blot^example^of^exogenous^GCase^(GBA1)^expression^ measured^from^cell^lysates.^Exogenous^GCase^DNA^was^transfected^into^HEK293T^cells^and^compared^ to^mCherry^transfection^control.^ FIGURE^18^(left^panel)^shows^an^in^vitro^assessment^of^secreted^exogenous^GCase^constructs^ comprised^of^different^example^signal^peptides.^Secreted^GCase^was^measured^from^the^supernatant^ of^DNA‐transfected^HEK293T^cell^cultures^and^quantified^by^western^blot^densitometry.^Right^panel^ shows^the^enzyme^activity^of^exogenous^secreted^GCase^of^GCase^constructs^comprised^of^different^ signal^peptides.^ FIGURE^19A^shows^an^example^expression^of^exogenous^human^GCase^(GBA1)^from^naked^mRNA^ transfected^with^lipofectamine^in^HEK293T^cells.^Bottom^panels^show^that^the^mRNA‐translated^ GCase^is^secreted^(supernatant^measures).^ FIGURE^19B^shows^an^example^expression^of^exogenous^human^GCase^(GBA1)^from^naked^mRNA^ transfected^with^Lipofectamine™^in^cultured^rat^primary^astrocytes.^ FIGURE^19C^shows^an^example^expression^of^exogenous^human^GCase^(GBA1)^over^48^hours^from^ naked^mRNA^transfected^with^Lipofectamine™^in^cultured^human^astrocytes.^ FIGURE^20^shows^an^example^exogenous^improved^human^GCase^expression^from^HEK293T^cells^ transfected^with^LNP‐mRNA‐GCase^(LNP^“O”)^compared^to^identical^mRNA‐GCase^transfected^using^ Lipofectamine™.^No^GCase^expression^was^detectable^from^transfection^controls.^ FIGURES^21A‐E^shows^representative^immunofluorescence^of^LNP‐mRNA‐GCase‐FLAG^(LNP^“O”)^ transfected^HEK293T^cells.^Culture^was^transfected^24^hrs,^fixed^with^4%^PFA,^and^stained^for^FLAG^ (GCase),^the^lysosomal^protein^LAMP1,^and^a^nuclear^stain^(DAPI),^revealing^co‐localized^expression^of^ FLAG^(GCase)^and^LAMP1,^indicating^GCase^localization^in^the^lysosome.^ FIGURES^21F‐J^shows^representative^immunofluorescence^of^LNP‐mRNA‐GCase‐FLAG^(LNP^“O”)^ transfected^HEK293T^cells.^Culture^was^transfected^24^hrs,^fixed^with^4%^PFA,^and^stained^for^FLAG^ (GCase),^the^hemichannel^connexin‐43^(CX‐43,^negative^control),^and^a^nuclear^stain^(DAPI),^revealing^ little^no^co‐localized^expression^of^FLAG^(GCase)^and^CX‐43.^ FIGURE^22A^shows^Human^GCase^secretion^in^human^iPSC^neurospheres^(hiNS).^hiNS^were^ transfected^with^300nM,^30nM,^or^3nM^of^LNP‐mRNA‐GCase^(LNP^“N”)^on^Day^0.^Supernatant^was^ sampled^at^subsequent^feeding^intervals^for^analysis^of^human^GCase^secretion^via^immunoassay.^n=3^ per^dilution,^per^timepoint^(paired);^lower^limit^of^detection^=^3.41^pg / mL.^ FIGURE^22B^shows^Human^GCase^secretion^in^iPSC^neurospheres.^hiNS^were^transfected^with^150nM,^ 15nM,^or^1.5nM^of^LNP‐mRNA‐GCase^(LNP^“O”)^on^Day^0.^Supernatant^was^sampled^at^subsequent^ feeding^intervals^for^analysis^of^human^GCase^secretion^via^immunoassay.^n=3^per^dilution,^per^ timepoint^(paired);^lower^limit^of^detection^=^3.41^pg / mL.^ FIGURE^22C^shows^Human^GCase^secretion^in^iPSC^neurospheres.^hiNS^were^transfected^with^30nM,^ 3nM,^or^0.3nM^of^LNP‐mRNA‐GCase^(LNP^“Q”)^on^Day^0.^Supernatant^was^sampled^at^subsequent^ feeding^intervals^for^analysis^of^human^GCase^secretion^via^immunoassay.^n=3^per^dilution,^per^ timepoint^(paired);^lower^limit^of^detection^=^3.41^pg / mL.^ FIGURE^23A‐D^shows^cross‐correction^of^human^GCase^in^hiNS^cultures.^hiNS^transfected^directly^ with^150nM^LNP‐mRNA‐GCase^(LNP^“O”)^stained^for^FLAG^(red,^GCase),^GFAP^(green,^astrocytes),^and^ MAP2^(white,^neurons).^ FIGURES^23E‐H^show^untransfected^hiNS^that^received^donor^supernatant^from^transfected^hiNS^ from^FIGURE^22A^reveals^widespread^intracellular^localization^of^GCase^(FLAG)^in^astrocytes^(GFAP)^ and^neurons^(MAP2).^ FIGURES^23I‐K^shows^an^untransfected^control^sphere^that^did^not^receive^donor^super^supernatant^ with^no^visible^GCase^(FLAG)^staining.^ FIGURE^24A‐C^shows^co‐localization^of^human^GCase^and^LAMP1^in^hiNS^cultures^transfected^with^ LNP‐mRNA(GCase)^(LNP^“O”).^hiNS^were^transfected^with^LNP‐mRNA^encoding^FLAG‐tagged^human^ GCase^(150nM^for^72^hours).^Neurospheres^were^fixed,^permeabilized,^and^stained^for^LAMP1^(white)^ and^FLAG^(red,^GCase).^ FIGURE^25^shows^in^vivo^human^GCase^expression^in^CSF,^plasma,^and^brain^samples^from^mice^ injected^with^LNP‐mRNA(GCase)(LNP^“N”).^0.5ug / hemisphere^LNP‐mRNA(GCase)(LNP^“N”)^was^ injected^ICV^bilaterally.^CSF,^plasma,^and^whole^brain^collected^from^injected^mice^was^tested^for^ human^GCase^via^MSD^U‐PLEX^immunoassay.^No^signal^was^detected^in^the^CSF^of^saline‐injected^ control^mice^(n=3^per^time^point,^not^shown).^Lower^limit^of^detection^=^3.41^pg / mL.^ FIGURE^26^shows^in^vivo^human^GCase^expression^in^CSF,^plasma,^and^brain^samples^from^mice^ injected^with^LNP‐mRNA(GCase)(LNP^“O”).^0.5ug / hemisphere^LNP‐mRNA(GCase)(LNP^“O”)^was^ injected^ICV^bilaterally.^CSF,^plasma,^and^whole^brain^collected^from^injected^mice^was^tested^for^ human^GCase^via^MSD^U‐PLEX^immunoassay.^No^signal^was^detected^in^the^CSF^of^saline‐injected^ control^mice^(n=3^per^time^point,^not^shown).^Lower^limit^of^detection^=^3.41^pg / mL.^ FIGURE^27^shows^in^vivo^human^GCase^expression^in^CSF,^plasma,^and^brain^samples^from^mice^ injected^with^LNP‐mRNA(GCase)(LNP^“P”).^0.5ug / hemisphere^LNP‐mRNA(GCase)(LNP^“P”)^was^ injected^ICV^bilaterally.^CSF,^plasma,^and^whole^brain^collected^from^injected^mice^was^tested^for^ human^GCase^via^MSD^U‐PLEX^immunoassay.^No^signal^was^detected^in^the^CSF^of^saline‐injected^ control^mice^(n=3^per^time^point,^not^shown).^Lower^limit^of^detection^=^3.41^pg / mL.^ FIGURE^28^shows^in^vivo^human^GCase^expression^in^CSF,^plasma,^and^brain^samples^from^mice^ injected^with^LNP‐mRNA‐GCase^(LNP^“Q”).^0.5ug / hemisphere^LNP‐mRNA(GCase)(LNP^“Q”)^was^ injected^ICV^bilaterally.^CSF,^plasma,^and^whole^brain^collected^from^injected^mice^was^tested^for^ human^GCase^via^MSD^U‐PLEX^immunoassay.^No^signal^was^detected^in^the^CSF^of^saline‐injected^ control^mice^(n=3^per^time^point,^not^shown).^Lower^limit^of^detection^=^3.41^pg / mL.^ FIGURE^29^shows^example^immunofluorescence^images^from^LNP‐mRNA‐GCase^injected^mice^(ICV)^ sacrificed^at^3^hours^or^48^hours^post‐injection^compared^to^an^un‐injected^control.^Images^from^LNP‐ mRNA‐GCase^injected^brains^reveal^punctate^immunostainings^from^neuronal^cell^body^layers^that^are^ absent^in^un‐injected^controls,^indicating^lysosomal^uptake.^All^images^are^from^cortical^layers^4‐5^ distal^to^the^ICV^injection^site^but^also^from^mCherry^positive^cells^in^the^corpus^callosum,^e.g.^from^ FIGURE^3^and^FIGURE^4,^suggesting^GCase^diffusion^and^uptake^by^cross‐correction.^ FIGURE^30^shows^exemplar^expression^of^GCase‐FLAG^(FLAG)^in^astrocytes^(GFAP)^in^the^corpus^ callosum^in^vivo^from^mice^injected^with^LNP‐mRNA(GCase)(LNP^“O”,^0.5ug^mRNA / hemisphere,^ bilateral^injection).^The^brain^was^harvested^48^hrs^post‐injection.^The^white^box^highlights^co‐ localization^of^FLAG^signal^in^a^GFAP‐positive^cell.^ FIGURE^31^shows^a^schematic^of^an^embodiment^of^representative^species^of^two^types^of^mRNA^ constructs^leading^to^expression^of^conventional^heavy^and^light^chain^antibodies^(top^panel)^and^non‐ conventional^Variable^domain^of^Heavy^chain^of^Heavy‐chain^antibodies^(VHH,^or^Nanobody).^The^ presence^or^absence^of^modular^components^of^the^final^mRNA^sequence^(shown^here^for^illustrative^ purposes^only)^can^be^individually^tuned.^ FIGURE^32^shows^trastuzumab^expression^from^supernatant^measured^from^HEK293T^culture^ transfected^with^heavy^and / or^light^chain^trastuzumab^mRNA^reveals^significant^antibody^expression^ when^heavy^and^light^chain^mRNA^sequences^are^co‐expressed^for^form^the^fully^assembled^antibody.^ FIGURE^33^shows^exemplar^expression^of^the^antibody^trastuzumab^(secreted)^from^supernatant^ measured^from^HEK293T^culture^transfected^with^pDNA^encoding^a^heavy:P2A:light^trastuzumab^ sequence^reveals^significant^antibody^expression^when^heavy^and^light^chain^mRNA^linked^in^series.^ FIGURE^34^shows^CSF^and^brain^expression^of^trastuzumab^from^mice^injected^with^1.5ug^ mRNA / hemisphere^of^LNP‐mRNA(trastuzumab)^(LNP^“S”).^Data^were^collected^using^an^MSD^U‐PLEX^ assay^again^human^IgG^to^detect^the^fully^assembled^trastuzumab^as^shown^in^FIGURE^30.^ ^ DETAILED^DESCRIPTION^ The^following^detailed^description^will^be^better^understood^when^read^in^conjunction^with^the^ appended^figures.^^For^the^purpose^of^illustrating^the^invention,^the^figures^demonstrate^embodiments^ of^the^present^invention,^however,^the^invention^is^not^limited^to^the^precise^arrangements,^examples,^ and^instrumentalities^shown.^ It^is^demonstrated^herein^that^delivery^of^a^lipid^nanoparticle^(“LNP”)^encapsulating^mRNA,^circular^ RNA^(circRNA)^or^self‐amplifying^RNA^(saRNA)^encoding^a^polypeptide,^antibody,^or^antigen‐binding^ polypeptide,^to^a^glial^cell^may^convert^the^glial^cell^into^a^“CNS^protein^bioreactor”^that^secretes^a^ polypeptide,^other^lysosomal^enzyme,^antibody^or^antigen‐binding^fragment^encoded^by^the^mRNA,^ circRNA,^or^saRNA.^^ The^method^discloses^herein^uses^a^consistent^subpopulation^of^neural^cells,^namely^glial^cells,^as^CNS^ protein^bioreactors^specifically^to^produce^and^secrete^proteins^for^brain‐wide^bio‐distribution^and^at^ therapeutic^concentrations.^^ Hereafter^in^the^Detailed^Description,^to^avoid^repetition,^“Therapeutic^Protein”^shall^be^interpreted^to^ mean^glucocerebrosidase^(Gcase),^lysosomal^enzyme,^antibody^or^antigen‐binding^protein^except^ when^otherwise^specified.^^Hereinafter,^“RNA”^will^be^understood^to^mean^“mRNA”^unless^otherwise^ specified.^ An^example^of^the^inventive^delivery^method^is^shown^in^FIGURE^1.^^With^reference^to^the^example^ depicted^in^FIGURE^1,^an^LNP^comprising^an^mRNA,^or^circRNA,^or^saRNA^that^encodes^a^secretable^ therapeutic^protein^such^as^GCase,^therapeutic^enzyme,^antibody,^or^antigen‐binding^protein^is^ injected^into^the^CNS^of^a^subject^(i.e.^intracerebroventricular^(ICV)^injection;^lumbar^intrathecal^ injection^(IT);^or^cisterna^magna^injection),^which^is^depicted^as^a^mouse^by^way^of^example,^but^ includes^any^mammalian^subject,^including^a^human.^The^LNP‐mRNA,^LNP‐circRNA^or^LNP‐saRNA^ may^be^taken^up^by^endocytosis^into^brain^cells^(e.g.,^glial^cell)^and^the^mRNA^is^translated^into^the^ Therapeutic^Protein,^antibody^or^antigen‐binding^protein^in^the^cytoplasm^of^the^cell^(FIGURE^2).^^The^ expressed^protein^is^subsequently^secreted^from^the^cell^into^an^interstitial^and / or^cerebrospinal^fluid^ (CSF),^allowing^the^protein^to^diffuse^to^a^target^site^and^exert^a^therapeutic^or^prophylactic^effect^in^ one^or^more^regions^of^the^central^nervous^system^(FIGURE^2).^As^further^demonstrated^herein,^the^ secreted^Therapeutic^Protein^can^exit^the^CNS^to^the^blood^and^is^capable^of^being^internalized^by^ peripheral^cells^throughout^the^body.^^The^principal^therapeutic^effect^of^Therapeutic^Protein^is^to^ metabolize^glucosylceramide.^^As^depicted,^in^some^advantageous^examples^of^the^disclosure,^the^ protein^secreted^by^the^glial^cell^is^diffusible,^thereby^effecting^brain‐wide^delivery^within^a^subject.^ The^secreted^protein^can^then^be^taken^up^by^transfected^glial^cells^and^un‐transfected^cells^in^the^ cytosol^and^lysosomes^via^cross‐correction^to^exert^its^therapeutic^effects^(FIGURE^2).^^ Accordingly,^in^one^aspect,^provided^herein^is^a^method^of^treating^or^preventing^a^central^nervous^ system^disease,^disorder,^trauma^or^injury^in^a^subject.^In^some^embodiments,^the^method^comprises^ contacting^a^lipid^nanoparticle^(LNP)^with^a^cell^of^the^central^nervous^system^of^the^subject.^In^some^ embodiments,^the^LNP^comprises^a^nucleic^acid^encoding^a^Therapeutic^Protein^or^functional^ fragment^thereof.^In^some^embodiments,^the^LNP^comprises^an^mRNA^encoding^for^any^lysosomal^ enzyme^that^has^therapeutic^benefit^in^a^lysosomal^storage^disorder.^In^some^embodiments,^the^LNP^ comprises^an^mRNA^encoding^an^antibody.^^In^some^embodiments,^the^LNP^comprises^an^mRNA^ encoding^an^antigen‐binding^protein.^^In^yet^other^embodiments,^the^LNP^comprises^a^circRNA^or^ saRNA^in^place^of^the^mRNA.^^In^some^embodiments,^the^Therapeutic^Protein^or^functional^fragment^ thereof^is^capable^of^secretion^from^the^cell^into^a^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ In^some^embodiments,^provided^herein^is^a^method^of^treating^or^preventing^a^central^nervous^system^ disease,^disorder,^trauma^or^injury^in^a^subject,^the^method^comprising:^contacting^a^LNP^with^a^cell^of^ the^central^nervous^system^of^the^subject,^the^LNP^comprising^a^nucleic^acid^encoding^a^Therapeutic^ Protein^or^functional^fragment^thereof,^the^Therapeutic^Protein^or^functional^fragment^thereof^being^ capable^of^secretion^from^the^cell^into^a^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ In^another^aspect,^provided^herein^is^a^lipid^nanoparticle.^In^some^embodiments,^the^LNP^comprises^a^ nucleic^acid^encoding^a^Therapeutic^Protein^or^functional^fragment^thereof.^^In^some^embodiments,^ the^Therapeutic^Protein^or^functional^fragment^thereof^is^being^capable^of^secretion^from^a^cell^of^the^ central^nervous^system^of^a^subject^into^a^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ In^some^embodiments,^provided^herein^is^a^LNP^comprising^a^nucleic^acid^encoding^a^Therapeutic^ Protein^or^functional^fragment^thereof,^the^Therapeutic^Protein^or^functional^fragment^thereof^being^ capable^of^secretion^from^a^cell^of^the^central^nervous^system^of^a^subject^into^a^interstitial^and / or^ cerebrospinal^fluid^of^the^subject.^ In^another^aspect,^provided^herein^is^an^LNP^comprising^a^circular^RNA^(“circRNA”)^encoding^a^ polypeptide.^In^some^embodiments,^the^polypeptide^is^capable^of^secretion^from^a^cell^of^the^central^ nervous^system^of^a^subject^into^an^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ In^some^embodiments,^provided^herein^is^an^LNP^comprising^a^circular^RNA^(“circRNA”)^encoding^a^ polypeptide,^the^polypeptide^being^capable^of^secretion^from^a^cell^of^the^central^nervous^system^of^a^ subject^into^an^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ In^another^aspect,^provided^herein^is^an^LNP^comprising^self‐amplifying^RNA^(“saRNA”)^encoding^a^ polypeptide.^In^some^embodiments,^the^polypeptide^is^capable^of^secretion^from^a^cell^of^the^central^ nervous^system^of^a^subject^into^an^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ In^some^embodiments,^provided^herein^is^an^LNP^comprising^a^self‐amplifying^RNA^(“saRNA”)^ encoding^a^polypeptide,^the^polypeptide^being^capable^of^secretion^from^a^cell^of^the^central^nervous^ system^of^a^subject^into^an^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ In^some^embodiments,^contacting^of^the^LNP^with^the^cell^results^in^the^nucleic^acid^entering^the^cell.^ In^some^embodiments,^contacting^of^the^LNP^with^the^cell^results^in^production^of^the^Therapeutic^ Protein^or^functional^fragment^thereof^in^the^cell.^ In^some^embodiments,^subsequent^to^the^production^of^the^Therapeutic^Protein^or^functional^ fragment^thereof,^the^Therapeutic^Protein^or^functional^fragment^thereof^is^secreted^from^the^cell^to^ an^interstitial^and / or^cerebrospinal^fluid^(CSF)^of^the^subject.^ In^some^embodiments,^the^Therapeutic^Protein^or^functional^fragment^thereof^is^diffusible^within^the^ interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ In^some^embodiments,^the^Therapeutic^Protein^or^functional^fragment^thereof^enters^un‐transfected^ cells^to^exert^therapeutic^effects.^ In^some^embodiments,^the^Therapeutic^Protein^or^functional^fragment^thereof^is^capable^of^being^ internalized^by^a^cell^of^the^CNS.^ In^some^embodiments,^the^cell^of^the^CNS^comprises^neurons,^glia,^neurovascular^cells,^and / or^ meningeal^cells.^ In^some^embodiments,^the^neurons^comprise^interneurons.^In^some^embodiments,^the^glia^comprise^ astrocytes,^oligodendrocytes,^microglia,^ependymal^cells,^and / or^radial^glia.^In^some^embodiments,^the^ neurovascular^cells^comprise^endothelial^cells,^pericytes,^and / or^smooth^muscle^cells.^ In^some^embodiments,^the^Therapeutic^Protein^or^functional^fragment^thereof^is^capable^of^being^ internalized^by^a^cell^of^outside^of^the^CNS.^^ Examples^of^cells^outside^of^the^CNS^that^are^contemplated^to^internalize^the^Therapeutic^Protein^or^ functional^fragment^thereof^include,^but^are^not^limited^to,^hematopoietic^cells,^epithelial^cells,^ endothelial^cells,^cardiac^cells,^vascular^smooth^muscle^cells,^cardiomyocytes,^skeletal^muscle^cells,^ hepatocytes,^lung^cells,^bone^cells,^skin^cells,^gastrointestinal^cells,^spleen^cells,^renal^cells,^stem^cells,^ mesenchymal^cells,^peripheral^neural^cells,^adipocytes,^beta^cells,^pituitary^cells,^synovial^lining^cells,^ ovarian^cells,^testicular^cells,^fibroblasts,^B^cells,^T^cells,^reticulocytes,^leukocytes,^macrophages,^ granulocytes^and^tumor^cells.^ In^some^embodiments,^the^secreted^Therapeutic^Protein^or^functional^fragment^thereof^is^present^in^ the^CSF,^ISF,^or^intracellular^compartments^of^the^subject^for^a^period^of^at^least^24^hours,^at^least^36^ hours,^or^at^least^48^hours.^ In^some^embodiments,^the^secreted^Therapeutic^Protein^or^functional^fragment^thereof^is^present^in^ the^CSF,^ISF,^or^intracellular^compartments^of^the^subject^for^a^period^of^at^least^1^week,^at^least^2^ weeks,^at^least^3^weeks,^at^least^4^weeks,^at^least^6^weeks,^or^at^least^8^weeks.^ In^some^embodiments,^the^secreted^Therapeutic^Protein^or^functional^fragment^thereof^is^present^in^ the^CSF,^ISF,^or^intracellular^compartments^of^the^subject^at^a^first^concentration^at^a^first^time^point^ and^at^a^second^concentration^at^a^later^second^time^point.^ In^some^embodiments,^the^first^time^point^is^about^3^hours^after^contacting^the^LNP^with^the^cell.^ In^some^embodiments,^the^second^time^point^is^about^48^hours^after^contacting^of^the^LNP^with^the^ cell.^ In^some^embodiments,^the^second^concentration^is^at^least^30%,^at^least^40%,^at^least^50%,^at^least^ 60%,^at^least^70%,^at^least^80%,^at^least^90%,^or^at^least^95%^of^the^first^concentration.^ In^some^embodiments,^the^contacting^is^in^vitro^or^in^vivo.^ In^some^embodiments,^the^cell^of^the^central^nervous^system^is^a^glial^cell.^A^glial^cell^can^for^example^ be^an^astrocyte,^^an^oligodendrocyte,^or^an^oligodendrocyte^precursor^cell.^ The^oligodendrocyte^can,^for^example,^be^a^pre‐oligodendrocytes,^an^(im)mature^oligodendrocytes^or^ a^myelinating^oligodendrocytes.^ In^some^embodiments,^the^cell^of^the^central^nervous^system^is^an^ependymal^cell.^An^ependymal^cell^ can^for^example^be^from^the^cerebral^ventricles^or^the^choroid^plexus.^ As^used^herein,^the^term^“glucocerebrosidase”^or^“GCase”^encompasses^glucocerebrosidases^from^any^ organism^and^variants^thereof.^^ In^some^embodiments,^the^glucocerebrosidase^is^a^human^glucocerebrosidase.^ As^used^herein,^the^term^“functional^fragment”^in^the^context^of^a^Therapeutic^Protein^or^polypeptide^ means^a^fragment^that^is^capable^of^having^one^or^more^activities^of^a^reference^protein^or^ polypeptide.^For^example,^a^functional^fragment^of^a^GCase^can^refer^to^a^GCase^fragment^that^retains^ at^least^one^activity^of^full^length^GCase^to^at^least^a^substantial^extent.^ In^some^embodiments,^the^GCase^comprises^an^amino^acid^sequence^of^at^least^at^least^80%,^at^least^ 85%,^at^least^90%,^at^least^95%,^or^at^least^98%^sequence^identity^to^SEQ^ID^NO:^1.^ The^term^“sequence^identity”,^in^the^context^of^nucleotide^or^amino^acid^sequences,^refers^to^the^ percentage^of^nucleotides^or^amino^acid^residues^that^are^identical^between^two^sequences^when^ aligned.^Alignment^may^be^in^such^a^way^to^provide^the^highest^level^of^matches^between^the^two^ sequences.^The^comparison^may^be^over^the^entire^length^of^a^sequence,^or^over^a^specific^portion^of^a^ sequence.^^Gapped^alignments^may^be^permitted.^Sequence^identity^may^be^determined^manually,^or^ by^a^sequence^comparison^algorithm.^When^determined^manually,^after^aligning^the^two^sequences,^ the^number^of^identical^positions^can^be^divided^by^the^number^of^nucleotides^or^amino^acid^residues^ in^the^reference^sequence.^Therefore,^for^example,^a^sequence^identity^of^at^least^90%^means^a^ sequence^is^identical^to^the^reference^sequence^at^least^90^out^of^100^positions^of^the^reference^ sequence.^Various^computer^programs^and^online^tools^may^be^used^to^determine^sequence^identity^ and^are^known^in^the^art.^For^example,^the^basic^local^alignment^search^tool^(“BLAST”15)^can^be^ available^through^the^National^Center^for^Biotechnology^Information^(“NCBI”).^When^using^computer^ programs^or^algorithms,^default^parameters^are^generally^used.^ In^some^embodiments,^the^GCase^or^functional^fragment^thereof^can^cross^a^central^nervous^system^ cellular^membrane^to^exert^an^extracellular^therapeutic^or^prophylactic^effect^within^one^or^more^ regions^of^the^central^nervous^system.^The^GCase^or^functional^fragment^thereof^may^comprise^an^ exogenous^secretion^signal^that^facilitates^secretion^thereof.^^Without^being^limiting,^the^secretion^ signal^may^be^located^at^the^amino^terminus^and^cause^the^translocation^of^newly^synthesized^protein^ through^the^endoplasmic^reticulum,^Golgi^network^and^to^the^cell^membrane^for^secretion^into^ interstitial^and / or^cerebrospinal^fluid.^The^GCase^or^functional^fragment^thereof^may^be^secreted^via^a^ “non‐classical”^pathway^that^is^not^reliant^on^the^inclusion^of^a^secretion^signal.^ In^some^embodiments,^the^GCase^further^comprises^an^amino^acid^sequence^of^at^least^at^least^80%,^at^ least^85%,^at^least^90%,^at^least^95%,^or^at^least^98%^sequence^identity^to^SEQ^ID^NO:^5.^ The^ability^of^a^protein^or^peptide^to^be^secreted^from^a^central^nervous^system^cell^can,^for^example,^ be^assessed^in^vitro^by^the^method^described^in^EXAMPLE^1,^EXAMPLE^4,^EXAMPLE^5,^and^ EXAMPLE^6^herein.^^Briefly,^the^assay^described^in^EXAMPLE^1,^EXAMPLE^4,^EXAMPLE^5,^and^ EXAMPLE^6^involves^culturing^HEK293T^cells^or^brain^glial^cells^(astrocytes)^or^human^induced^ pluripotent^stem^cell^(iPSC)^neurosphere^cultures^and^treating^the^cell^cultures^with^LNP‐mRNA^ coding^for^GCase.^^GCase^secretion^is^then^assayed^by^collecting^the^cell^supernatant^and^quantifying^ the^protein^amount,^for^example,^via^immunoblotting^or^ELISA.^^The^supernatant^will^be^collected^at^ various^time^points^to^estimate^the^peak^production / secretion^timeline.^^The^in^vitro^assay^will^ typically^be^followed^up^with^in^vivo^studies^in^rodents^to^confirm^protein^secretion.^^Such^in^vivo^ studies^are^described^in^the^Examples^section^herein.^ The^GCase^or^functional^fragment^thereof^can^comprise^one^or^more^amino^acid^variations / mutations^ to^for^example^increase^its^half‐life^in^the^central^nervous^system.^ For^some^proteins^where^intracellular^localization^after^secretion^is^desirable,^a^sequence^coding^for^a^ cell‐penetrating^peptide^(CPP)^such^as^TAT^peptide^may^be^included.^^Genetically^engineered^mRNA^ sequences^could^therefore^include^both^a^signal^sequence^and^a^CPP^sequence^to^induce^secretion^and^ promote^cellular^uptake^of^the^protein^by^a^target^cell,^respectively.^ In^some^embodiments,^the^GCase^or^fragment^thereof^is^capable^of^being^internalized^by^cells^in^the^ CNS,^such^as^neurons^(including^interneurons),^glia^(such^as^astrocytes,^oligodendrocytes,^microglia,^ ependymal^cells,^radial^glia),^neurovascular^cells^(endothelial^cells,^pericytes,^smooth^muscle^cells),^ and^meningeal^cells.^ The^GCase^or^functional^fragment^thereof^can^comprise^further^domains,^for^example,^to^enhance^its^ activity^in^the^brain,^and / or^to^improve^its^therapeutic^effect.^^ In^some^embodiments,^the^GCase^further^comprises^a^cell‐penetrating^peptide.^ In^embodiments,^the^mRNA,^circRNA,^or^saRNA^encode^a^secretory^protein.^ As^used^herein,^the^terms^“polypeptide”,^“peptide”^and^“protein”^are^used^interchangeably^and^refer^to^ a^polymer^of^at^least^two^amino^acids.^ The^secretory^protein^may^comprise^a^secretion^signal^that^facilitates^secretion^thereof.^^The^protein^ may^already^possess^a^secretion^signal^sequence^(e.g.,^growth^factors^and^cytokines)^or^may^be^ genetically^modified^to^include^a^secretion^signal^sequence.^^^ Without^being^limiting,^the^secretion^signal^may^be^located^at^the^amino^terminus^of^the^secretory^ protein^and^cause^the^translocation^of^newly^synthesized^protein^through^the^endoplasmic^reticulum,^ Golgi^network^and^to^the^cell^membrane^for^secretion^into^interstitial^and / or^cerebrospinal^fluid.^ Alternatively,^in^one^example^of^the^disclosure,^the^secretory^protein^is^secreted^via^a^“non‐classical”^ pathway^that^is^not^reliant^on^the^inclusion^of^a^secretion^signal.^^Moreover,^secretory^proteins^may^be^ naturally^released^by^central^nervous^system^cells,^such^as^astrocyte^or^oligodendrocyte^or^an^ ependymal^cell^of^the^cerebral^ventricles^or^the^choroid^plexus^under^specific^environmental^ conditions.^ The^ability^of^a^protein^or^peptide^to^be^secreted^from^a^central^nervous^system^cell^can^be^assessed^in^ vitro^by^the^method^described^in^EXAMPLE^4,^EXAMPLE^5,^and^EXAMPLE^6^herein.^^Briefly,^the^assay^ involves^culturing^brain^glial^cells^(primary^rodent^astrocytes^or^human^astrocytes)^or^human^induced^ pluripotent^stem^cell^neurosphere^cultures^and^treating^the^cell^cultures^with^LNP‐mRNA,^LNP‐ circRNA,^or^LNP‐saRNA^coding^for^the^candidate^protein.^^Secretion^of^the^protein^is^then^assayed^by^ collecting^the^cell^supernatant^and^quantifying^the^protein^amount,^for^example,^via^immunoblotting,^ ELISA^or^a^U‐PLEX™^assay.^^The^supernatant^is^collected^at^various^time^points^to^estimate^the^peak^ production / secretion^timeline.^^The^in^vitro^assay^will^typically^be^followed^up^with^in^vivo^studies^in^ rodents^to^confirm^protein^secretion.^^Such^in^vivo^studies^are^described^in^the^Examples^section^ herein.^ For^some^proteins^where^intracellular^localization^after^secretion^is^desirable,^a^sequence^coding^for^a^ cell‐penetrating^peptide^(CPP)^such^as^TAT^peptide^may^be^included.^^Genetically^engineered^mRNA,^ circRNA^and^saRNA^sequences^could^therefore^include^both^a^signal^sequence^and^a^CPP^sequence^to^ induce^secretion^and^promote^cellular^uptake^of^the^protein^by^a^target^cell,^respectively.^ The^secretory^protein^may^treat^or^prevent^a^central^nervous^system^disease,^disorder,^trauma^or^ injury.^^The^secretory^protein^may^also^treat^or^prevent^conditions^such^as^aging,^preventative^ medicine^and / or^be^used^as^part^of^a^personalized^medicine^regime.^Typically,^the^protein^is^secreted^ from^the^cell^of^the^central^nervous^system^into^the^cerebrospinal^fluid^of^the^subject^and^diffuses^to^a^ target^site.^The^secretory^protein^may^also^be^used^as^a^diagnostic^agent.^Accordingly,^the^protein^or^ polypeptide^encoded^by^the^mRNA,^circRNA^or^saRNA^can^be^a^therapeutic^polypeptide,^a^prophylactic^ polypeptide,^and / or^a^diagnostic^polypeptide.^ In^some^embodiments,^the^polypeptide^comprises^a^therapeutic^polypeptide.^In^some^embodiments,^ the^polypeptide^comprises^a^prophylactic^polypeptide.^In^some^embodiments,^the^polypeptide^ comprises^a^diagnostic^polypeptide.^ It^should^be^appreciated^that^a^combination^of^two^or^more^secretory^proteins^may^be^used^to^treat^or^ prevent^a^central^nervous^system^disease,^disorder,^trauma^or^injury.^One^or^more^mutations^can^be^ introduced^to^a^secretory^protein^to,^for^example,^increase^its^half‐life^in^the^central^nervous^system.^^ Further,^certain^secretory^proteins^may^be^used^to^treat^or^prevent^more^than^one^disease^indication.^ The^secretory^protein^may^also^be^modified^post‐translationally.^ The^term^“antibody”^as^used^herein^means^any^immunoglobulin^molecule^that^is^capable^of^specific^ binding^of^a^target^through^at^least^one^antigen‐binding^site.^As^used^herein,^the^term^encompasses^ conventional^full‐length^antibodies^and^derivatives^or^configurations^thereof,^including^but^not^limited^ to^monoclonal^antibodies,^humanized^antibodies,^chimeric^antibodies,^multi‐specific^antibodies,^ diabodies,^triabodies,^minibodies,^single^domain^antibodies,^intrabodies,^and^covalently^modified^ variants^(e.g.^glycosylation).^ The^term^“antigen‐binding^fragment”^as^used^herein^means^any^polypeptide^that^can^bind^specifically^ to^an^epitope^of^an^antigen.^Examples^of^antigen‐binding^fragments^include^but^are^not^limited^to^ HCAb^(heavy^chain^antibody),^VNAR^(variable^new^antigen^receptor),^VHH,^Fab^(fragment^antigen^ binding),^F(ab’),^F(ab’)2,^Fd^fragments,^Fv^fragments,^scFv^(single^chain^fragment^variable),^single^ domain^antibodies,^and^Fc^(fragment^crystallizable).^^ The^antibody^or^antigen‐binding^fragment^can^be^multi‐specific,^meaning^they^can^bind^specifically^to^ more^than^one^epitope.^A^bispecific^antigen‐binding^fragment,^for^example,^can^comprise^two^different^ variable^domains^capable^of^binding^to^different^antigens^or^different^epitopes^of^the^same^antigen.^ The^antibody^or^antigen‐binding^fragment^can^be^a^single^polypeptide.^The^antibody^or^antigen‐ binding^fragment^can^comprise^more^than^one^polypeptide.^For^example,^the^antibody^or^antigen‐ binding^fragment^can^comprise^a^light^chain^and^a^heavy^chain.^ In^some^embodiments,^the^antibody^or^antigen‐binding^fragment^comprise^a^light^chain.^In^some^ embodiments,^the^antibody^comprises^a^heavy^chain.^In^some^embodiments,^the^antibody^or^antigen‐ binding^fragment^comprise^a^light^chain^and^a^heavy^chain.^ The^antibody^or^antigen^binding^fragment^can^comprise^one^or^more^further^domains.^For^example,^ the^antibody^or^antigen‐binding^fragment^can^comprise^a^secretion^signal^that^facilitates^secretion^ thereof.^The^antibody^or^antigen‐binding^fragment^may^already^possess^a^secretion^signal^sequence^or^ may^be^genetically^modified^to^include^a^secretion^signal^sequence.^^Without^being^limiting,^the^ secretion^signal^may^be^located^at^the^amino^terminus^of^the^secretory^protein^and^cause^the^ translocation^of^newly^synthesized^protein^through^the^endoplasmic^reticulum,^Golgi^network^and^to^ the^cell^membrane^for^secretion^into^interstitial^and / or^cerebrospinal^fluid.^^ The^antibody^or^antigen^binding^fragment^can^comprise^single‐domain^antibodies^(sdAB)^like^variable^ domain^of^heavy‐chain^antibodies^(VHH)^or^variable^new^antigen^receptors^(VNAR).^While^ conventional^antibodies^are^made^of^two^heavy^and^two^light^chains,^sdABs^like^VHH^are^composed^of^ the^variable^domain^of^Heavy‐chain^antibodies^(HCAb),^themselves^non‐conventional^antibodies^from^ camelids.^VHH^sdABs^are^roughly^ten^times^smaller^than^conventional^antibodies^with^a^molecular^ weight^of^approximately^15^kDa.^VNARs^are^also^composed^of^the^variable^domain^of^HCAb,^derived^ from^sharks^and^cartilaginous^fishes.^The^small^size^of^sdAbs^may^also^allow^for^binding^of^regions^of^a^ target^protein^otherwise^inaccessible^by^conventional^antibodies.^However,^sdABs^have^a^much^ shorter^half‐life^than^conventional^antibodies,^reducing^their^use^for^some^therapeutic^applications.^ The^antibody^or^antigen^binding^fragment^can^further^comprise^a^cell‐penetrating^peptide^(CPP)^such^ as^TAT^peptide^to^facilitate^internalization^by^a^cell^of^the^CNS,^such^as^neurons^(including^ interneurons),^glia^(such^as^astrocytes,^oligodendrocytes,^microglia,^ependymal^cells,^radial^glia),^ neurovascular^cells^(endothelial^cells,^pericytes,^smooth^muscle^cells),^progenitor^cells^and^meningeal^ cells.^An^internalized^antibody^or^antigen‐binding^fragment^can^bind^an^intracellular^target.^ The^Therapeutic^Protein^or^functional^fragment^thereof,^therapeutic^enzyme,^antibody,^or^antigen^ binding^fragment^may^treat^or^prevent^a^central^nervous^system^disease,^disorder,^trauma^or^injury.^^ The^LNP^comprising^the^nucleic^acid^encoding^the^Therapeutic^Protein^or^functional^fragment^thereof,^ therapeutic^enzyme,^antibody,^or^antigen^binding^fragment,^may^also^be^used^in^other^applications^ besides^the^treatment^and / or^prevention^of^a^disease,^disorder,^trauma^or^injury.^^The^LNP^comprising^ the^nucleic^acid^encoding^the^Therapeutic^Protein^or^functional^fragment,^therapeutic^enzyme,^ antibody,^or^antigen^binding^fragment,^thereof^may^be^used^to^treat^conditions^such^as^aging,^ preventative^medicine^and / or^as^part^of^a^personalized^medicine^regime.^^In^further^embodiments,^the^ LNP^comprising^the^nucleic^acid^encoding^the^Therapeutic^Protein^or^functional^fragment^thereof,^ therapeutic^enzyme,^antibody,^or^antigen^binding^fragment^may^be^used^in^a^diagnostic^application.^ Non‐limiting^examples^of^central^nervous^system^diseases^or^disorders^are^Neuronopathic^Lysosomal^ Storage^Disorders:^Gaucher^Disease^Type^1,^Gaucher^Disease^Type^2,^Gaucher^Disease^Type^3,^ Sphingolipidoses^(e.g.^Niemann‐Pick^Disease^Types^A^and^C,^Krabbe^Disease,^Metachromatic^ Leukodystrophy,^Fabry^Disease,^Farber^Disease,^Multiple^Sulfatase^Deficiency),^GM1^Gangliosidoses^ (e.g.^Type^I,^Type^II,^Type^III),^GM2^Gangliosidoses^(e.g.^Tay‐Sachs^Disease,^Sandhoff^Disease),^ Mucopolysaccharidoses^(e.g.^MPSI,^MPSII,^MPS^III,^MPS^VII),^Neuronal^Ceroid^Lipofuscinoses^(CLN,^e.g.^ CLN1^Santavuori‐Haltia^Disease,^CLN2^Jansky‐Bielschowsky^Disease,^CLN3^Batten^Disease,^CLN4^Kufs^ Disease,^CLN5,^CLN6,^CLN7,^CLN8,^CLN10,^CLN11,^CLN12,^CLN13,^CLN14)^^Mucolipidosis^Type^II,^ Mucolipidosis^Type^III,^Mucolipidosis^Type^IV,^and^Wolman^Disease;^Neurodegenerative^disorders:^ Parkinson’s^Disease^and^related^synucleinopathies,^Alzheimer’s^Disease,^Lewy^Body^Dementia,^ frontotemporal^dementia,^^Vascular^Dementia,^Multiple^Sclerosis,^and^Amyotrophic^Lateral^Sclerosis;^ Traumatic^Brain^Injury^and^Spinal^Cord^injury;^and^Cerebrovascular^Diseases^(e.g.^ischemic^and^ hemorrhagic^stroke,^Transient^Ischemic^attack,^Brain^Aneurysm,^Arteriovenous^Malformation,^ Cavernous^Malformation,^Moyamoya^Disease).^ It^should^be^appreciated^that^the^LNP^comprising^the^nucleic^acid^encoding^the^Therapeutic^Protein^or^ functional^fragment,^therapeutic^enzyme,^antibody,^or^antigen^binding^fragment^thereof^disclosed^ herein^may^be^used^to^treat^or^prevent^more^than^one^disease^indication.^ Nucleic^acid^ The^term^“nucleic^acid”^or^“nucleic^acid^molecule”^refers^to^covalently^linked^sequence^of^nucleotides,^ such^as^ribonucleotides^and^deoxyribonucleotides,^and^encompass^analogs^and^derivatives^of^natural^ nucleotides^and^modifications.^A^nucleic^acid^molecule^may^be^formed^by^a^single^type^of^nucleotides^ (e.g.^DNA^or^RNA),^or^it^may^be^a^hybrid^of^different^nucleotides,^for^example,^a^DNA / RNA^hybrid.^A^ nucleic^acid^molecule^may^be^single‐^stranded,^double‐stranded,^or^partially^double‐stranded.^For^ example,^a^nucleic^acid^molecule^may^have^a^stem‐loop^structure,^where^the^stem^portion^is^double‐ stranded^and^the^loop^portion^is^single‐stranded.^A^nucleic^acid^may^be^in^different^conformations,^ such^as^linear^or^circular.^ In^some^embodiments,^the^nucleic^acid^comprises^a^sequence^of^at^least^60%,^at^least^70%,^at^least^ 75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ID^NO:^2.^ In^some^embodiments,^the^nucleic^acid^further^comprises^a^nucleotide^sequence^of^at^least^60%,^at^ least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^ SEQ^ID^NO:^3^or^4.^ In^some^embodiments,^the^nucleic^acid^further^comprises^a^nucleotide^sequence^of^at^least^60%,^at^ least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^ SEQ^ID^NO:^6.^ In^some^embodiments,^the^nucleic^acid^further^comprises^a^nucleotide^sequence^of^at^least^60%,^at^ least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^ SEQ^ID^NO:^7.^ In^some^embodiments,^the^nucleic^acid^comprises^a^nucleotide^sequence^of^at^least^60%,^at^least^70%,^ at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ID^NO:^ 8.^ In^some^embodiments,^the^nucleic^acid^comprises^an^RNA.^ In^some^embodiments,^the^nucleic^acid^comprises^an^mRNA.^ In^some^embodiments,^the^nucleic^acid^comprises^a^circular^RNA^(circRNA).^ In^some^embodiments,^the^nucleic^acid^comprises^a^self‐amplifying^RNA^(saRNA).^ In^the^case^of^the^nucleic^acid^encoding^an^antibody,^antibody^component,^or^antigen‐binding^protein,^ there^are^one,^two,^or^more^nucleic^acids.^The^at^least^one^nucleic^acid^encoding^the^antibody^or^ antigen‐binding^fragment^can^be^one^nucleic^acid^molecule.^The^at^least^one^nucleic^acid^encoding^the^ antibody^or^antigen‐binding^fragment^can^be^two^nucleic^acid^molecules.^The^at^least^one^nucleic^acid^ encoding^the^antibody^or^antigen‐binding^fragment^can^be^three^nucleic^acid^molecules.^The^at^least^ one^nucleic^acid^encoding^the^antibody^or^antigen‐binding^fragment^can^be^more^than^three^nucleic^ acid^molecules.^ The^at^least^one^nucleic^acid^can^comprise^nucleic^acid^molecules^that^encodes^different^polypeptides,^ such^as^a^light^chain^and^a^heavy^chain.^^ In^some^embodiments,^the^at^least^one^nucleic^acid^comprises^a^nucleic^acid^that^encodes^a^light^chain.^ In^some^embodiments,^the^at^least^one^nucleic^acid^comprises^a^nucleic^acid^that^encodes^a^heavy^ chain.^In^some^embodiments,^the^at^least^one^nucleic^acid^comprises^a^nucleic^acid^that^encodes^a^light^ chain^and^a^nucleic^acid^that^encodes^a^heavy^chain.^^ Where^the^at^least^one^nucleic^acid^comprises^more^than^one^nucleic^acid,^the^more^than^one^nucleic^ acid^can^be^encapsulated^at^various^ratios.^ In^some^embodiments,^the^at^least^one^nucleic^acid^comprises^an^mRNA^that^encodes^a^light^chain.^In^ some^embodiments,^the^at^least^one^nucleic^acid^comprises^an^mRNA^that^encodes^a^heavy^chain.^In^ some^embodiments,^the^at^least^one^nucleic^acid^comprises^an^mRNA^that^encodes^a^light^chain^and^an^ mRNA^that^encodes^a^heavy^chain.^ In^some^embodiments,^the^nucleic^acid^that^encodes^the^light^chain^and^the^nucleic^acid^that^encodes^ the^heavy^chain^are^at^a^ratio^of^between^about^1:1^and^about^1:4^(w / w).^In^some^embodiments,^the^ nucleic^acid^that^encodes^the^light^chain^and^the^nucleic^acid^that^encodes^the^heavy^chain^are^at^a^ ratio^of^about^1:1^(w / w).^In^some^embodiments,^the^nucleic^acid^that^encodes^the^light^chain^and^the^ nucleic^acid^that^encodes^the^heavy^chain^are^at^a^ratio^of^about^1:2^(w / w).^In^some^embodiments,^the^ nucleic^acid^that^encodes^the^light^chain^and^the^nucleic^acid^that^encodes^the^heavy^chain^are^at^a^ ratio^of^about^1:3^(w / w).^In^some^embodiments,^the^nucleic^acid^that^encodes^the^light^chain^and^the^ nucleic^acid^that^encodes^the^heavy^chain^are^at^a^ratio^of^about^1:4^(w / w).^ In^some^embodiments,^the^nucleic^acid^that^encodes^the^light^chain^and^the^nucleic^acid^that^encodes^ the^heavy^chain^are^linked^in^series^at^a^ratio^of^1:1^using^a^2A^linker,^such^as^P2A,^T2A,^E2A,^F2A.^In^ some^embodiments,^the^nucleic^acid^that^encodes^the^light^chain^and^the^nucleic^acid^that^encodes^the^ heavy^chain^are^linked^in^series^at^a^ratio^of^1:2^using^2A^linkers,^such^as^P2A,^T2A,^E2A,^F2A.^In^some^ embodiments,^the^nucleic^acid^that^encodes^the^light^chain^and^the^nucleic^acid^that^encodes^the^heavy^ chain^are^linked^in^series^at^a^ratio^of^1:3^using^2A^linkers,^such^as^P2A,^T2A,^E2A,^F2A.^In^some^ embodiments,^the^nucleic^acid^that^encodes^the^light^chain^and^the^nucleic^acid^that^encodes^the^heavy^ chain^are^linked^in^series^at^a^ratio^of^1:4^using^2A^linkers,^such^as^P2A,^T2A,^E2A,^F2A.^ The^at^least^one^nucleic^acid^can^comprise^a^nucleic^acid^that^encodes^more^than^one^polypeptide.^For^ example,^the^nucleic^acid^can^be^a^single^nucleic^acid^molecule^that^encodes^a^heavy^chain^and^a^light^ chain.^^ As^used^herein,^the^term^“circular^RNA”^or^“circRNA”,^refers^to^a^circularized^RNA^polynucleotide.^The^ circRNA^can^encode^and^express^a^polypeptide,^such^as^a^secretory^protein.^The^term^“self‐amplifying^ RNA”^or^“saRNA”,^refers^to^a^RNA^polynucleotide^that^comprises^genes^necessary^for^self‐replication^ of^the^polynucleotide.^Examples^of^such^gens^include,^but^are^not^limited^to,^non‐structural^genes^(e.g.^ nsP1‐4^genes)^for^translation^of^replicase^proteins^and^formation^of^an^RNA‐dependent^RNA^ polymerase.^ The^circRNA^and^saRNA^as^used^herein^encompasses^both^modified^and^unmodified^RNA.^^In^one^ embodiment,^the^RNA^comprises^one^or^more^coding^and^non‐coding^regions.^^The^RNA^can^be^ purified^from^natural^sources,^produced^using^recombinant^expression^systems^and^optionally^ purified,^or^may^be^chemically^synthesized.^^Modifications^to^the^RNA^can^improve^immunogenicity,^ stability,^and^translational^efficiency^and^fidelity^of^the^RNA^and^thus^increase^the^amount^of^protein^ produced^from^the^RNA.^^^ In^some^embodiments,^the^nucleic^acid^comprises^an^mRNA^that^encodes^and^expresses^a^Therapeutic^ Protein^or^functional^fragment^thereof,^therapeutic^enzyme,^antibody,^or^antigen^binding^fragment.^ In^some^embodiments,^the^nucleic^acid^comprises^a^circular^RNA^or^a^self‐amplifying^RNA^that^ encodes^and^expresses^a^Therapeutic^Protein^or^functional^fragment^thereof,^therapeutic^enzyme,^ antibody,^or^antigen^binding^fragment.^ In^some^embodiments,^the^mRNA^comprises^a^sequence^of^at^least^60%,^at^least^70%,^at^least^75%,^at^ least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ID^NO:^2.^ In^some^embodiments,^the^mRNA^further^comprises^a^nucleotide^sequence^of^at^least^60%,^at^least^ 70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ ID^NO:^3^or^4.^ In^some^embodiments,^the^mRNA^further^comprises^a^nucleotide^sequence^of^at^least^60%,^at^least^ 70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ ID^NO:^6.^ In^some^embodiments,^the^mRNA^further^comprises^a^nucleotide^sequence^of^at^least^60%,^at^least^ 70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ ID^NO:^7.^ In^some^embodiments,^the^mRNA^comprises^a^nucleotide^sequence^of^at^least^60%,^at^least^70%,^at^ least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ID^NO:^8.^ ^The^term^“messenger^RNA”^or^“mRNA”^as^used^herein^encompasses^both^modified^and^unmodified^ mRNA,^as^well^as^circular^RNA,^self‐amplifying^RNA,^and^any^other^type^of^RNA^that^could^be^ translated^to^produce^a^protein.^In^one^embodiment,^the^mRNA^comprises^one^or^more^coding^and^ non‐coding^regions.^^The^mRNA^can^be^purified^from^natural^sources,^produced^using^recombinant^ expression^systems^and^optionally^purified,^or^may^be^chemically^synthesized.^^Modifications^to^the^ mRNA^can^improve^immunogenicity,^stability,^and^translational^efficiency^and^fidelity^of^the^mRNA^ and^thus^increase^the^amount^of^protein^produced^from^the^mRNA.^^^ In^those^embodiments^in^which^an^mRNA^is^chemically^synthesized,^the^mRNA^can^comprise^ nucleoside^analogs^such^as^analogs^having^chemically^modified^bases^or^sugars,^and / or^backbone^ modifications.^^In^some^embodiments,^an^mRNA^is^or^comprises^natural^nucleosides^(e.g.,^adenosine,^ guanosine,^cytidine,^uridine);^nucleoside^analogs^(e.g.,^2‐aminoadenosine,^2‐^thiothymidine,^inosine,^ pyrrolo‐pyrimidine,^3‐methyl^adenosine,^5‐methylcytidine,^C‐5^propynyl‐^cytidine,^C‐5^propynyl‐ uridine,^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,^O(6)‐methylguanine,^2^thiocytidine,^pseudouridine,^and^5‐ methylcytidine);^chemically^modified^bases;^biologically^modified^bases^(e.g.,^methylated^bases);^ intercalated^bases;^modified^sugars^(e.g.,^2′‐fluororibose,^ribose,^2′‐deoxyribose,^sN‐phosphoramidite^ linkages).^ The^mRNAs^of^the^disclosure^may^be^synthesized^according^to^any^of^a^variety^of^known^methods.^^For^ example,^mRNAs^in^certain^embodiments^may^be^synthesized^via^in^vitro^transcription^(IVT).^^Briefly,^ IVT^is^typically^performed^with^a^linear^or^circular^DNA^template^containing^a^promoter,^a^pool^of^ ribonucleotide^triphosphates,^a^buffer^system^that^may^include^DTT^and^magnesium^ions,^and^an^ appropriate^RNA^polymerase^(e.g.,^T3,^T7^or^SP6^RNA^polymerase),^DNAse^I,^pyrophosphatase,^and / or^ RNAse^inhibitor.^ In^some^embodiments,^in^vitro^synthesized^mRNA^may^be^purified^before^encapsulation^in^an^LNP^to^ remove^undesirable^impurities^including^various^enzymes^and^other^reagents^used^during^mRNA^ synthesis.^ The^present^disclosure^may^be^used^to^formulate^and^encapsulate^mRNAs^of^a^variety^of^lengths.^In^ some^embodiments,^the^present^disclosure^may^be^used^to^formulate^and^encapsulate^in^vitro^ synthesized^mRNA^ranging^from^about^0.1‐20^kb,^about^1‐20^kb,^about^1‐15^kb,^about^1‐10^kb,^about^ 5‐20^kb,^about^5‐15^kb,^about^5‐12^kb,^about^5‐10^kb,^about^8‐20^kb,^or^about^8‐15^kb^in^length.^^ Alternatively,^the^peptide^or^protein^produced^from^the^mRNA^may^be^between^about^500^Da^and^ about^200^kDa^ Typically,^mRNA^synthesis^includes^the^addition^of^a^“cap”^on^the^5′^end,^and^a^“tail”^on^the^3′^end.^^The^ presence^of^the^cap^is^important^in^providing^resistance^to^nucleases^found^in^most^eukaryotic^cells.^^ The^presence^of^a^“tail”^serves^to^protect^the^mRNA^from^exonuclease^degradation.^ In^some^embodiments,^mRNAs^include^a^5′^and / or^3′^untranslated^region.^^^In^some^embodiments,^a^5′^ untranslated^region^includes^one^or^more^elements^that^affect^an^mRNA's^stability^or^translation,^for^ example,^an^iron^responsive^element.^^In^some^embodiments,^a^5′^untranslated^region^may^be^between^ about^50^and^500^nucleotides^in^length.^ In^some^embodiments,^a^3′^untranslated^region^includes^one^or^more^of^a^polyadenylation^signal,^a^ binding^site^for^proteins^that^affect^an^mRNA's^stability^of^location^in^a^cell,^or^one^or^more^binding^ sites^for^miRNAs.^^In^some^embodiments,^a^3′^untranslated^region^may^be^between^50^and^500^ nucleotides^in^length^or^longer.^ While^mRNA^provided^from^in^vitro^transcription^reactions^may^be^desirable^in^certain^embodiments,^ other^sources^of^mRNA^are^contemplated,^such^as^mRNA^produced^from^bacteria,^fungi,^plants,^and / or^ animals.^ For^embodiments^where^the^mRNA^encodes^for^an^antibody^composed^of^a^heavy^and^a^light^chain,^ varying^ratios^of^mRNA^encoding^for^light^chain:heavy^chain^(w / w)^can^be^encapsulated^within^the^ LNP,^for^example^1:1,^or^1:2,^or^1:4.^^ For^embodiments^where^the^mRNA^encodes^for^an^antibody^composed^of^a^heavy^and^a^light^chain,^ the^mRNA^encoding^for^heavy^and^light^chain^are^linked^in^series^using^a^2A^linker.^ The^lipid^nanoparticle^(LNP)^described^herein^can^cause^uptake^of^encapsulated^^m‐^RNA^into^the^ cytoplasm^of^a^cell^of^the^central^nervous^system.^Such^LNP^compositions^are^shown^herein^to^be^ particularly^efficacious^in^the^delivery^of^mRNA^to^a^cytoplasm^of^a^cell^of^the^central^nervous^system^ where^it^can^be^translated^into^a^secretory^protein^that^is^ultimately^secreted^into^the^interstitial^ and / or^cerebrospinal^fluid.^^ In^some^embodiments,^the^LNP^comprises^an^ionizable^lipid.^In^some^embodiments,^the^LNP^ comprises^a^non‐cationic^helper^lipid.^In^some^embodiments,^the^LNP^comprises^a^sterol.^In^some^ embodiments,^the^LNP^comprises^a^hydrophilic^polymer‐lipid^conjugate.^ As^used^herein,^the^term^“encapsulation”,^“encapsulated”^or^the^like,^with^reference^to^incorporating^a^ nucleic^acid^within^a^LNP^refers^to^any^association^of^the^nucleic^acid^with^any^lipid^component^or^ compartment^of^the^lipid^nanoparticle.^^In^one^example^of^the^disclosure,^the^nucleic^acid^is^present^in^ the^core^of^the^LNP.^More^than^one^nucleic^acid^may^be^encapsulated.^For^example,^an^LNP^may^ encapsulate^a^nucleic^acid^encoding^a^Therapeutic^Protein^or^fragment^thereof,^together^with^one^or^ more^other^nucleic^acid^molecules^encoding^one^or^more^different^polypeptides.^ ^In^one^example^of^the^disclosure,^the^circRNA^or^saRNA^is^present^in^the^core^of^the^LNP.^More^than^ one^circRNA^or^saRNA^may^be^encapsulated.^For^example,^a^LNP^may^encapsulate^a^first^circRNA^ encoding^a^first^polypeptide,^together^with^a^second^circRNA^encoding^a^second^polypeptide.^ An^ionizable^lipid^as^described^herein^may^be^an^ionizable,^cationic,^amino^lipid.^^The^ionizable,^ cationic,^amino^lipid^may^have^a^pKa^such^that^it^is^positively^charged^at^low^pH^and^near^neutral^at^ physiological^pH.^^This^allows^for^electrostatic^interactions^between^the^lipid^and^the^negatively^ charged^mRNA^during^initial^formulation.^^Since^the^ionizable,^cationic,^amino^lipid^is^near^neutral^at^ physiological^pH,^toxicity^is^reduced.^^Without^being^limited^by^theory,^after^cellular^uptake^by^ endocytosis,^the^acidic^environment^of^the^endosome^leads^to^an^increase^in^the^net^positive^charge^of^ the^ionizable,^cationic.^amino^lipids,^which^promotes^fusion^with^the^anionic^lipids^of^the^endosomal^ membrane^and^subsequent^membrane^destabilization^and^release^of^the^mRNA^into^the^cytoplasm^of^ the^CNS^cell^for^translation^into^the^secretory^protein.^^As^noted^previously,^the^translated^protein^is^ subsequently^routed^to^the^cell^membrane^via^the^endoplasmic^reticulum^and^Golgi^network^for^ secretion^into^interstitial^and / or^cerebrospinal^fluid.^^Alternatively,^the^ionizable,^cationic,^amino^lipid^ may^be^an^ionizable,^amino^cationic^lipids^as^described^in^WO^2022 / 24657116.^^Alternatively,^the^ ionizable,^cationic,^amino^lipid^may^have^a^pKa^below^7.0.^ Examples^of^suitable^ionizable,^cationic^aminolipids^are^disclosed,^for^example,^in^PCT^Publication^Nos.^ WO2020252589^and^W02021000041.^ In^some^embodiments,^the^ionizable^lipid^is^DODMA^(l,2‐dioleyloxy‐3‐^dimethylaminopropane),^DLin‐ MC3‐DMA^(O‐(Z,Z,Z,Z‐heptatriaconta‐6,9,26,29‐tetraen‐^19‐yl)‐4‐(N,N‐dimethylamino)),^DLin‐KC2‐ DMA^(2‐dilinoleyl‐4‐dimethylaminoethyl‐^[l,3]‐dioxolane),^butanoic^acid,^BOCHD‐C3‐DMA^(4‐ (dimethylamino)‐,9‐(2‐^octylcyclopropyl)‐l‐[8‐(2^octylcyclopropyl)^octyl]nonyl^ester),^or^C12‐200.^^ The^neutral^form^of^the^ionizable,^amino,^cationic^lipid^has^a^calculated^logarithm^of^the^partition^ coefficient^between^water^and^1‐octanol^(i.e.,^a^cLogP)^greater^than^8.^^In^some^embodiments,^the^ ionizable,^cationic,^amino^lipid^has^a^pKa^that^is^between^5.0^and^7.0,^or^more^typically^between^6.0^ and^6.8.^ As^used^herein,^“non‐cationic^helper^lipid”^is^a^neutral^or^an^anionic^structural^lipid^that^is^capable^of^ formulation^in^a^lipid^nanoparticle.^^ As^used^herein,^“anionic^lipid”^is^a^lipid^that^is^negatively^charged^at^physiological^pH.^^A^non‐limiting^ example^is^a^phosphatidylglycerol^lipid^such^as^1,2‐dioleoyl‐sn‐glycero‐3‐phosphoglycerol^(DOPG).^^ As^used^herein,^“neutral^lipid”^is^a^structural^lipid^that^is^neutral^(including^net^neutral)^at^ physiological^pH^and^that^typically^includes^a^lipid^selected^from^sphingomyelin,^a^ phosphatidylcholine^lipid^or^mixtures^thereof.^^The^term^“neutral^lipid”^includes^zwitterionic^lipids.^^ In^some^embodiments,^the^neutral^lipid^is^selected^from^sphingomyelin,^ distearoylphosphatidylcholine^(DSPC),^dioleoylphosphatidylcholine^(DOPC),^1‐palmitoyl‐2‐^oleoyl‐ phosphatidylcholine^(POPC)^and^dipalmitoyl‐phosphatidylcholine^(DPPC).^^In^certain^embodiments,^ the^neutral^lipid^is^DOPC,^DSPC^or^sphingomyelin.^^In^one^embodiment,^the^neutral^lipid^is^DOPC.^^The^ neutral^lipid^content^may^include^mixtures^of^two^or^more^types^of^different^neutral^lipids.^^ Alternatively,^a^“neutral^lipid”^may^be^a^cylindrically‐shaped^lipid.^^A^cylindrically‐shaped^lipid^is^ generally^understood^to^refer^to^a^phospholipid^that,^because^of^its^optimized^shape,^forms^a^bilayer.^^ A^“neutral^lipid”^may^be^distearoylphosphatidylcholine^(DSPC).^^A^“neutral^lipid”^may^have^a^ phosphatidylethanolamine^content^that^is^less^than^2^mol%.^^A^“neutral^lipid”^may^be^sphingomyelin,^a^ phosphatidylcholine^lipid^or^mixtures^thereof.^^A^“neutral^lipid”^may^be^selected^from^sphingomyelin,^ distearoylphosphatidylcholine^(DSPC),^dioleoylphosphatidylcholine^(DOPC),^1‐palmitoyl‐2‐^oleoyl‐ phosphatidylcholine^(POPC)^and^dipalmitoyl‐phosphatidylcholine^(DPPC);^or^mixtures^of^two^or^more^ types^of^different^neutral^lipids.^ The^LNP^further^includes^a^sterol^in^some^embodiments.^^The^term^“sterol”^refers^to^a^naturally^ occurring^or^synthetic^compound^having^a^gonane^skeleton^and^that^has^a^hydroxyl^moiety^attached^to^ one^of^its^rings,^typically^the^A‐ring.^ Examples^of^sterols^include^cholesterol,^or^a^cholesterol^derivative,^the^latter^referring^to^a^cholesterol^ molecule^having^a^gonane^structure^and^one^or^more^additional^functional^groups.^ The^cholesterol^derivative^may^be^selected^from^one^or^more^of^the^following:^β‐sitosterol,^3‐ sitosterol,^campesterol,^stigmasterol,^fucosterol,^or^stigmastanol,^dihydrocholesterol,^ent‐cholesterol,^ epi‐cholesterol,^desmosterol,^cholestanol,^cholestanone,^cholestenone,^cholesteryl‐2′‐hydroxyethyl^ ether,^cholesteryl‐4′‐^hydroxybutyl^ether,^3β[N‐(N′N′‐dimethylaminoethyl)carbamoyl^cholesterol^(DC‐ Chol),^24(S)‐^hydroxycholesterol,^25‐hydroxycholesterol,^25(R)‐27‐hydroxycholesterol,^22‐ oxacholesterol,^23‐^oxacholesterol,^24‐oxacholesterol,^cycloartenol,^22‐ketosterol,^20‐hydroxysterol,^ 7‐^hydroxycholesterol,^19‐hydroxycholesterol,^22‐hydroxycholesterol,^25‐hydroxycholesterol,^7‐^ dehydrocholesterol,^5α‐cholest‐7‐en‐3β‐ol,^3,6,9‐trioxaoctan‐1‐ol‐cholesteryl‐3e‐ol,^ dehydroergosterol,^dehydroepiandrosterone,^lanosterol,^dihydrolanosterol,^lanostenol,^lumisterol,^ sitocalciferol,^calcipotriol,^coprostanol,^cholecalciferol,^lupeol,^ergocalciferol,^22‐^dihydroegocalciferol,^ ergosterol,^brassicasterol,^tomatidine,^tomatine,^ursolic^acid,^cholic^acid,^chenodeoxycholic^acid,^ zymosterol,^diosgenin,^fucosterol,^fecosterol^or^a^salt^or^ester^thereof.^ In^one^embodiment,^the^LNP^comprises^a^hydrophilic‐polymer^lipid^conjugate^capable^of^ incorporation^into^the^LNP.^^The^conjugate^includes^a^lipid^covalently^attached^(optionally^via^a^linker^ group)^to^a^polymer^chain^that^is^hydrophilic.^^Examples^of^hydrophilic^polymers^include^ polyethyleneglycol^(PEG),^polyvinylpyrrolidone,^polyvinylmethylether,^polyhydroxypropyl^ methacrylate,^polyhydroxypropylmethacrylamide,^polyhydroxyethyl^acrylate,^polymethacrylamide,^ polydimethylacrylamide,^polymethyloxazoline,^polyethyloxazoline,^polyhydroxyethyloxazoline,^ polyhydroxypropyloxazoline,^polysarcosine^and^polyaspartamide.^^In^one^embodiment,^the^ hydrophilic‐polymer^lipid^conjugate^is^a^PEG‐lipid^conjugate.^^The^hydrophilic^polymer^lipid^conjugate^ may^also^be^a^naturally‐occurring^or^synthesized^oligosaccharide‐containing^molecule,^such^as^ monosialoganglioside^(GM1).^^The^ability^of^a^given^hydrophilic‐polymer^lipid^conjugate^to^enhance^ the^circulation^longevity^of^the^LNPs^herein^could^be^readily^determined^by^those^of^skill^in^the^art^ using^known^methodologies.^^A^PEG‐lipid^conjugate^may^be^1,2‐dimyristoyl‐rac‐glycero‐3‐ methoxypolyethylene^glycol^(DMG‐PEG).^ ^In^some^embodiments,^the^LNP^comprises^0.5‐2^percent^PEG‐lipid,^optionally,^for^example^1.5^to^2.5^ mol^percent,^1‐2^mol^percent,^0.5^to^1.5^mol^percent.^^ In^some^embodiments,^the^lipid^nanoparticle^comprises^10‐40^mol^percent^non‐cationic^helper^lipid.^ For^example,^the^lipid^nanoparticle^may^comprise^15‐35^mol^percent,^20‐30^mol^percent,^10‐20^mol^ percent,^15‐25^mol^percent,^20‐30^mol^percent,^30‐40^mol^percent.^^In^some^embodiments,^the^lipid^ nanoparticle^comprises^10^mol^percent,^15^mol^percent,^20^mol^percent,^25^mol^percent,^30^mol^ percent,^35^mol^percent,^or^40^mol^percent^neutral^lipid.^ In^some^embodiments,^the^lipid^nanoparticle^comprises^40‐50^mol^percent^ionizable^amino^lipid,^ optionally^45‐50^mol^percent,^for^example,^45‐46^mol^percent,^46‐47^mol^percent,^47‐48^mol^percent,^ 48‐49^mol^percent,^or^49‐50^mol^percent^for^example^about^45^mol^percent,^45.5^mol^percent,^46^mol^ percent,^46.5^mol^percent,^47^mol^percent,^47.5^mol^percent,^48^mol^percent,^48.5^mol^percent,^49^mol^ percent,^or^49.5^mol^percent.^ In^some^embodiments,^the^lipid^nanoparticle^comprises^38‐39.5^mol^percent^sterol,^optionally^38^mol^ percent,^38‐39^mol^percent,^39.5^mol^percent,^or^39^mol^percent.^^ LNP^may^be^manufactured^by^a^variety^of^related^techniques.^^Typically,^a^stream^of^aqueous^nucleic^ acid^and^a^stream^of^lipidic^components^in^organic^phase^are^combined^at^appropriate^pressures^and^ speeds,^and^within^a^confined^space.^^NanoAssemblr™^LNP^manufacturing^systems^are^available^from^ Cytiva^(Vancouver,^Canada).^ In^some^embodiments,^the^LNP^comprising^mRNA^is^part^of^a^pharmaceutical^composition.^^The^ pharmaceutical^composition^can^comprise^at^least^one^pharmaceutically^acceptable^excipient.^The^ pharmaceutical^composition^may^provide^a^prophylactic^(preventative),^ameliorative^or^a^therapeutic^ benefit.^^The^pharmaceutical^composition^will^be^administered^at^any^suitable^dosage.^The^type^of^ administration^used^to^introduce^the^pharmaceutical^composition^includes^any^route^that^delivers^the^ LNP^comprising^the^nucleic^acid^to^a^central^nervous^system^(CNS)^site^in^which^the^extracellular^ space^comprises^the^interstitial^and / or^cerebrospinal^fluid^cerebrospinal^fluid.^^The^site^includes^the^ brain^and / or^spinal^cord.^^The^administration^methods^include^but^are^not^limited^to^ intracerebroventricular^(ICV)^injection,^lumbar^intrathecal^injection,^cisterna^magna^injection^and^the^ use^of^a^catheter^(e.g.,^intrathecal^catheter)^to^introduce^the^LNP‐mRNA^to^the^brain^or^spinal^cord^ (see^FIGURES^3‐6,^12).^ Pharmaceutical^compositions^as^described^herein^may^be^administered^to^a^subject.^^As^used^herein,^a^ “subject”^may^be^a^human,^non‐human^primate,^rat,^mouse,^cow,^horse,^pig,^sheep,^goat,^dog,^cat,^etc.^^ The^subject^may^be^suspected^of^having^or^at^risk^for^having^a^central^nervous^system^disease,^ disorder,^trauma^or^injury,^are^known^to^those^of^ordinary^skill^in^the^art.^^Some^examples^are^ Neuronopathic^Lysosomal^Storage^Disorders:^Gaucher^Disease^Type^1,^Gaucher^Disease^Type^2,^ Gaucher^Disease^Type^3,^Sphingolipidoses^(e.g.^Niemann‐Pick^Disease^Types^A^and^C,^Krabbe^Disease,^ Metachromatic^Leukodystrophy,^Fabry^Disease,^Farber^Disease,^Multiple^Sulfatase^Deficiency),^GM1^ Gangliosidoses^(e.g.^Type^I,^Type^II,^Type^III),^GM2^Gangliosidoses^(e.g.^Tay‐Sachs^Disease,^Sandhoff^ Disease),^Mucopolysaccharidoses^(e.g.^MPSI,^MPSII,^MPS^III,^MPS^VII),^Neuronal^Ceroid^Lipofuscinoses^ (CLN,^e.g.^CLN1^Santavuori‐Haltia^Disease,^CLN2^Jansky‐Bielschowsky^Disease,^CLN3^Batten^Disease,^ CLN4^Kufs^Disease,^CLN5,^CLN6,^CLN7,^CLN8,^CLN10,^CLN11,^CLN12,^CLN13,^CLN14)^^Mucolipidosis^ Type^II,^Mucolipidosis^Type^III,^Mucolipidosis^Type^IV,^and^Wolman^Disease;^Neurodegenerative^ disorders:^Parkinson’s^Disease^and^related^synucleinopathies,^Alzheimer’s^Disease,^Lewy^Body^ Dementia,^frontotemporal^dementia,^^Vascular^Dementia,^Multiple^Sclerosis,^and^Amyotrophic^Lateral^ Sclerosis;^Traumatic^Brain^Injury^and^Spinal^Cord^injury;^and^Cerebrovascular^Diseases^(e.g.^ischemic^ and^hemorrhagic^stroke,^Transient^Ischemic^attack,^Brain^Aneurysm,^Arteriovenous^Malformation,^ Cavernous^Malformation,^Moyamoya^Disease).^ In^some^embodiments,^contacting^of^the^LNP^with^the^cell^results^in^the^mRNA,^circRNA^or^saRNA^ entering^the^cell.^In^some^embodiments,^contacting^of^the^LNP^with^the^cell^results^in^production^of^the^ polypeptide^in^the^cell.^In^some^embodiments,^subsequent^to^the^production^of^the^polypeptide,^the^ polypeptide^is^secreted^from^the^cell^to^an^interstitial^and / or^cerebrospinal^fluid^(CSF)^of^the^subject.^ In^some^embodiments,^the^polypeptide^is^diffusible^within^the^interstitial^and / or^cerebrospinal^fluid^ of^the^subject.^In^some^embodiments,^the^polypeptide^enters^un‐transfected^cells^to^exert^therapeutic^ effects.^In^some^embodiments,^the^polypeptide^is^capable^of^being^internalized^by^a^cell^of^the^CNS.^In^ some^embodiments,^the^cell^of^the^CNS^comprises^neurons,^glia,^neurovascular^cells,^and / or^meningeal^ cells.^In^some^embodiments,^the^neurons^comprise^interneurons.^In^some^embodiments,^the^glia^ comprise^astrocytes,^oligodendrocytes,^microglia,^ependymal^cells,^and / or^radial^glia.^In^some^ embodiments,^the^neurovascular^cells^comprise^endothelial^cells,^pericytes,^and / or^smooth^muscle^ cells.^In^some^embodiments,^the^secreted^polypeptide^is^present^in^the^CSF,^ISF,^or^intracellular^ compartments^of^the^subject^for^a^period^of^at^least^24^hours,^at^least^36^hours,^or^at^least^48^hours.^ In^some^embodiments,^the^secreted^polypeptide^is^present^in^the^CSF,^ISF,^or^intracellular^ compartments^of^the^subject^for^a^period^of^at^least^1^week,^at^least^2^weeks,^at^least^3^weeks,^at^least^4^ weeks,^at^least^6^weeks,^or^at^least^8^weeks.^In^some^embodiments,^the^secreted^polypeptide^is^present^ in^the^CSF,^ISF,^or^intracellular^compartments^of^the^subject^at^a^first^concentration^at^a^first^time^point^ and^at^a^second^concentration^at^a^later^second^time^point.^In^some^embodiments,^the^first^time^point^ is^about^3^hours^after^contacting^the^LNP^with^the^cell.^ In^some^embodiments,^the^second^time^point^is^about^48^hours^after^contacting^of^the^LNP^with^the^ cell.^In^some^embodiments,^the^second^concentration^is^at^least^30%,^at^least^40%,^at^least^50%,^at^ least^60%,^at^least^70%,^at^least^80%,^at^least^90%,^or^at^least^95%^of^the^first^concentration.^In^some^ embodiments,^the^contacting^is^in^vitro^or^in^vivo.^In^some^embodiments,^the^cell^of^the^central^ nervous^system^is^a^glial^cell.^A^glial^cell^can^for^example^be^an^astrocyte,^^an^oligodendrocyte,^or^an^ oligodendrocyte^precursor^cell.^The^oligodendrocyte^can,^for^example,^be^a^pre‐oligodendrocytes,^an^ (im)mature^oligodendrocytes^or^a^myelinating^oligodendrocytes.^In^some^embodiments,^the^cell^of^the^ central^nervous^system^is^an^ependymal^cell.^An^ependymal^cell^can^for^example^be^from^the^cerebral^ ventricles^or^the^choroid^plexus.^ The^LNP‐circRNA^or^LNP‐saRNA^of^the^present^disclosure^can^be^used^to^treat^or^prevent^a^central^ nervous^system^disease,^disorder,^trauma^or^injury.^^The^LNP^of^the^present^disclosure^may^also^be^ used^to^treat^or^prevent^conditions^such^as^aging,^preventative^medicine^and / or^be^used^as^part^of^a^ personalized^medicine^regime.^Further,^the^LNP^of^the^present^disclosure^may^be^used^in^other^ applications,^such^as^diagnostic^applications.^The^polypeptide^encoded^by^the^circRNA^or^saRNA^in^the^ lipid^nanoparticle^can^be^a^secretory^protein.^The^secretory^protein^can^cross^a^central^nervous^system^ cellular^membrane^to^exert^an^extracellular^therapeutic^or^prophylactic^effect^within^one^or^more^ regions^of^the^central^nervous^system.^In^some^embodiments,^the^LNP^is^used^in^the^manufacture^of^a^ medicament.^In^some^embodiments,^the^LNP^is^used^for^the^treatment^of^central^nervous^system^ disease.^Various^features^and^embodiments^of^the^present^invention^will^now^be^described^by^way^of^ non‐limiting^examples.^ ^ EXAMPLES^ Example^1.^^Methods^&^Materials^ ICV^microinjections^ All^experimental^protocols^were^approved^by^The^University^of^British^Columbia^Committee^on^ Animal^Care^and^conducted^in^compliance^with^guidelines^provided^by^the^Canadian^Council^of^Animal^ Care.^^Adult^C57Bl6^mice^(P40‐P60)^were^anesthetized^under^1‐2%^isoflurane^and^positioned^on^a^ stereotaxic^frame.^^A^small^hole^(diameter^1^mm)^was^drilled^in^the^skull^to^allow^access^to^the^brain^ (−0.5^mm^anterior / posterior^(AP)^and^±1.0^mm^medial / lateral^(ML)^from^bregma^and^‐1.8^mm^ dorsal / ventral^(DV)^‐^see^FIGURE^12).^^A^glass^micropipette^(tip^diameter^40^µm)^was^connected^to^a^ Hamilton™^syringe^and^LNP‐mRNAs^were^injected^using^an^infusion^pump^(Harvard^Apparatus™,^ Holliston,^MA)^at^a^rate^of^200^nl / minute.^^The^total^volume^injected^was^1^µL^of^LNP‐mRNA^(0.05^–^1^mg^ mRNA / ml^in^sterile^PBS).^^LNP‐mRNA^preparations^were^injected^sequentially^in^the^lateral^ventricle^of^ both^hemispheres.^^After^needle^retraction,^the^skin^on^the^skull^was^sutured,^and^mice^were^then^single‐housed.^ ^For^mCherry^imaging^experiments,^mice^were^kept^for^two^days^before^transcardial^perfusion^with^PBS^and^4%^paraformaldehyde^for^brain^collection.^^For^FGF‐21^measurements^in^ cerebrospinal^fluid^(CSF),^mice^were^kept^for^3^or^48^hours^after^injection,^and^CSF^was^then^collected^via^ a^cisterna^magna^puncture^(see^FIGURE^12).^ IT^injections^ All^experimental^protocols^were^approved^by^The^University^of^British^Columbia^Committee^on^ Animal^Care^and^conducted^in^compliance^with^guidelines^provided^by^the^Canadian^Council^of^Animal^ Care.^^Adult^C57Bl6^mice^(P40‐P60)^mice^were^anesthetized^under^1‐2%^isoflourane^and^fur^around^ the^lower^spine^was^removed^using^an^electric^shaver.^^The^mouse^was^positioned^over^a^15^mL^ conical^tube^under^the^abdomen^and^with^a^nose^cone^for^continuous^1‐2%^isoflourane^ administration.^LNP‐mRNAs^(5^µL)^was^injected^using^a^Hamilton^syringe^(10^µL)^between^L5‐L6.^ LNP^formulations^ The^LNPs^were^prepared^by^dissolving^mRNA^in^25^mM^sodium^acetate,^pH^4.0,^while^the^lipid^ components^at^the^mole^%^specified^were^dissolved^in^absolute^ethanol.^^The^lipids^in^ethanol^and^the^ mRNA^in^buffer^were^combined^in^a^1:3^volume^by^volume^ratio^using^a^T‐junction^with^dual‐syringe^ pumps.^^The^solutions^were^pushed^through^the^t‐junction^at^a^combined^flow^rate^of^20^mL / min^(5^ mL / minute^for^the^lipid‐containing^syringe,^15^mL / minute^for^the^mRNA‐containing^syringe).^^The^ mixture^was^subsequently^dialyzed^overnight^against^at^least^~100^volumes^of^1×^phosphate^buffered^ saline,^pH^7.4^using^Spectro / Por^dialysis^membranes^(molecular^weight^cut‐off^12^000–14^000^Da).^^ The^LNPs^were^concentrated^as^required^with^an^Amicon^Ultra™^10^000^MWCO^(molecular^weight^ cut‐off),^regenerated^cellulose^concentrator.^^ Lipid^nanoparticles^with^the^different^formulations^set^forth^in^TABLE^1^below^were^assayed.^^The^ LNPs^are^composed^of^4^lipid^components:^(1)^an^ionizable^lipid^(for^example,^nor‐MC3,^ALC‐0315,^ ionizable^lipid^1,^or^Compound^22);^(2)^a^non‐cationic^helper^lipid^(for^example,^DSPC^or^DOPG,^or^egg^ sphingomyelin^(ESM));^(3)^a^sterol^(for^example,^cholesterol);^and^(4)^a^polymer^lipid^conjugate^(for^ example,^PEG‐DMG),^as^set^out^in^TABLE^1^below.^^The^LNPs^were^prepared^with^mRNA^coding^for^ fluorescent^reporter^protein^mCherry^or^Fibroblast^Growth^factor^21^(FGF‐21)^NM_020013.4^or^ Erythropoietin^(EPO)^NM_000799^or^gaussian^Luciferase^(GLuc)^(SEQ^ID.^NO.^16)^or^ glucocerebrosidase^(GCase)^(SEQ^ID^No.8)^loaded^into^LNPs.^^Fluorescent^lipid^dye^DiO^was^included^in^ some^LNP^formulations^to^allow^tracking^of^LNP^diffusion^in^the^brain^independent^of^reporter^protein^ expression.^^For^some^experiments^(LNPs^A,^B,^C,^and^D),^we^used^the^lipophilic^dye^DiO^(DiOC18(3)),^ which^is^a^green^fluorescent,^lipophilic^carbocyanine^dye^that^was^incorporated^into^the^LNP.^^The^ nitrogen‐to‐phosphate^charge^ratio^was^6.^ For^the^expression^and^production^of^antibodies,^the^formulations^set^forth^in^TABLE^1^below^will^be^ used^with^mRNA^encoding^for^the^monoclonal^antibody^Trastuzumab.^The^nucleotide^sequences^for^ Trastuzumab^include^a^sequence^coding^for^the^light^chain,^and^a^sequence^coding^for^the^heavy^chain.^ These^two^mRNA^sequences^were^formulated^either^with^varying^ratios^of^light^chain:^heavy^chain,^for^ example^1:1,^or^1:2,^or^1:4^(w / w),^or^in^a^linear^arrangement^where^the^heavy^and^light^chain^ sequences^were^linked^using^a^P2A^linker^(detailed^below).^^The^sequences^for^the^antibody^mRNA^are^ set^out^in^^Haryadi^R,^Ho^S,^Kok^YJ,^Pu^HX,^Zheng^L,^et^al.^(2015)^Optimization^of^Heavy^Chain^and^Light^ Chain^Signal^Peptides^for^High^Level^Expression^of^Therapeutic^Antibodies^in^CHO^Cells.^PLOS^ONE^ 10(2):^e0116878.^^ Compound^22^ TABLE^1:^Lipid^nanoparticle^(LNP)^formulations^^ LNP^ Cargo^(mRNA)^ LNP^Components^ Molar^ratios^(mol / mol)^ G^ mCherry^mRNA^ nMC3 / ESM / Chol / PEG‐DMG^ 20 / 40 / 38.5 / 1.5^ The^ionizable,^amino,^cationic^lipid^nMC3^referred^to^in^TABLE^1^above^is^the^lipid^referred^to^as^nor‐ MC3^described^at^page^8^of^WO^2022 / 246571.^^The^ionizable,^amino,^cationic^lipid,^is^Compound^22^ from^WO^2024 / 065043.^The^lipid^ALC‐0315^is^from^WO2018081480.^Ionizable^Lipid^1^was^provided^ by^NanoVation^Therapeutics,^Vancouver,^Canada.^ Imaging^ Brains^were^sliced^in^300^µm‐thick^sections^prior^to^immunostaining^and^imaging^on^a^confocal^ microscope^(Zeiss™).^Individual^cell^types^were^labelled^using^common^cell^markers^(rabbit^anti‐Olig2^ 1:200^for^oligodendrocytes;^rat^anti‐GFAP^1:500^for^astrocytes^and^ependymal^cells).^^Goat^anti‐rabbit^ (or^anti‐rat)^secondary^antibodies^conjugated^to^Alexa‐647™^dye^were^imaged^using^the^622^nm^laser^ line.^^The^lipophilic^dye^DiO^(incorporated^into^the^LNP)^was^imaged^using^the^488^nm^laser^line.^^The^ mCherry^fluorescent^protein^was^imaged^using^the^561^nm^laser^line.^Green^was^imaged^using^the^488^ nm^laser^line.^ Cell^culture^ HEK293T^cells^were^maintained^in^Dulbecco’s^Modified^Eagle^Medium^(DMEM)^supplemented^with^ 10%^fetal^bovine^plasma^(FBS)^and^1%^penicillin / streptomycin^(P / S)^at^37°C^and^5%^CO2^for^a^ maximum^of^20^passages.^^ Primary^astrocyte^cultures^were^obtained^from^whole^brain^cultures^of^E18^rats.^Whole^brains^were^ homogenized,^filtered^using^a^0.45µm^strainer,^and^maintained^in^Minimum^Essential^Media^(MEM)^ supplemented^with^7%^FBS,^1%^P / S^and^0.5%^glucose.^To^detach^microglia,^cultures^were^agitated^at^ 120^RPM^for^4^hours.^Experiments^were^performed^at^passage^number^3‐5.^ Immortalized^human^astrocytes^(Innoprot™^P10251‐IM‐tGFP)^were^seeded^for^transfection^in^a^poly‐ D‐lysine^coated^48‐well^culture^plate^at^the^manufacturer^recommended^seeding^density.^ For^GCase^inhibition^experiments,^conduritol^B^epoxide^was^added^to^cell^culture^media^to^a^working^ concentration^of^0.1‐100µM.^ Human^iPSC‐derived^neurosphere^cultures^ To^assay^for^the^duration^of^protein^expression,^we^used^a^neurosphere^culture^system.^Human^ induced^pluripotent^stem^cells^(iPSCs)^were^grown^into^neurosphere^according^to^published^protocol.^ This^allows^for^testing^of^LNP‐RNA‐induced^protein^production^in^vitro^in^cells^of^human^origin,^and^in^ cells^derived^into^neuronal^and^astrocytic^phenotypes.^This^also^allows^for^convenient^longitudinal^ imaging^of^fluorescent^protein^expression.^^ Peripheral^blood^was^drawn^from^a^healthy^female^individual^carrying^the^APOE^alleles^ε3 / ε3^and^ human^iPSCs^(hiPSCs)^were^generated^from^erythroid^progenitor^cells.^hiPSCs^were^grown^on^ Matrigel^(BD^Biosciences,^Franklin^Lakes,^USA)^coated^6‐well^plates^in^mTeSRTM^Plus^(STEMCELL^ Technologies,^Vancouver,^Canada).^Cells^were^fed^every^2‐3^days^and^used^for^the^induction^of^ neuronal^progenitor^cells^(NPCs)^by^a^dual^SMAD^inhibition^protocol.^Briefly,^hiPSCs^were^plated^in^6‐ well^plates^in^Basal^Neural^Maintenance^media^(BNMM)^supplemented^with^10^mM^SB‐431542^ (Tocris,^Bristol,^United^Kingdom)^and^0.5^mM^LDN‐193189^(Tocris)^for^7^days,^followed^by^a^3‐week^ differentiation^in^Neural^Stem^Cell^Media^(BNMM^+^20ng / mL^bFGF)^with^media^changes^every^2‐3^ days.^ To^generate^human^iPSC‐derived^neurospheres^(hiNS),^P2^NPCs^were^plated^in^Matrigel™^coated^6‐ well^plates^using^Neurosphere^Expansion^Media^(NEX;^DMEM / F12^media^+^Neurobasal^Media,^1%^N2,^ 2%^B27,^1%^GlutaMax™,^ITS‐A,^NEAA^and^P / S,^20ng / mL^bFGF).^To^start^hiNS^formation,^NPCs^were^ lifted^using^Accutase™^(STEMCELL^Technologies)^and^plated^in^single^wells^of^an^Akura^384^ ImagePro™^(InSphero,^Schlieren,^Switzerland)^at^a^density^of^6*104^NPCs^per^well^in^NEX.^The^plate^ was^tilted^on^a^stand^at^a^30‐degree^angle^in^the^incubator^over^night^to^facilitate^neurosphere^ formation.^After^7^days^post‐plating,^the^media^was^changed^to^Neurosphere^Differentiation^Media^ (NDM;^BrainPhys^Neuronal^Medium^+^2%^SM1,^1%^N2A,^P / S^and^cAMP,^0.2^µM^ascorbic^acid,^ 0.01µg / mL^BDNF^and^GDNF)^fed^every^2‐3^days^by^full^media^change^and^switched^to^Neurosphere^ Maintenance^Media^(NMM;^NDM^without^cAMP^and^ascorbic^acid)^28^days^after^plating.^ Human^iPSC^neurosphere^cultures^were^transfected^with^LNP‐mRNA‐mCherry^at^300nM,^30nM,^3nM,^ and^0.3nM^mRNA^at^43^days^in^vitro^and^imaged^initially^from^4^hours^to^24^hours^post^transfection.^ Subsequent^images^were^acquired^after^regular^feedings^at^48‐72^hours^intervals.^For^LNP‐ circRNA(NeonGreen),^neon^Green^was^imaged^daily^for^multiple^weeks^to^evaluate^the^time^course^of^ LNP‐circRNA‐induced^translation^into^a^functional^fluorescent^protein.^Images^were^acquired^as^ three‐dimensional^z‐stacks,^and^mCherry^or^NeonGreen^fluorescence^were^measured^from^the^ maximum‐intensity^projection^image.^ CSF^collection^ CSF^was^collected^as^previously^described^[6].^^Briefly,^glass^capillaries^were^pulled^and^trimmed^so^ the^inner^diameter^is^approx.^0.5mm.^^Mice^were^anaesthetized^using^the^3‐Component^anesthetic^ (Fentanyl™^0.05mg / kg,^Midazolam^5^mg / kg,^Dexmedetomidine^0.5mg / kg).^^Eye^ointment^was^applied,^ and^fur^was^shaved^from^the^animal’s^neck^and^skull.^^Mice^were^fixed^on^a^stereotaxic^frame^to^form^ angle^approximately^135o^from^the^body,^and^a^sagittal^incision^was^made^inferior^to^the^occiput.^^ Subcutaneous^tissue^was^separated^to^expose^the^dura^mater^of^the^cisterna^magna^(see^FIGURE^12),^ and^the^dura^mater^was^pierced^with^the^capillary^tube^to^draw^CSF^(2‐10^µL).^^The^CSF^collected^in^ the^capillary^was^flushed^into^a^clean^tube^and^frozen^immediately^at^‐80oC^for^future^analysis.^ FGF‐21^and^EPO^measurements^ CSF^was^collected^from^mice^3‐^or^48‐^hours^after^LNP‐mRNA(FGF‐21)^or^LNP‐mRNA(EPO)^injection.^ The^presence^of^FGF‐21^or^EPO^was^quantified^using^a^highly^sensitive^electrochemiluminescence^ ELISA^(U‐PLEX™)^for^FGF‐21^or^for^EPO^^from^Meso^Scale^Discovery™,^USA^(MSD).^ GCase^measurements^ To^generate^a^custom^human^GCase^“U‐PLEX”^immunoassay,^an^unconjugated,^carrier‐free^antibody^ pair^and^lyophilized^GCase^calibrator^was^purchased^commercially^(Abcam;^ab309266);^the^antibody^ pair^was^then^conjugated^in‐house^with^capture^(biotin)^and^detector^(SulfoTag™;^R91AO‐1)^moieties.^ MSD^GOLD^96‐well^Small^Spot^Streptavidin^SECTOR^plates^(L45SA‐1)^were^used^for^capture^antibody^ immobilization.^Briefly,^we^pre‐washed^plates^with^0.05%^PBST^before^incubation^with^capture^ antibody^(1.0^µg / mL)^for^1^hour^at^room^temperature^with^agitation.^Next,^we^incubated^the^coated^ plate^with^diluted^samples^(1:50^for^CSF,^1:10^for^plasma,^1:5^for^brain^homogenate)^or^calibrator^ overnight^at^4°C.^We^then^incubated^the^plate^with^detector^antibody^(0.125^µg / mL)^for^1^hour^at^ room^temperature,^washed^the^plate,^and^then^added^150uL^MSD^Gold^Read^Buffer^B.^The^plate^was^ read^using^a^MESO^QuickPlex^SQ™^120MM^instrument.^All^incubations^took^plate^using^a^temperature‐ controlled^microplate^shaker^at^700^rpm^with^at^least^three^PBST^washes^between^each^step.^To^ generate^the^GCase^calibration^curve,^the^calibrator^(lyophilized,^recombinant^GCase^protein^ resuspended^at^11200^pg / mL)^was^diluted^4‐fold^seven^times^and^curve^generation / quantification^ was^performed^using^the^MSD^Discovery^Workbench^software.^All^samples^including^the^calibrators^ were^run^in^technical^duplicate^with^blanks^and^positive^controls^on^each^plate^to^assess^inter‐plate^ consistency.^ Plasmid^DNA^preparation^and^transfection^ Plasmids^were^synthesized^by^Twist^Bioscience^(San^Francisco,^CA)^and^midiprepped^using^PureLink™^ HiPure™^Plasmid^Midiprep^Kit™^(Thermofisher^Scientific).^For^transfection^experiments,^cells^were^ seeded^in^coated^microwell^plates^24^hours^prior^to^transfection,^to^a^confluency^of^50‐70%.^1^µg^ plasmid^DNA^was^transfected^using^Lipofectamine^3000^according^to^manufacturer^protocol^in^ plasma‐free^media^for^6^or^24^hours.^^ RNA^preparation^and^transfection^ Briefly,^a^plasmid^DNA^backbone^was^generated^with^the^desired^nucleic^acid^sequence^downstream^ of^a^T7^promoter,^linearized^using^the^restriction^enzyme^BspQI,^and^used^as^a^template^for^in^vitro^ transcription^(IVT).^IVT,^5’^capping,^and^3’^poly(A)^tailing^was^performed^using^reagents^ manufactured^by^Hongene^Biotech^with^UBC^RNA^&^Formulation^Core^optimized^protocols.^For^ transfection,^cells^were^seeded^in^coated^microwell^plates^24^hours^prior^to^transfection,^to^a^ confluency^of^50‐70%.^2^µg^total^RNA^was^transfected^using^Lipofectamine^MessengerMAX™^ according^to^manufacturer^protocol^in^plasma‐free^media^for^3^or^24^hours.^GCase^mRNA^was^ obtained^through^The^University^of^British^Columbia^RNA^and^Formulation^Core,^(rfc@msl.ubc.ca)^ For^LNP‐mRNA^transfections,^2^µg^encapsulated^mRNA^was^diluted^in^plasma‐free^media^and^added^ directly^to^individual^wells^for^3^or^24^hours.^ Western^blot^ Cell^lysate,^cell^supernatant,^and^mouse^brain^homogenate^were^harvested^for^Western^blot^analysis^ as^follows:^2^mL^of^total^cell^culture^supernatant^was^collected^and^cell^debris^removed^using^two^ rounds^of^centrifugation.^Cleared^supernatant^was^concentrated^(four^sequential^spins^of^500^µL)^ using^an^Amicon^Ultra‐0.5^Centrifugal^Filter^Unit^with^a^10^kDa^retention^cutoff^to^a^final^volume^of^ <200^µL.^Cell^lysate^was^collected^by^scraping^cells^in^ice‐cold^1X^PBS,^centrifuging^the^cell^pellet^ (700xg,^15^minutes,^4°C),^and^resuspending^the^pellet^in^ice‐cold^RIPA^buffer^for^30^minutes^on^ice^ with^inversion^every^10^minutes.^Lysate^was^clarified^using^one^round^of^centrifugation.^5^mg^of^whole^ brain^tissue^was^homogenized^in^500‐1000µl^RIPA^buffer^using^an^electric^homogenizer^followed^by^ constant^inversion^at^4°C.^Lysate^was^clarified^using^two^rounds^of^centrifugation.^ Lysate^and^supernatant^samples^were^quantified^using^DC^Protein^Assay^(Bio‐Rad).^20^µg^cell^lysate^or^ supernatant^was^loaded^on^a^4‐20%^gradient^SDS‐PAGE^gel^(Bio‐Rad)^and^electrophoretically^ separated.^Proteins^were^transferred^to^a^PVDF^membrane^using^semi‐dry^transfer^for^30^minutes^(20^ V).^The^membrane^was^then^incubated^in^blocking^buffer^(TBST^+^5%^milk^power)^for^30^minutes^and^ incubated^overnight^in^primary^antibody^diluted^in^blocking^buffer^at^4°C.^The^next^day,^membranes^ were^washed^with^1X^TBST^before^incubation^with^secondary^antibody^(HRP‐conjugated)^at^a^1:2000‐ 1:5000^dilution^for^1^hour^at^room^temperature.^Membranes^were^imaged^using^the^C‐DiGit™^Blot^ Scanner^(LI‐COR).^ GLuc^luminescence^Assay^ Gaussia^luciferase^luminescence^was^measured^using^a^Pierce^Gaussia^Luciferase^Glow^Assay^Kit^ (Thermofisher,^16160).^Cell^culture^supernatant^was^collected^from^neurospheres^and^secreted^GLuc^ was^measured^using^a^SpectraMAX™^M2^plate^reader,^read^at^480nm.^^ Glucocerebrosidase^activity^assays^ GCase^activity^assays^were^performed^according^to^manufacturer^protocol^(abab273339)^using^cell^ lysate^or^cell^supernatant^(5^µg^total^protein / well).^Fluorescence^was^measured^at^360 / 445nm^and^a^ standard^curve^was^used^to^determine^substrate^turnover^per^minute.^ Trastuzumab^construct^generation^and^detection^^ In^vitro^Delivery^and^Sample^Collection:^HEK293T^and^rat^primary^astrocytes^were^transfected^with^ pDNA^or^mRNA^encoding^the^heavy^and^light^chains^of^Trastuzumab^(for^both^mRNA^and^pDNA^ transfections,^a^2:1^ratio^of^heavy:light^chain^was^used).^In^Some^experiments,^the^heavy^and^light^ chains^were^fused^in^series^using^a^P2A^linker^^containing^an^enhancer‐^(sequence:^CSG)^and^furin^ cleavage^sequence^(sequence:^RKRR),^total^linker^sequence:^RKRRGSGATNFSLLKQAGDVEENPGP^(SEQ^ ID^No.^13).^^Full^length^trastuzumab^heavy:P2A:light^(including^Furin^+^P2A)^is^(SEQ^ID^No.^14).^^ Plasma‐free^supernatant^was^collected^from^transfected^cells.^The^supernatant^was^diluted^in^a^range^ of^1:5^to^1:200,^with^the^optimal^dilution^determined^based^on^variables^such^as^transfection^volume^ and^the^quantity^of^RNA^delivered.^Detection^of^human^IgG1^was^performed^via^ELISA^(ThermoFisher,^ #EHIGG1)^or^MSD^Immunoassay^(MSD,^#K150JLD).^For^human‐derived^samples,^such^as^HEK293T^ supernatant,^untransfected^controls^were^utilized^to^quantify^background^IgG1^levels,^specifically^non‐ Trastuzumab^IgG1.^ In^vivo^Delivery^and^Sample^Collection:^Mice^received^bilateral^stereotactic^injections^of^1.5^μg^LNP‐ mRNA^per^hemisphere.^Mice^were^euthanized^at^24^and^48^hours^post‐injection.^Cerebrospinal^fluid^ (CSF)^was^collected^and^snap‐frozen^until^analysis.^Blood^was^collected^via^cardiac^puncture,^plasma^ was^separated^by^centrifugation^and^snap‐frozen^until^analysis.^Whole^brains^were^rapidly^dissected,^ snap‐frozen^in^liquid^nitrogen,^and^stored^at^‐80°C^until^analysis.^For^analysis,^whole^brain^tissue^was^ homogenized^in^lysis^buffer^using^a^Qiagen^TissueLyser™^(two^cycles^of^30^seconds^at^30^Hz^with^a^60‐ second^incubation^on^ice^between^cycles),^followed^by^a^1:5^dilution^in^assay^buffer.^For^brain^lysate,^ total^concentration^is^adjusted^to^pg / mg^tissue^post^hoc.^ Quantification^of^Human^IgG1:^Human^IgG1^protein^concentrations^in^all^samples^were^quantified^ using^a^commercially^available^enzyme‐linked^immunosorbent^assay^(ELISA;^ThermoFisher,^ #EHIGG1)^or^a^multiplexed^immunoassay^(MSD,^#K150JLD),^according^to^the^manufacturers’^ protocols.^ VHH^measurements^ Brains^and^CSF^are^collected^from^mice^at^3^or^24^or^48^hours^(or^other^time^points)^after^LNP‐ mRNA(VHH‐B9)^injection.^Immunofluorescence,^immunoblotting^or^ELISA^are^used^to^assay^the^ presence^and^measure^the^concentration^of^VHH‐B9^in^brain^sections,^in^CSF^or^in^brain^lysate.^In^ cases^where^the^RNA^sequence^also^encodes^a^Flag^or^3xFLAG^epitope^tag^appended^to^the^C‐terminus.^ Quantitative^reverse^transcriptase^PCR^(qRT‐PCR)^ Two‐step^qRT‐PCR^is^performed^according^to^manufacturer^protocol^(New^England^Biolabs).^Briefly,^ total^RNA^is^isolated^from^cell^lysate^or^mouse^brain^homogenate^and^reverse^transcribed^to^generate^ cDNA^(LunaScript^RT^SuperMix^Kit,^#E3010).^cDNA^is^serially^diluted^to^a^final^concentration^of^1:100^ and^used^directly^as^a^template^for^qRT‐PCR^(Luna^Universal^qPCR^Master^Mix).^Relative^quantitation^ is^performed^according^to^the^2‐ΔΔCt^method^using^GAPDH,^β‐actin,^or^β‐tubulin^as^a^housekeeping^ gene.^qPCR^primers^aredesigned^using^Primer3^software.^ Immunofluorescence^ Sterile^glass^coverslips^were^inserted^into^12‐well^culture^plates,^and^HEK293T^and^astrocyte^cell^ cultures^were^plated^to^a^final^confluency^of^50‐70%^prior^to^transfection,^as^described^above.^After^ transfection,^plates^were^washed^with^1X^PBS^and^fixed^with^4%^paraformaldehyde^for^20^minutes^at^ room^temperature.^Fixed^cells^were^then^permeabilized^using^0.1%^Triton‐X^100^for^5^minutes^at^ room^temperature.^Plates^were^blocked^with^blocking^buffer^(1X^PBS^+^5%^bovine^plasma^albumin)^ for^1^hour^at^room^temperature^before^incubation^overnight^at^4°C^in^primary^antibody^diluted^in^ blocking^buffer.^The^next^day,^plates^were^washed^with^1X^PBS^and^incubated^in^secondary^antibody^ diluted^in^blocking^buffer^for^1^hour^at^room^temperature.^After^incubation,^plates^were^ counterstained^with^DAPI^or^Hoescht^(1:20,000)^for^10^minutes^at^room^temperature.^Coverslips^were^ removed^from^culture^plates^with^forceps^and^mounted^on^glass^slides^with^mounting^media.^ Coverslips^were^imaged^using^confocal^microscopy.^For^tissue^immunostainings,^fixed^slices^were^ incubated^in^rabbit^anti‐human^GCase^primary^antibody^for^4^days,^followed^by^incubation^in^anti‐ rabbit^secondary^antibody^conjugated^to^AlexaFLuor^647™^for^confocal^imaging.^ Example^2.^LNP‐mRNA^leads^to^broad^expression^of^a^reporter^protein^in^the^brain^and^ spinal^cord^ To^test^for^the^feasibility^of^using^brain^cells^to^induce^brain‐wide^diffusion^of^secretory^proteins,^the^ inventors^first^investigated^the^distribution^pattern^of^LNP‐mRNA^and^downstream^protein^ expression^following^intracerebroventricular^(ICV)^injections.^^LNPs^containing^the^lipophilic^dye^DiO^ and^carrying^mRNA^encoding^for^mCherry^were^injected^bilaterally^in^the^lateral^ventricles^of^adult^ (p40‐60)^mice.^^After^2^days^to^allow^for^the^mCherry^protein^expression^and^accumulation,^brains^ were^collected^and^imaged.^ The^distribution^pattern^of^the^DiO^dye^contained^in^the^LNP^formulation^showed^that^LNPs^had^ spread^throughout^the^corpus^collosum,^a^major^white^matter^tract^in^the^brain.^^Importantly,^robust^ expression^of^the^mCherry^fluorescent^protein^reporter^was^readily^observed^along^the^corpus^ callosum,^as^well^as^along^the^ventricular^wall^and^the^choroid^plexus^within^the^ventricles.^^The^ reporter^protein^was^expressed^over^a^spread^of^>3^mm^in^the^anterior‐posterior^axis,^and^>4^mm^ laterally^(FIGURE^3).^^Closer^examination^of^the^cell^types^producing^mCherry^within^the^white^matter^ revealed^expression^in^two^glial^cell^types:^oligodendrocytes,^Olig2+^cells^responsible^for^myelinating^ neuronal^axons,^as^well^as^in^astrocytes,^immunostained^for^GFAP^(FIGURE^4).^^Furthermore,^ ependymal^cells^lining^the^ventricle^wall^as^well^as^choroid^plexus^ependymal^cells^were^found^to^ express^mCherry^(FIGURE^5).^^This^shows^that^LNP‐mRNA^injection^can^be^used^to^achieve^extensive^ production^of^exogenous^proteins^within^cells^of^the^CNS.^ In^a^separate^experiment^LNP‐mRNA^encoding^for^mCherry^was^injected^intrathecally^in^the^lumbar^ region^between^L5‐L6.^^Robust^expression^of^mCherry^was^observed^along^the^entire^length^of^the^ spinal^cord^(FIGURE^6).^^This^shows^that^LNP‐mRNA^injection^can^be^used^to^achieve^extensive^ production^of^exogenous^proteins^within^cells^of^the^CNS^via^multiple^delivery^routes.^ In^a^separate^experiment,^LNP‐mRNA^encoding^for^mCherry^was^injected^ICV^into^mice^encapsulated^ with^various^LNP^formulations^where^the^ionizable^lipids,^helper^lipid,^and^molar^ratios^of^the^four^ lipid^components^were^altered.^FIGURE^7^shows^mCherry^expression^in^coronal^brain^slices^of^the^ white^matter^tract^of^the^corpus^callosum^at^2^days^post‐injection,^where^mCherry^was^used^to^ visualize^LNP‐mRNA^transfection^of^five^different^LNP^formulations^(LNP^“D”,^LNP^“E”,^LNP^“F”,^LNP^ “G”,^and^LNP^“H”^from^TABLE^1.^These^data^show^that^widespread^LNP^distribution^and^mRNA^ express^can^be^achieved^throughout^the^corpus^callosum^and^ventricular^system^using^different^LNP^ formulations.^ Example^3.^LNP‐mRNA^expression^of^a^secretory^protein^leads^to^widespread^diffusion^ in^the^brain^ The^broad^distribution^across^white^matter^tracts^as^well^as^in^structures^responsible^for^CSF^ production^suggests^LNP‐mRNA^could^be^harnessed^to^produce^secretory^proteins^that^would^then^ diffuse^across^the^CNS.^^The^inventors^tested^this^‘bioreactor’^approach^by^injecting^LNP(1)[or^K]‐ mRNA‐FGF‐21^and^LNP(2)^[or^L]‐mRNA‐FGF‐21^as^set^out^in^TABLE^1^(obtained^from^NanoVation^ Therapeutics^Inc.™)^of^LNP‐mRNA^encoding^for^Fibroblast^growth^factor^21^(FGF‐21,^RNA^ Technologies^and^Therapeutics,^Montreal,^QC),^a^secreted^growth^factor^that^is^primarily^produced^by^ hepatocytes^and^therefore^normally^virtually^absent^in^the^brain.^^Either^three^or^48^hours^after^ bilateral^intracerebroventricular^injection^in^the^brain,^CSF^was^collected^from^the^cisterna^magna^(the^ largest^CSF‐filled^cistern^in^the^brain)^and^quantified^for^the^presence^of^FGF‐21^using^a^U‐PLEX^assay.^^ CSF^FGF‐21^concentrations^were^significantly^higher^for^LNP(1)[or^K]‐mRNA‐FGF‐21^(2,790.3^pg / mL)^ and^LNP(2)^[or^L]‐mRNA‐FGF‐21^(23,765^pg / mL)^over^un‐injected‐^(Control:^164.3^pg / mL)^and^ injected^controls^(LNP‐mRNA‐EPO:^28.6^pg / mL)^(FIGURE^8).^^The^levels^of^FGF21^in^the^CSF^were^ elevated^for^a^period^of^at^least^48^hours^(FIGURE^9);^LNP‐mRNA‐FGF‐21^CSF^collect^3hr^post‐ injection:^30,740^pg / mL;^48hr:^27,719^pg / mL).^^Additionally,^the^levels^of^FGF‐21^in^the^CSF^varied^ with^the^LNP‐mRNA^concentrations^injected,^in^a^dose‐dependent^manner^(FIGURE^9);^LNP‐mRNA‐ FGF‐21^1mg / mL^dose:^30,740^pg / mL;^0.5mg / mL^dose:^30,576^pg / mL;^0.2mg / mL^dose:^31,604^ pg / mL;^0.05mg / mL^dose:^1,213^pg / mL).^^In^a^separate^experiment,^LNP‐mRNA^encoding^for^ Erythropoietin^(EPO)^was^injected^intracerebroventricular.^^Either^three^or^48^hours^after^bilateral^ ventricular^injection^in^the^brain,^CSF^was^collected^from^the^cisterna^magna^and^quantified^for^the^ presence^of^EPO^using^a^U‐PLEX™^assay.^^EPO^was^found^in^the^CSF^at^high^levels^(FIGURE^10);^un‐ injected^Control:^8.5^pg / ml;^injection^control^LNP‐FGF‐21:^15.0^pg / ml;^LNP‐mRNA‐EPO:^396,899^ pg / ml).^^The^levels^of^EPO^in^the^CSF^were^elevated^for^a^period^of^at^least^48^hours^(FIGURE^11);^ LNP‐mRNA‐EPO^3hr:^396,899^pg / mL;^48hr:^144,286^pg / mL).^^Additionally,^the^levels^of^EPO^in^the^ CSF^varied^with^the^LNP‐mRNA^concentrations^injected,^in^a^dose‐dependent^manner^(FIGURE^11);^ LNP‐mRNA‐EPO^1mg / mL^dose:^396,899^pg / mL;^0.5^mg / mL^dose:^218,629^pg / mL).^^The^data^shows^ that^secretory^proteins,^such^as^growth^factors^or^erythropoietin,^can^diffuse^across^the^entire^brain^ and^reach^sites^of^action^that^are^distal^to^the^actual^site^of^LNP‐mRNA^uptake^and^expression.^^ Surprisingly,^the^data^shows^that^protein^production,^secretion^and^diffusion^across^the^entire^brain^is^ consistent^over^a^period^of^at^least^48^hours.^ Example^4.^LNP‐mRNA^and^circular^RNA^leads^to^long‐term^protein^production^in^human^ neural^cells^derived^from^induced^pluripotent^stem^cells^(iPSC).^ Conventional^cell^culture^methods^do^not^allow^for^long^term^evaluation^of^protein^production^due^to^ changing^cell^growth^and^viability,^do^not^recapitulate^the^cell‐cell^interactions^observed^in^the^brain,^ do^not^integrate^the^variety^of^cell^types^found^in^the^brain,^and^are^not^of^human^origin.^We^therefore^ used^a^3D^neurosphere^culture^model^composed^of^neural^cells^derived^from^human^iPSC^(hiPSC).^ These^neurospheres^contain^cells^with^neuronal^and^astrocytic^phenotypes,^are^of^human^origin,^and^ stable^for^longitudinal^imaging^over^a^period^of^several^weeks.^This^therefore^allows^for^better^ investigation^of^the^time^course^of^protein^production^induced^by^LNP‐RNA.^^ hiPSC‐derived^neurospheres^(40^days^in^vitro)^were^exposed^to^LNP‐mRNA(mCherry)^(FIGURE^13).^ Neurospheres^were^then^imaged^for^mCherry^fluorescence^5^hours^after^transfection,^and^ subsequently^at^various^time^points.^mCherry^production^could^be^observed^over^a^period^of^>25 / 30^ days,^demonstrating^that^RNA^translation^into^a^functional^protein^of^interest^can^occur^in^a^sustained^ manner^in^human^cells.^ For^circRNA,^neurospheres^were^imaged^for^NeonGreen^fluorescence^5^hours^after^transfection,^and^ subsequently^at^various^time^points.^NeonGreen^production^could^be^observed^over^a^period^of^>15^ days,^demonstrating^that^circRNA^translation^into^a^functional^protein^of^interest^can^occur^in^a^ sustained^manner^in^human^cells^(FIGURE^14).^^ To^test^whether^hiNS^cultures^can^be^used^to^study^secreted^proteins^as^a^surrogate^model^system^for^ the^LNP‐mRNA^strategy^described^here,^the^inventors^transfected^LNP‐mRNA^encoding^for^gaussia^ luciferase^(GLuc),^a^secreted^variant^of^luciferase^that^can^be^used^as^a^reporter^for^LNP‐mRNA^ delivery^and^protein^translation.^hiNS^cultures^were^transfected^with^LNP‐mRNA(GLuc)^and^the^ supernatant^was^subsequently^imaged^for^luciferase^luminescence.^GLuc^luminescence^in^the^ supernatant^increased^over^the^first^48^hours^post^transfection^in^a^dose‐dependent^manner,^and^ persisted^for^at^least^96^hours^(FIGURE^15).^Example^ 5^Measuring^ exogenous^GCase^ expression^ and^ secretion^ in^ in^ vitro^models^using^DNA^sequence^optimization^ Glucocerebrosidase^(GCase)^is^an^endogenous^lysosomal^enzyme^that^metabolizes^glucocerebroside^ encoded^by^the^GBA1^gene.^To^test^for^the^functional^expression^of^exogenous^human^GCase,^the^ inventors^used^a^DNA^strategy^to^assess^functional^GCase^protein^expression,^secretion,^and^activity^in^ HEK293^cells^and^astrocyte^cultures^in^vitro.^DNA^sequences^encoding^for^full‐length^GCase^(based^on^ the^canonical^coding^sequence^(CDS)^of^the^endogenous^transcript)^will^be^synthesized^and^inserted^ into^mammalian^expression^vectors^(e.g.^pcDNA3.1).^The^endogenous^human^GCase^coding^sequence^ was^tested^against^codon‐optimized^sequences^generated^using^open‐source^algorithms,^to^optimize^ downstream^mRNA^secondary^structure,^guanine:cytosine^content,^transcript^stability^and^translation^ speed^and^efficiency.^In^some^cases,^in^addition^to^the^coding^sequence^for^human^GCase,^the^ expression^plasmid^will^encode^a^3xFLAG^epitope^tag^(SEQ^ID^No.^18)^was^appended^to^the^C‐terminus^ purely^for^experimental^detection^purposes^and^to^differentiate^exogenously^expressed^GCase^from^ the^endogenous^protein.^^FLAG^is^a^widely^used^epitope^tag^that^is^unlikely^to^affect^protein^function^or^ stability^when^appended^to^the^C‐terminus.^Each^expression^plasmid^was^transfected^into^HEK293T^ (non‐neural)^and / or^astrocyte^cell^lines^to^validate^exogenous^expression^of^GCase.^DNA^transfections^ were^be^performed^transiently^using^standard^transfection^reagents^(e.g.^Lipofectamine^3000™)^and^ positive^protein^expression^readouts^will^be^determined^by^immunoblotting^or^ELISA.^In^HEK293T^ transfected^with^a^DNA^construct^for^GCase^containing^a^3xFLAG^epitope^tag,^we^observed^an^increase^ in^exogenous^GCase^(aka^GBA1)^expression^levels^(FIGURE^17)^relative^to^endogenous^GCase.^ mCherry^reporter^plasmid^were^co‐transfected^as^negative^controls.^Functional^GCase^enzymatic^ activity^was^measured^using^commercially^available^kits^(FIGURE^18).^^ The^human^GCase^sequence^contains^an^endogenous^signal^“leader”^amino^acid^sequence^ (MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASG,^SEQ^ID^No.^5))^that^can^target^the^protein^for^ secretion^to^the^extracellular^space.^Secretion^of^GCase^protein^is^assessed^from^culture^media^ supernatant^as^shown^in^FIGURES^16,^17,^18,^19^and^20.^Substituting^the^endogenous^leader^ sequence^with^either^engineered^sequences,^or^sequences^identified^from^endogenous^secreted^ proteins,^was^found^to^alter^GCase^protein^secretion^(FIGURE^18,^first^graph).^Importantly,^including^ such^sequences^could^alter^GCase^enzyme^activity.^Therefore,^functional^GCase^activity^was^confirmed^ from^supernatant^samples^(FIGURE^18,^second^graph)^possessing^engineered^sequence^additions^as^ in^FIGURE^18A.^ Example^6.^mRNA^transcript^optimization^and^GCase^enzyme^expression^in^vitro^ As^an^important^confirmation^that^in^vitro^transcribed^(IVT)^mRNA^could^yield^functional^and^secreted^ GCase,^the^mRNA^constructs^were^generated^for^GCase.^All^constructs^included^pseudouridine^ substitutions^throughout^the^mRNA^sequence,^as^well^as^exogenous^caps,^5’^and^3’^UTRs,^and^polyA^ tails^(FIGURE^16).^These^mRNA^transcript^components^described^in^FIGURE^16^were^serially^ substituted^to^test^for^improved^mRNA^stability^and^half‐life,^translation^speed,^and^translation^ efficiency.^mRNA^transcript^modifications^were^compared^against^the^native^human^coding^sequence^ that^included^capping^structure^and^UTRs^utilized^in^the^COVID‐19^vaccine^and^testing^in^in^vitro^as^in^ illustrated^in^FIGURES^18,^19,^20,^21,^and^22.^These^engineered^mRNA^transcripts^for^GCase^were^ generated^IVT^and^transfected^into^HEK293T,^rodent^primary^astrocyte^and^hiNS^cultures^using^ Lipofectamine™^or^LNPs^as^described^in^Table^1.^GCase^protein^expression^and^secretion^was^assayed^ by^collecting^cell^lysate^and^culture^supernatant,^respectively,^and^the^protein^amount^was^quantified^ via^immunoblotting^or^ELISA^^(results^shown^in^FIGURE^19).^The^subcellular^localization^of^GCase^ was^assessed^in^HEK293T^cells,^with^significant^accumulation^in^lysosomes^(FIGURE^21).^GCase^ enzyme^activity^was^also^assessed^using^commercial^kits^as^in^FIGURE^18.^^The^supernatant^was^ collected^at^various^time^points^(days^to^weeks^post‐transfection)^to^estimate^the^peak^ production / secretion^timeline^of^GCase^in^hiNS^of^different^LNP^constructs^(FIGURE^22).^While^the^ objective^of^the^assay^is^focused^on^whether^the^protein^is^successfully^translated,^it^also^served^as^a^ quantifiable^assay^for^optimal^LNP‐mRNA^formulation^and^concentration^and^to^test^the^ production / secretion^efficiency^of^various^mRNA^constructs^over^a^period^of^>15^days.^ To^test^for^M6P‐dependent^cross^correction^of^secreted^GCase,^supernatant^was^collected^from^LNP‐ mRNA(GCase)^transfected^hiNS^and^applied^to^untransfected^(i.e.^LNP‐mRNA‐GCase^naïve)^hiNS^ cultures^to^assess^reuptake.^Indeed,^applying^supernatant^containing^secreted^GCase^to^naïve^hiNS^led^ to^clear^intracellular^localization^of^GCase^as^identified^by^FLAG^immunostaining^(FIGURE^23).^ GCase^expression^from^LNP‐mRNA^transfection^was^also^tested^to^assess^localization^to^the^lysosome^ in^hiNS^cultures.^Co‐staining^for^GCase^(FLAG)^with^the^lysosomal^protein^LAMP1^revealed^significant^ co‐localized^of^the^two^proteins,^indicating^that^GCase^synthesized^from^LNP‐mRNA^would^translocate^ to^the^lysosome^(FIGURE^24).^ Example^7.^LNP‐mRNA^expression^of^secretory^GCase^leads^to^widespread^diffusion^in^ the^brain^^ The^distribution^of^LNP‐mRNA‐mCherry^across^white^matter^tracts^and^lateral^ventricles^as^described^ in^Example^2,^and^the^CSF^expression^of^secreted^FGF‐21^and^EPO^from^LNP‐mRNAs^in^Example^3,^ indicate^LNP‐mRNAs^encoding^for^secreted^GCase^would^engender^GCase^expression^in^at^least^the^ white^matter^tracts^and^ventricles,^but^likely^with^brain‐wide^distribution.^^LNP‐mRNA‐GCase^was^ tested^using^formulations^set^out^in^TABLE^1.^LNP‐mRNA‐GCase^constructs^were^injected^bilaterally^ ICV,^followed^by^varying^time^points^days‐^to^weeks‐post^injection^for^GCase^expression^and^activity^ analyses.^CSF^was^collected^from^the^cisterna^magna^to^assess^secretory^GCase^in^the^CSF^and^ISF.^ Animals^were^then^either^perfused^for^whole‐brain^IHC^to^assess^GCase^distribution^in^the^brain,^or^ flash^frozen^at^‐80oC^to^perform^western^blot,^ELISA,^and^activity^analyses^for^GCase^protein.^LNP‐ mRNA‐GCase^injection^led^to^broad^GCase^expression^in^the^brain^compared^to^saline^control^or^LNP‐ mRNA‐FGF21^controls^(not^detectable),^as^detected^from^CSF^and^plasma^samples^collected^at^least^3‐,^ 24‐,^48‐,^and^96‐hours^post‐injection^and^measured^by^a^custom^MSD^U‐PLEX^as^described^in^ Example^1.^^Results^are^shown^in^FIGURES^25‐28).^In^some^examples,^GCase^constructs^contained^a^ FLAG^epitope^tag^for^specific^labelling^with^primary^antibodies.^^^ Experiments^are^repeated^in^intrathecal^lumbar^administration.^ For^GCase^immunofluorescence^experiments,^staining^was^performed^to^assess^the^cell^tropism^for^ astrocytes^(GFAP)^and^GCase^expression,^secretion,^and^activity^was^compared^against^un‐injected^ and^injection^controls^(saline^only)^and^LNP‐mRNA^transfection^controls^(LNP‐mRNA‐mCherry^and^ LNP‐mRNA‐FGF21).^Results^are^shown^in^FIGURES^29^and^30.^ For^GCase^immunofluorescence^experiments,^staining^is^performed^to^assess^the^cell^tropism^of^GCase^ localization^for^neurons^(NeuN),^microglia^(Iba1),^oligodendrocytes^(Olig2,^and^CC1),^and^vascular^ cells^(αSMA^for^smooth^muscle,^NG2^for^pericytes,^CD31^for^endothelial^cells).^GCase^expression,^ secretion,^and^activity^is^compared^against^un‐injected^and^injection^controls^(saline^only)^and^LNP‐ mRNA^transfection^controls^(LNP‐mRNA‐mCherry^and^LNP‐mRNA‐FGF21).^^Example^8.^LNP‐mRNA^expression^of^secretory^GCase^restores^enzyme^deficiency^ in^a^mouse^model^of^neuronopathic^Gaucher^Disease^^ The^therapeutic^efficacy^of^LNP‐mRNA‐GCase^is^tested^in^a^well‐characterized^model,^GBA‐D409V,^for^ neuronopathic^Gaucher^Disease^Types^2^and^3.^These^GBA‐D409V^mice^express^a^D409V^mutant^ GCase^protein,^with^loxP^sites^flanking^exons^6^through^8^for^CRE‐recombinase‐dependent^GBA1^gene^ knockout.^The^D409V^mutation^leads^to^significant^reductions^in^GCase^activity^in^the^brain.^These^ animals^are^also^used^as^a^model^for^Parkinson’s^disease.^^ GBA‐D409V^mice^are^injected^with^lead^LNP‐mRNA‐GCase^constructs^as^described^in^Example^2^^as^ well^as^saline^only^(injection^control)^and^LNP‐mRNA‐mCherry^and^LNP‐mRNA‐FGF21^as^LN‐mRNA^ transfection^controls.^LNP‐mRNAs^are^injected^bilaterally^ICV,^and^brains^are^collected^and^flash‐ frozen^for^tissue^analyses^for^days^to^weeks^post^injection.^GCase^activity^is^assessed^using^ commercially^available^kits^and^compared^against^the^aforementioned^controls^and^is^found^to^be^ elevated^in^D409V^mice^injected^with^LNP‐mRNA‐GCase.^^ Further,^homozygous^GBA‐D409V^animals^are^crossed^with^a^CRE‐recombinase^mouse^line^to^ generate^constitutive^brain^GBA1^knockout^mice.^In^these^mice,^survivability^studies^are^performed^to^ compare^the^therapeutic^efficacy^of^LNP‐mRNA‐GCase^constructs^in^animals^completely^lacking^native^ GCase^expression^compared^to^appropriate^injection^controls.^Example^ 9.^ In^ vitro^method^ to^ assess^whether^an^ antibody^ encoded^ by^ the^mRNA^ is^capable^ of^ production^ and^ secretion^ from^ a^ cell^ of^ the^ central^ nervous^ system^ into^cerebrospinal^fluid^ An^in^vitro^primary^culture^assay^was^used^to^test^the^ability^of^cells^of^the^central^nervous^system^ (e.g.,^HEK293^cells,^rat^primary^astrocytes)^to^produce^and^secrete^antibodies^from^an^LNP‐mRNA.^In^ parallel^experiments,^human^astrocytes^were^cultured^using^common^methods.^^ HEK293T^was^treated^with^DNA^or^LNP‐mRNA^coding^for^the^protein.^Production^of^the^antibody^was^ assayed^by^collecting^the^cell^lysate^and^quantifying^the^antibody^amount,^via^immunoblotting,^ELISA^ or^U‐PLEX^assay.^^^ Secretion^of^the^antibody^was^assayed^by^collecting^the^cell^supernatant^and^quantifying^the^antibody^ concentration^via^U‐PLEX^assay^described^in^Example^1.^The^supernatant^is^collected^at^various^time^ points^to^estimate^the^peak^production / secretion^timeline.^While^the^objective^of^the^assay^is^focused^ on^whether^the^antibody^is^successfully^secreted,^it^also^serves^as^a^quantifiable^assay^for^optimal^ LNP‐mRNA^formulation^and^concentration^and^to^test^the^production / secretion^efficiency^of^various^ mRNA^constructs.^ To^assess^the^feasibility^of^using^mRNA^to^produce^monoclonal^antibody,^we^transfected^HEK293T^ cells^with^mRNA^encoding^for^the^heavy^(SEQ^ID^NO.^10)^^and / or^light^chains^(SEQ^ID^No.12)^of^ trastuzumab.^Compared^to^no^mRNA^transfection^controls,^trastuzumab^heavy^and^light^only^ fragments^were^detectable^using^an^electrochemiluminescence^U‐PLEX^ELISA^from^MSD^that^is^ specific^for^human^IgG.^However,^we^observed^a^significant^increase^in^trastuzumab^detected^when^ heavy^and^light^chain^mRNAs^were^co‐transfected^at^a^2:1^(heavy:light)^ratio,^indicating^the^U‐PLEX^ IgG^MSD^assay^used^is^optimized^to^detect^the^fully^assembled^trastuzumab^antibody^(SEQ^ID^No.^ 14)(FIGURE^32).^ To^test^whether^the^heavy^and^light^chain^sequences^could^be^assembled^linearly^in^a^single^mRNA^ construct,^we^linked^the^two^sequences^using^a^P2A^linker^(detailed^in^Example^1)^strategy^and^ measured^trastuzumab^expression^in^the^cell^culture^supernatant.^HEK293T^cells^were^transfected^ with^pDNA^encoding^for^the^linear^heavy:P2A:light^sequence^(FIGURE^31),^and^supernatant^was^ collected^24‐^and^48^hrs^post‐transfection^(FIGURE^33).^^ ^ Example^10.^in^vivo^LNP‐mRNA‐induced^production^of^trastuzumab^in^cells^of^brain.^ Following^successful^LNP‐mRNA‐induced^production^of^proteins^like^mCherry,^FGF21,^EPO,^or^GCase^ in^brain^cells,^we^injected^LNPs^encapsulating^mRNA^encoding^for^a^monoclonal^antibody.^As^a^proof‐ of‐concept,^we^tested^the^production^of^an^antibody^representative^of^conventional^Heavy^chain^and^ Light^chain‐containing^antibodies,^trastuzumab.^Trastuzumab^is^composed^of^two^pairs^of^light^and^ heavy^chains.^We^generated^a^linear^mRNA^sequence^encoding^for^one^copy^of^heavy^(SEQ^ID^No.^10)^ and^light^chain^(SEQ^ID^NO.^12)^(1:1^ratio)^joined^by^a^P2A^linker^(SEQ^ID^No.^13)^(described^in^ Example^1).^^ Unlinked^mRNA^sequences^encapsulated^at^varying^ratios^of^light:heavy^chain^(1:1,^1:2,^1:4,^w / w)^and^ co‐encapsulated^into^lipid^nanoparticle^formulations^as^illustrated^in^FIGURE^31.^^Results^not^shown.^ In^Vivo,^CSF^collected^is^from^the^cisterna^magna^of^mice^injected^with^LNP‐mRNA(trastuzumab)^ (bilateral^1.5ug^mRNA / hemisphere)^either^24‐^or^48‐hours^post‐injection^had^significant^levels^of^ fully^matured^humanized^trastuzumab^at^48^hours^compared^to^saline^injection^controls^detected^ using^a^huma^IgG^MSD^assay.^Trastuzumab^is^also^detected^from^whole‐brain^lysate^samples^(CSF,^ISF,^ and^intracellular^trastuzumab)^at^the^same^time^points.^ Example^11.^in^vivo^LNP‐mRNA‐induced^production^of^VHH^single‐domain^antibody^in^ cells^of^the^brain.^ Following^successful^LNP‐mRNA‐induced^production^of^proteins^like^mCherry,^FGF21^or^EPO^in^brain^ cells,^we^are^testing^LNPs^encapsulating^mRNA^sequences^that^encode^for^a^single‐domain^VHH^ antibody.^As^a^proof‐of‐concept,^we^are^testing^the^production^of^an^antibody^representative^of^VHH^ single‐domain^antibodies,^VHH‐B9.^Following^injection^in^mouse^brain^(ICV,^intrathecal^lumbar^or^ intracisternal),^brains^are^collected,^fixed,^and^sliced,^at^various^time^points^to^evaluate^the^presence^of^ VHH‐B9^using^immunohistochemistry.^Co‐staining^with^cell^type^markers^(GFAP,^Olig2,^etc…)^allows^ for^identification^of^the^cell^types^producing^VHH‐B9.^Such^imaging^demonstrates^the^spatial^ distribution^of^the^cells^producing^VHH‐B9^within^the^brain.^In^some^cases,^in^addition^to^the^coding^ sequence^for^VHH‐B9,^the^RNA^sequence^encodes^a^Flag^(SEQ^ID^No.^17)^or^3xFLAG^epitope^tag^(SEQ^ ID^No.^18^)^appended^to^the^C‐terminus^purely^for^experimental^detection^purposes;^importantly,^Flag^ is^a^widely‐used^epitope^tag^and^is^unlikely^to^affect^protein^function^or^stability^when^appended^to^ the^C‐terminus.^Example^12.^ In^ vitro^method^ to^ assess^whether^a^protein^ encoded^by^ the^ circRNA^ is^capable^of^secretion^from^a^cell^of^the^central^nervous^system^into^cerebrospinal^fluid^ An^in^vitro^primary^culture^assay^will^be^used^to^test^the^ability^of^cells^of^the^central^nervous^system^ (e.g.,^astrocytes^and^oligodendrocytes^(OL))^to^produce^and^secrete^proteins^from^an^LNP‐mRNA.^In^ parallel^experiments,^primary^astrocytes^and^primary^OL^are^cultured^using^common^methods^from^ perinatal^mixed^glial^cultures^(commonly^known^as^the^McCarthy^and^deVelis^method).^Briefly,^brains^ from^P0‐P3^mice^are^collected,^cortices^are^dissected^and^cells^are^mechanically^dissociated^through^trituration.^After^successive^washing^steps,^cells^are^plated^and^cultured^for^3‐6^days^at^37°C,^5‐8%^CO2.^This^gives^rise^to^a^“mixed^glial^culture’,^composed^of^a^bottom^layer^of^astrocytes,^on^top^of^which^is^found^a^layer^of^oligodendrocyte^progenitor^cells^(OPCs)^as^well^as^loosely^adherent^microglia.^ Shaking^the^plate^induces^detachment^of^microglia,^leaving^astrocytes^and^OPC.^Further^differential^ shaking^steps^then^lead^to^OPC^detachment.^These^OPCs^are^subsequently^cultured^separately^and^ differentiated^into^mature^OL^using^OL‐specific^culture^media.^In^parallel,^the^remaining^layer^of^ astrocytes^is^passaged^several^times^to^obtain^astrocyte‐^rich^cultures.^The^purity^of^astrocyte^and^OL^ cultures^can^be^confirmed^using^GFAP^and^O4 / MBP^immunostaining,^respectively.^ Astrocytes^and^OL^cultures^will^be^used^to^assay^the^secretion^of^proteins.^Primary^cells^(astrocytes^or^ OL)^are^treated^with^LNP‐mRNA^coding^for^the^protein.^Secretion^of^the^protein^will^be^assayed^by^ collecting^the^cell^supernatant^and^quantifying^the^protein^amount,^via^immunoblotting,^ELISA^or^U‐ PLEX^assay.^The^supernatant^will^be^collected^at^various^time^points^to^estimate^the^peak^ production / secretion^timeline.^While^the^objective^of^the^assay^is^focused^on^whether^the^protein^is^ successfully^secreted,^it^may^also^serve^as^a^quantifiable^assay^for^optimal^LNP‐circRNA^formulation^ and^concentration^and^to^test^the^production / secretion^efficiency^of^various^mRNA^constructs.^ Example^13.^LNP‐circRNA^leads^to^broad^and^sustained^expression^of^a^reporter^protein^ in^the^brain^and^spinal^cord^ To^test^for^the^feasibility^of^using^brain^cells^to^induce^brain‐wide^diffusion^of^secretory^proteins^using^ circRNA,^we^investigated^the^distribution^pattern^of^LNP‐circRNA‐induced^protein^expression^ following^intracerebroventricular^(ICV)^injections.^LNPs^carrying^circRNA^encoding^for^NeonGreen^ are^injected^bilaterally^in^the^lateral^ventricles^of^adult^(p40‐60)^mice.^^Brains^were^collected^and^ imaged^after^2^days^to^allow^for^the^NeonGreen^protein^expression^and^accumulation.^Results^not^ shown.^ In^a^separate^experiment^LNP‐circRNA^encoding^for^NeonGreen^is^injected^intrathecally^in^the^lumbar^ region^between^L5‐L6,^and^the^distribution^and^time^course^of^NeonGreen^expression^is^being^ investigated.^ Example^14.^LNP‐circRNA^leads^to^sustained^secreted^delivery^of^a^protein^of^interest^to^ the^CNS.^ LNP‐circRNA^is^tested^to^assess^induced^secretion^of^proteins^from^brain^cells^to^the^brain^interstitial^ fluid^and^cerebrospinal^fluid.^At^various^time^points^after^LNP‐circRNA(FGF21)^or^LNP‐circRNA(EPO)^ injection^(ICV,^intrathecal^lumbar^or^intracisternal),^the^CSF^is^collected^and^the^presence^of^FGF21^or^ EPO^in^the^CSF^is^measured^using^immunoblotting^or^ELISA.^Brain^lysates^of^various^brain^regions^are^ collected,^and^immunoblotting^or^ELISA^are^performed^to^measure^the^presence^of^FGF21^or^EPO^in^ brain^regions^distal^to^the^injection^site.^^ Example^15.^LNP‐saRNA^leads^to^broad^and^sustained^expression^of^a^reporter^protein^in^ the^brain^and^spinal^cord^ To^test^for^the^feasibility^of^using^brain^cells^to^induce^brain‐wide^diffusion^of^secretory^proteins^using^ saRNA,^the^distribution^pattern^of^LNP‐saRNA‐induced^protein^expression^following^ intracerebroventricular^(ICV)^injections^is^investigated.^LNPs^carrying^saRNA^encoding^for^mCherry^ are^injected^bilaterally^in^the^lateral^ventricles^of^adult^(p40‐60)^mice.^^After^2^days^to^allow^for^the^ mCherry^protein^expression^and^accumulation,^brains^are^collected^and^imaged.^ The^distribution^pattern^of^the^LNP‐mRNA‐induced^mCherry^expression^is^investigated.^^ In^a^separate^experiment^LNP‐saRNA^encoding^for^mCherry^is^injected^intrathecally^in^the^lumbar^ region^between^L5‐L6.^^Expression^distribution^and^time^course^of^mCherry^is^^investigated^in^the^CNS.^ Example^16.^LNP‐saRNA^leads^to^sustained^secreted^delivery^of^a^protein^of^interest^to^ the^CNS.^ LNP‐saRNA^delivery^is^tested^for^its^ability^to^induce^secretion^of^proteins^from^brain^cells^to^the^brain^ interstitial^fluid^and^cerebrospinal^fluid.^At^various^time^points^after^LNP‐saRNA(FGF21)^or^LNP‐ saRNA(EPO)^injection^(ICV,^intrathecal^lumbar^or^intracisternal),^the^CSF^is^collected^and^measured^ for^the^presence^of^FGF21^or^EPO^in^the^CSF^using^immunoblotting^or^ELISA.^Brain^lysates^of^various^ brain^regions^are^also^be^collected^and^immunoblotting^or^ELISA^are^performed^to^inform^on^the^ presence^of^FGF21^or^EPO^in^brain^regions^distal^to^the^injection^site.^^ List^of^Sequences^ SEQ^Sequence^ Type^^ Description^^ ^ SEQ^Sequence^ Type^^ Description^^ ^ SEQ^Sequence^ Type^^ Description^^ ^ SEQ^Sequence^ Type^^ Description^^ ^ ^ SEQ^Sequence^ Type^^ Description^^ ^ SEQ^Sequence^ Type^^ Description^^ ^ SEQ^Sequence^ Type^^ Description^^ ^ SEQ^Sequence^ Type^^ Description^^ ^ SEQ^Sequence^ Type^^ Description^^ ^ SEQ^Sequence^ Type^^ Description^^ ^ exogenouscaps, an s,an poy a s

Claims

We^claim^ 1.^^A^lipid^nanoparticle^(LNP)^comprising^at^least^one^nucleic^acid^encoding^a^glucocerebrosidase^ (GCase)^or^functional^fragment^thereof,^capable^of^secretion^from^the^cell^into^an^interstitial^and / or^ cerebrospinal^fluid^of^a^subject^after^the^LNP^has^been^in^contact^with^a^central^nervous^system^cell.^ 2.^The^lipid^nanoparticle^of^claim^1^wherein^the^GCase^or^functional^fragment^thereof,^further^ comprises^a^secretion^signal,^and^the^lipid^nanoparticle^further^comprises^an^amino^lipid^cationic^at^ neutral^pH^and^a^generally^cylindrical‐shaped^non‐cationic^helper^lipid.^ 3.^The^lipid^nanoparticle^of^any^one^of^claims^1‐2,^wherein^the^GCase^or^functional^fragment^thereof^is^ present^in^the^CSF^of^the^subject^at^a^first^concentration^at^a^first^time^point^and^at^a^second^ concentration^at^a^later^second^time^point.^ 4.^The^lipid^nanoparticle^of^claim^3,^wherein^the^first^time^point^is^about^3^hours,^and^wherein^the^ second^time^point^is^about^48^hours^after^contacting^of^the^LNP^with^the^cell,^and^wherein^the^second^ concentration^is^at^least^30%,^at^least^40%,^at^least^50%,^at^least^60%,^at^least^70%,^at^least^80%,^at^ least^90%,^or^at^least^95%^of^the^first^concentration.^^ 5.^The^lipid^nanoparticle^of^any^one^of^claims^3^or^4,^wherein^the^GCase^^or^functional^fragment^thereof^ is^present^in^the^CSF^of^the^subject^for^a^period^of^at^least^1^week,^at^least^2^weeks,^at^least^3^weeks,^at^ least^4^weeks,^at^least^6^weeks,^or^at^least^8^weeks.^ 6.^The^lipid^nanoparticle^of^any^one^of^claims^1‐5,^wherein^the^GCase^or^functional^fragment^thereof^is^ present^in^the^CSF^of^the^subject^for^a^period^of^at^least^24^hours,^at^least^36^hours,^or^at^least^48^ hours.^ 7.^The^lipid^nanoparticle^of^any^one^of^claims^1‐5,^wherein^the^GCase^or^functional^fragment^comprises^ an^amino^acid^sequence^of^at^least^80%,^at^least^85%,^at^least^90%,^at^least^95%,^or^at^least^98%^ sequence^identity^to^SEQ^ID^NO:^1.^ 8.^The^lipid^nanoparticle^of^any^one^of^claims^2‐5,^wherein^the^secretion^signal^comprises^an^amino^ acid^sequence^of^at^least^80%,^at^least^85%,^at^least^90%,^at^least^95%,^or^at^least^98%^sequence^ identity^to^SEQ^ID^NO:^5.^^ 9.^The^lipid^nanoparticle^of^any^one^of^claims^1‐5.^wherein^the^nucleic^acid^comprises^a^nucleotide^ sequence^of^at^least^60%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^ least^95%^sequence^identity^to^SEQ^ID^NO:^2.^ 10.^The^lipid^nanoparticle^of^any^one^of^claims^1‐5,^wherein^the^nucleic^acid^further^comprises^a^ nucleotide^sequence^of^at^least^60%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^ 90%,^or^at^least^95%^sequence^identity^to^SEQ^ID^NO:^3^or^4.^ 11^The^lipid^nanoparticle^of^any^one^of^claims^1‐5,^wherein^the^nucleic^acid^further^comprises^a^ nucleotide^sequence^of^at^least^60%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^least^95%^sequence^identity^to^SEQ^ID^NO:^6.^ 12.^The^lipid^nanoparticle^of^any^one^of^claims^1‐5,^wherein^the^nucleic^acid^further^comprises^a^ nucleotide^sequence^of^at^least^60%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^ 90%,^or^at^least^95%^sequence^identity^to^SEQ^ID^NO:^7.^ 13.^The^lipid^nanoparticle^of^any^one^of^claims^1‐5,^wherein^the^nucleic^acid^comprises^a^nucleotide^ sequence^of^at^least^60%,^at^least^70%,^at^least^75%,^at^least^80%,^at^least^85%,^at^least^90%,^or^at^ least^95%^sequence^identity^to^SEQ^ID^NO:^8.^^ 14.^The^lipid^nanoparticle^of^any^one^of^claims^1‐5,^wherein^the^cell^of^the^central^nervous^system^is^a^ glial^cell^selected^from^an^astrocyte^or^oligodendrocyte^or^an^ependymal^cell^of^the^cerebral^ventricles^ or^the^choroid^plexus.^ 15.^^A^lipid^nanoparticle^comprising^at^least^one^nucleic^acid^encoding^a^polypeptide^or^an^antibody^or^ antigen‐binding^fragment,^wherein^the^polypeptide,^antibody^or^antigen‐binding^fragment^is^capable^ of^secretion^from^a^central^nervous^system^cell^to^which^it^has^been^administered^into^interstitial^ and / or^cerebrospinal^fluid^of^a^subject.^ 16.^The^lipid^nanoparticle^of^claim^15^wherein^the^polypeptide,^antibody^or^antigen‐binding^fragment^ further^comprises^a^secretion^signal.^ 17.^The^lipid^nanoparticle^of^any^one^of^claims^15‐16,^wherein^the^at^least^one^nucleic^acid^comprises^a^ nucleic^acid^encoding^a^light^chain^and^a^nucleic^acid^encoding^a^heavy^chain.^ 18.^The^lipid^nanoparticle^of^claim^17,^wherein^the^nucleic^acid^encoding^the^light^chain^and^the^ nucleic^acid^encoding^the^heavy^chain^are^at^a^ratio^of^about^1:1^to^1:4^(w / w).^ 19.^The^lipid^nanoparticle^of^any^one^of^claims^15‐16,^wherein^the^secreted^polypeptide,^antibody^or^ antigen‐binding^fragment^is^present^in^the^CSF^of^the^subject^at^a^first^concentration^at^a^first^time^ point^and^at^a^second^concentration^at^a^later^second^time^point.^ 20.^The^lipid^nanoparticle^of^claim^19,^wherein^the^second^concentration^is^at^least^30%,^at^least^40%,^ at^least^50%,^at^least^60%,^at^least^70%,^at^least^80%,^at^least^90%,^or^at^least^95%^of^the^first^ concentration.^^ 21.^The^lipid^nanoparticle^of^claim^19^or^20,^wherein^the^first^time^point^is^about^3^hours^after^ contacting^the^LNP^with^the^cell.^ 22.^The^lipid^nanoparticle^of^any^one^of^claims^19‐21,^wherein^the^second^time^point^is^about^48^hours^ after^contacting^of^the^LNP^with^the^cell.^ 23.^The^lipid^nanoparticle^of^any^one^of^claims^19‐22,^wherein^the^secreted^polypeptide,^antibody^or^ antigen‐binding^fragment^is^present^in^the^CSF^of^the^subject^for^a^period^of^at^least^1^week,^at^least^2^ weeks,^at^least^3^weeks,^at^least^4^weeks,^at^least^6^weeks,^or^at^least^8^weeks.^ 24.^The^lipid^nanoparticle^of^any^one^of^claims^15‐23,^wherein^the^lipid^nanoparticle^comprises^an^ionizable^lipid.^ 25.^The^lipid^nanoparticle^of^claim^24^wherein^the^ionizable^lipid^is^an^amino^lipid^cationic^at^neutral^ pH.^ 26.^The^lipid^nanoparticle^of^claim^24^or^25,^wherein^the^ionizable^lipid^has^a^pKa^between^5.0^and^7.0.^ 27.^The^lipid^nanoparticle^of^any^one^of^claims^24‐26,^wherein^the^lipid^nanoparticle^further^comprises^ a^non‐cationic^helper^lipid.^^ 28.^The^lipid^nanoparticle^of^claim^27,^wherein^the^non‐cationic^helper^lipid^is^a^generally^cylindrical‐ shaped^lipid.^ 29.^The^lipid^nanoparticle^of^claim^27^or^28,^wherein^the^non‐cationic^helper^lipid^is^ distearoylphosphatidylcholine^(DSPC)^or^dioleoylphosphatidylglycerol^(DOPG)^or^^ sphingomyelin.^ 30.^The^lipid^nanoparticle^of^claim^27^or^28,^wherein^the^non‐cationic^helper^lipid^has^a^ phosphatidylethanolamine^content^that^is^less^than^2^mol%.^ 31.^The^lipid^nanoparticle^of^any^one^of^claims^24‐30,^wherein^the^lipid^nanoparticle^further^comprises^ a^sterol.^ 32.^The^lipid^nanoparticle^of^any^one^of^claims^24‐31,^wherein^the^lipid^nanoparticle^further^comprises^ a^polymer^lipid^conjugate.^^ 33.^The^lipid^nanoparticle^of^any^one^of^claim^1‐32,^wherein^the^lipid^nanoparticle^comprises^(a)^an^ ionizable,^amino,^cationic^lipid;^(b)^a^non‐cationic^helper^lipid;^(c)^a^sterol;^and^(d)^a^polymer^lipid^ conjugate,^and^wherein^the^molar^ratio^of^ionizable^lipid / non‐cationic^helper^lipid / sterol / polymer^ lipid^conjugate^is^20‐50 / 10‐40 / 38‐39.5 / 0.5‐2.0.^ 34.^The^lipid^nanoparticle^of^claim^1‐32,^wherein^the^molar^ratio^of^ionizable^lipid / non‐cationic^helper^ lipid / sterol / polymer^lipid^conjugate^is^20 / 40 / 38‐39.5 / 0.5‐2.0.^ 35.^The^lipid^nanoparticle^of^claim^33,^wherein^the^molar^ratio^of^ionizable^lipid / non‐cationic^helper^ lipid / sterol / polymer^lipid^conjugate^is^50 / 10 / 38‐39.5 / 0.5‐2.0.^ 36.^The^lipid^nanoparticle^of^any^one^of^claims^15‐35^wherein^the^nucleic^acid^is^RNA.^ 37.^The^lipid^nanoparticle^of^claim^36^wherein^the^nucleic^acid^is^mRNA.^ 38.^The^lipid^nanoparticle^of^claim^36^wherein^the^nucleic^acid^is^circular^RNA^(“circRNA”)^or^self‐ amplifying^RNA^(“saRNA”).^ 39.^The^lipid^nanoparticle^of^any^one^of^claims^1‐38,^wherein^the^cell^of^the^central^nervous^system^is^a^ glial^cell^selected^from^an^astrocyte^or^oligodendrocyte^or^an^ependymal^cell^of^the^cerebral^ventricles^ or^the^choroid^plexus.^ 40.^The^lipid^nanoparticle^of^claim^any^one^of^claims^1‐39,^wherein^the^lipid^nanoparticle^is^part^of^a^ pharmaceutical^formulation.^^41.^The^lipid^nanoparticle^of^any^one^of^claims^1‐40^for^use^in^treating^or^preventing^a^central^nervous^ system^disease,^disorder,^trauma^or^injury^in^the^subject.^ 42.^The^lipid^nanoparticle^of^claim^41,^wherein^the^central^nervous^system^disease,^disorder,^trauma^ or^injury^is^Gaucher^Disease^Type^1,^Gaucher^Disease^Type^2,^Gaucher^Disease^Type^3,^Parkinson’s^ Disease^and^related^synucleinopathies,^Alzheimer’s^Disease,^Lewy^Body^Dementia,^Multiple^Sclerosis,^ or^any^combination^thereof.^ 43.^A^method^of^treating,^preventing^or^diagnosing^a^central^nervous^system^disease,^disorder,^trauma^ or^injury^in^a^subject,^the^method^comprising:^contacting^a^lipid^nanoparticle^(LNP)^with^a^cell^of^the^ central^nervous^system^of^the^subject,^^ the^lipid^nanoparticle^comprising^at^least^one^nucleic^acid^encoding^a^polypeptide^capable^of^secretion^ from^the^cell^into^an^interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ 44.^The^method^of^claim^43^wherein^the^polypeptide^is^an^antibody^or^antigen‐binding^fragment.^ 45.^The^method^of^claim^43,^wherein^the^polypeptide^is^glucocerebrosidase^(GCase)^or^a^functional^ fragment^thereof.^ 46.^The^method^of^claim^43,^wherein^the^nucleic^acid^is^an^mRNA.^ 47.^The^method^of^claim^43,^wherein^the^nucleic^acid^is^a^circular^RNA^(“circRNA”)^or^a^self‐amplifying^ RNA^(“saRNA”).^ 48.^The^method^of^claim^43,^wherein^the^contacting^of^the^LNP^with^the^cell^results^in^the^least^one^ nucleic^acid^entering^the^cell^and^production^of^the^polypeptide^in^the^cell.^ 49.^The^method^of^claim^43,^wherein^subsequent^to^the^production^of^the^polypeptide,^the^polypeptide^ is^secreted^from^the^cell^to^the^interstitial^and / or^cerebrospinal^fluid^(CSF)^of^the^subject.^ 50.^The^method^of^claim^49,^wherein^the^secreted^polypeptide^is^present^in^the^CSF^of^the^subject^for^a^ period^of^at^least^24^hours,^at^least^36^hours,^or^at^least^48^hours.^ 51.^The^method^of^claim^49,^wherein^the^secreted^polypeptide^is^present^in^the^CSF^of^the^subject^for^a^ period^of^at^least^1^week,^at^least^2^weeks,^at^least^3^weeks,^at^least^4^weeks,^at^least^6^weeks,^or^at^ least^8^weeks.^^ 52.^The^method^of^any^one^of^claims^50‐51,^wherein^the^secreted^polypeptide^is^present^in^the^CSF^of^ the^subject^at^a^first^concentration^at^a^first^time^point^and^at^a^second^concentration^at^a^later^second^ time^point.^ 53.^The^method^of^claim^52,^wherein^the^first^time^point^is^about^3^hours^after^contacting^the^LNP^with^ the^cell.^ 54.^The^method^of^claim^52,^wherein^the^second^time^point^is^about^48^hours^after^contacting^the^LNP^ with^the^cell.^ 55.^The^method^of^claim^52,^wherein^the^second^concentration^is^at^least^30%,^at^least^40%,^at^least^50%,^at^least^60%,^at^least^70%,^at^least^80%,^at^least^90%,^or^at^least^95%^of^the^first^concentration.^ 56.^The^method^of^claim^43‐55,^wherein^the^cell^of^the^central^nervous^system^is^selected^from^one^or^ more^of:^ ^(a)^a^glial^cell;^and^^ ^(b)^an^ependymal^cell.^ 57.^The^method^of^claim^56,^wherein^the^glial^cell^is^selected^from^an^astrocyte,^an^oligodendrocyte,^or^ an^oligodendrocyte^precursor^cell.^ 58.^The^method^of^claim^57,^wherein^the^ependymal^cell^is^from^the^cerebral^ventricles^or^the^choroid^ plexus.^ 59.^The^method^of^any^one^of^^claims^43‐58,^wherein^the^polypeptide^is^diffusible^within^the^ interstitial^and / or^cerebrospinal^fluid^of^the^subject.^ 60.^The^method^of^any^one^of^^claims^43‐59,^wherein^the^lipid^nanoparticle^comprises^^ ^an^ionizable,^cationic,^amino^lipid.^ 61.^The^method^of^claim^43‐60,^wherein^the^LNP^comprises^a^generally^cylindrically‐shaped^lipid.^^ 62.^The^method^of^claim^58^wherein^the^cylindrically‐shaped^lipid^comprises^ distearoylphosphatidylcholine^(DSPC)^or^dioleoylphosphatidylglycerol^(DOPG)^or^sphingomyelin.^ 63.^The^method^of^any^one^of^claims^43‐62,^wherein^the^LNP^comprises^a^phosphatidylethanolamine^ content^that^is^less^than^2^mol%.^ 64.^The^method^of^any^one^of^claims^43‐63^wherein^the^LNP^further^comprises^a^sterol.^ 65.^The^method^of^any^one^of^claims^43‐64^wherein^the^LNP^further^comprises^a^polymer^lipid^ conjugate.^ 66.^The^method^of^any^one^of^claims^43‐65,^wherein^the^LNP^comprises^(a)^an^ionizable^lipid;^(b)a^ cylindrical^lipid^(c)^a^sterol;^and^(d)^a^polymer^lipid^conjugate^in^a^molar^ratio^of^20‐50 / 10‐40 / 38‐ 39.5 / 0.5‐2.0.^ 67.^^The^method^of^claim^66,^wherein^the^molar^ratio^of^ionizable,^amino,^cationic^lipid / non‐cationic^ helper^lipid / sterol / polymer^lipid^conjugate^is^20 / 40 / 38‐39.5 / 0.5‐2.0^or^50 / 10 / 38‐39.5 / 0.5‐2.0.^ 68.^The^method^of^any^one^of^t^claims^43‐67,^wherein^contacting^of^the^LNP^with^the^cell^comprises^ administering^the^LNP^via^injection^via^an^intracerebroventricular^(ICV)^or^lumbar^intrathecal^route,^ cisterna^magna^route^or^via^a^catheter^to^the^central^nervous^system^of^the^subject.^ 69.^The^method^of^any^one^of^claims^43‐68,^wherein^the^polypeptide,^antibody^or^antigen‐binding^ fragment^further^comprise^a^secretion^signal.^ 70.^The^method^of^any^one^of^claims^43‐69,^wherein^the^at^least^one^nucleic^acid^comprises^a^nucleic^ acid^encoding^a^heavy^chain^and^a^nucleic^acid^encoding^a^light^chain.^ 71.^The^method^of^any^one^of^^claims^43‐70,^wherein^the^at^least^one^nucleic^acid^comprises^a^nucleic^acid^that^encodes^more^than^one^polypeptide.^ 72.^The^method^of^any^one^of^^claims^43‐71,^wherein^the^polypeptide^comprises^trastuzumab^ 73.^The^method^of^any^one^of^^claims^43‐72,^wherein^the^central^nervous^system^disease,^disorder,^ trauma^or^injury^is^Gaucher^Disease^Type^1,^Gaucher^Disease^Type^2,^Gaucher^Disease^Type^3,^ Parkinson’s^Disease^and^related^synucleinopathies,^Alzheimer’s^Disease,^Lewy^Body^Dementia,^ Multiple^Sclerosis,^or^any^combination^thereof.^ 74.^The^method^of^any^one^of^claims^43‐73,^wherein^the^cell^of^the^CNS^comprises^neurons;^ interneurons;^glia;^astrocytes;^oligodendrocytes;^microglia;^ependymal^cells;^radial^glia;^neurovascular^ cells^including^endothelial^cells,^pericytes,^and^smooth^muscle^cells;^progenitor^cells;^and / or^ meningeal^cells.^^ 75.^The^method^of^any^one^of^claims^43‐74,^wherein^the^contacting^is^in^vitro^or^in^vivo.^ 76.^The^use^of^the^lipid^nanoparticle^of^any^one^of^claims^1‐42^for^the^treatment^of^central^nervous^ system^disease.^ 77.^Use^of^the^lipid^nanoparticle^of^any^one^of^claims^1‐42^^for^the^manufacture^of^a^medicament^for^a^ central^nervous^system^disease,^wherein^the^central^nervous^system^disease,^disorder,^trauma^or^ injury^is^Gaucher^Disease^Type^1,^Gaucher^Disease^Type^2,^Gaucher^Disease^Type^3,^Parkinson’s^ Disease^and^related^synucleinopathies,^Alzheimer’s^Disease,^Lewy^Body^Dementia,^Multiple^Sclerosis,^ or^any^combination^thereof.^ 78.^A^method^of^treating,^preventing^or^diagnosing^a^central^nervous^system^disease,^disorder,^trauma^ or^injury^in^a^subject,^the^method^comprising:^^contacting^a^lipid^nanoparticle^(LNP)^of^any^one^of^ claims^1‐42^with^a^cell^of^the^central^nervous^system^of^the^subject,^the^lipid^nanoparticle^comprising^ at^least^one^nucleic^acid^encoding^a^polypeptide^capable^of^secretion^from^the^cell^into^an^interstitial^ and / or^cerebrospinal^fluid^of^the^subject.^ 79.^The^use^of^a^lipid^nanoparticle^of^any^one^of^claims^1^to^42^of^for^inducing^exogenous^expression^of^ GCase^in^a^glial^cell^of^a^subject.^ 80.^The^method^of^claims^43‐78^^where^the^contacting^^of^the^LNP^with^the^cell^is^accomplished^by^ administration^into^the^lateral^ventricle^of^one^or^both^cerebral^hemispheres.^ 81.^A^glial^cell^comprising^the^nucleic^acid^of^any^one^of^claims^1‐15.^ 82.^A^glial^cell^expressing^exogenous^mRNA^encoding^GCase.^^ 83.^The^lipid^nanoparticle^of^claims^1‐42^wherein^the^antibody^is^trastuzumab.^ 84.^A^glial^cell^expressing^exogenous^mRNA^encoding^trastuzumab.^ 85.^The^lipid^nanoparticle^of^claim^1,^wherein^the^first^time^point^is^about^3^hours,^and^wherein^the^ second^time^point^is^about^24^hours^after^contacting^of^the^LNP^with^the^cell,^and^wherein^the^second^concentration^higher^than^the^first^concentration.^^ ^

Images