The Onpattro story and the clinical translation
`of nanomedicines containing nucleic acid-based
`drugs
`
`The regulatory approval of Onpattro, a lipid nanoparticle-based short interfering RNA drug for the treatment of
`polyneuropathies induced by hereditary transthyretin amyloidosis, paves the way for clinical development of many
`nucleic acid-based therapies enabled by nanoparticle delivery.
`Akin Akinc, Martin A. Maier, Muthiah Manoharan, Kevin Fitzgerald, Muthusamy Jayaraman,
`Scott Barros, Steven Ansell, Xinyao Du, Michael J. Hope, Thomas D. Madden, Barbara L. Mui,
`Sean C. Semple, Ying K. Tam, Marco Ciufolini, Dominik Witzigmann, Jayesh A. Kulkarni, Roy van der Meel
`and Pieter R. Cullis
`
`+
`
`+
`
`Endosomal
`membrane
`
`+ –
`
`–
`
`–+
`
`–
`
`+
`–
`
`+
`
`–
`
`pH ࣈ 5
`
`+
`–
`
`ApoE-binding
`cell surface receptor
`(e.g. LDLR)
`
`+
`
`+
`
`+
`
`P
`
`RISC
`
`Lipoprotein
`particle
`
`Recruitment
`of ApoE
`
`ApoE
`
`Fenestration
`
`pH 7.4
`
`Blood compartment
`
`Space of Disse
`
`Hepatocyte
`
`Fig. 1 | Integrated model of lipid nanoparticle (LNP)-mediated delivery of siRNA to hepatocytes in vivo.
`Key steps include the dissociation of PEG-lipids from the particle surface, recruitment of endogenous
`ApoE to the LNP surface, trafficking of LNPs through fenestrated endothelium and binding to low density
`lipoprotein receptors and other ApoE-binding receptors on hepatocytes, internalization of LNPs via
`endocytosis, protonation of the ionizable lipid due to the low pH in the endosome, interaction of the
`protonated ionizable lipid with negatively charged endogenous lipids, which results in the destabilization
`of the endosomal membrane, and release of siRNA into the cytoplasm, where it can engage with the
`RNAi machinery. RISC, RNA-induced silencing complex. LDLR, low density lipoprotein receptor.
`
`target tissue; by contrast, LNP formulations
`of nucleic acid-based drugs must also
`facilitate intracellular delivery of these
`macromolecules into target cells.
`Here, we describe the successful
`preclinical development and clinical
`translation of patisiran (trade name
`Onpattro), which is an LNP formulation of
`
`siRNA for the treatment of polyneuropathies
`resulting from the hereditary disease
`transthyretin-mediated amyloidosis
`(hATTR). This drug acts by inhibiting the
`synthesis of the transthyretin (TTR) protein
`in the liver. The positive results of a global
`phase 3 study3 resulted in FDA approval of
`Onpattro in August 2018. The success of
`
`Nanomedicines resulting from the
`
`application of nanotechnology to
`medicine are having an increasing
`impact on the treatment of disease. This
`applies particularly to nanomedicines using
`lipid nanoparticle (LNP) drug delivery
`systems as there are now more than ten
`US Food and Drug Administration (FDA)
`approved pharmaceuticals employing
`LNPs to deliver drugs to disease sites
`(Table 1). Most of these nanomedicines
`are formulations of cancer drugs that offer
`the benefits of reduced toxicity and/or
`enhanced efficacy compared to the ‘free’
`drug1. Due to the clinical success of LNP-
`based drug delivery systems, we now have
`a good understanding of the requirements
`for successful clinical translation of LNP
`systems for delivery of small molecules.
`Translational criteria include a size
`range of 100 nm or less, highly efficient
`encapsulation techniques, a low surface
`charge, robust, scalable manufacturing
`processes and adequate product stability2.
`It is of great interest to extend LNP
`technology to delivery of nucleic acid-
`based drugs, such as short interfering
`RNA (siRNA), messenger RNA (mRNA)
`and gene editing constructs. Unmodified
`nucleic acid-based drugs face particular
`delivery problems, because they are readily
`broken down in biological fluids, do not
`accumulate in target tissues and cannot
`penetrate into target cells even if they get
`to the desired tissues. Unfortunately, many
`of the techniques developed for generating
`clinically viable LNP formulations of
`small molecule drugs cannot be applied to
`nucleic acid polymers owing to their large
`size and negative charge. Further, LNP
`formulations of small molecule drugs have
`only to release drug cargo after arrival in the
`
`1084
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`comment
`
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`

`

`Table 1 | LNP drugs that have received regulatory approval from the FDA or eMA
`Name
`encapsulated drug
`Indication
`Ambisome
`Amphotericin b
`Fungal infections Leishmaniasis
`
`company
`Gilead
`
`Johnson& Johnson
`
`year approved
`1990 (Europe)
`1997 (USA)
`1995 (USA)
`Kaposi’s sarcoma
`1999 (USA)
`Ovarian cancer
`2003 (Europe)
`breast Cancer
`1996 (Europe), 1996 (USA) Galen
`Kaposi’s sarcoma
`Daunorubicin
`2000 (Europe)
`Cephalon
`breast cancer
`Doxorubicin
`1995 (USA)
`Enzon
`Aspergillosis
`Amphotericin b
`1996 (USA)
`Intermune
`Invasive aspergillosis
`Amphotericin b
`2000 (USA)
`QLT
`Wet macular degeneration
`Verteporfin
`2012 (USA)
`Spectrum Pharma
`Acute lymphoblastic leukemia
`Vincristine
`2015 (USA)
`Ipsen biopharma
`metastatic pancreatic cancer
`Irinotecan
`2017 (USA)
`Jazz Pharma
`Acute lymphocytic leukemia
`Daunorubicin, Cytarabine
`siRNA targeting transthyretin Transthyretin induced amyloidosis (hATTR) 2018 (USA), 2018 (Europe) Alnylam Pharmaceuticals
`
`Doxorubicin
`
`Doxil/Caelyx
`
`DaunoXome
`myocet
`Abelcet
`Amphotec
`Visudyne
`marqibo
`Onyvide
`Vyxeos
`Onpattro
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`% Residual factor VII
`
`2nd generation LNP
`(DLin-MC3-DMA)
`
`1st generation LNP
`(DLinDMA)
`
`siRNA into LNPs can then be achieved by
`rapid mixing of lipids in ethanol with siRNA
`in aqueous media at low pH (pH 4) using
`a readily scalable manufacturing process.
`These LNP systems, which have a novel
`‘solid core’ structure5, display low surface
`charge at physiological pH and are relatively
`non-toxic and non-immunogenic.
`Diameters of 100 nm or less could be
`achieved by incorporating polyethylene
`glycol (PEG)-lipids that associate with the
`surface of the LNP. LNP size can then be
`regulated by adjusting the proportion of
`surface PEG lipid to core lipid to generate
`sizes over the range 20–100 nm6. The
`presence of a PEG coating on the LNP
`surface has the disadvantage of inhibiting
`interactions with target cells and thus
`reducing intracellular delivery. This problem
`was overcome by using PEG-lipids with
`relatively short C14 acyl chains. Such PEG-
`lipids remain associated with the particles
`during formulation and under storage
`conditions; however, in the presence of a
`lipid sink (for example, lipoprotein particles
`in plasma), the PEG-lipids can exchange
`out of the LNP, thereby generating an
`unshielded particle that can engage with
`target cells to enable uptake7.
`The development of LNP siRNA systems
`with high loading efficiencies, defined size
`and low surface charge satisfied the basic
`criteria for clinical potential; however, the
`potency of these systems for gene silencing
`in hepatocytes remained to be characterized
`and optimized. As the in vitro potency of an
`LNP nanomedicine rarely correlates with
`in vivo performance, we moved directly to
`an in vivo model to optimize gene silencing
`properties. LNPs containing siRNA against
`factor VII (FVII) were administered to mice
`
`0
`0.0001
`
`0.001
`
`0.01
`
`0.1
`FVII siRNA dose (mg kg–1)
`
`1
`
`10
`
`Fig. 2 | LNP siRNA systems containing 2nd generation ionizable aminolipids exhibit greatly improved
`potency for silencing factor VII (FVII) in the liver. The data presented shows dose-dependent silencing
`of FVII following i.v. injection of LNP encapsulating siRNA against FVII in a mouse model. 2nd generation
`LNP containing heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-mC3-DmA)
`are more than two orders of magnitude more potent than 1st generation LNP containing 1,2-dilinoleyl-
`N,N-dimethyl-3-aminopropane (DLinDmA).
`
`Onpattro heralds the arrival of a new class of
`medicines based on nucleic acid polymers.
`In particular, subsequent studies of closely
`related LNP systems containing much larger
`mRNA cargos indicate that LNP delivery
`technology can potentially enable most
`forms of nucleic acid-based therapies.
`
`Preclinical development
`The basic features required of an LNP
`siRNA system with the potential for clinical
`translation include efficient encapsulation
`of siRNA into an LNP with low surface
`charge, a diameter of 100 nm or less and the
`ability to deliver encapsulated siRNA to the
`cytoplasm of hepatocytes in vivo following
`
`intravenous administration. With regard to
`encapsulation, nucleic acid polymers can
`be readily associated with lipidic particles
`containing permanently positively charged
`lipids; however, such positively charged
`systems induce pronounced toxicity in vivo
`due to immune activation (activation of
`complement and coagulation pathways
`as well as cytokine stimulation) and
`cytotoxicity. To circumvent this problem,
`we developed ionizable cationic lipids
`that possess an amine function with an
`acid dissociation constant (pKa) of ~6.5
`(ref. 4). These lipids are positively charged
`at acidic pH values, but nearly neutral at
`physiological pH. Efficient encapsulation of
`
`NAtuRe NANotechNoLogy | VOL 14 | DECEmbER 2019 | 1084–1087 | www.nature.com/naturenanotechnology
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`

`

`***
`
`10
`
`15
`
`*
`
`25
`
`30
`
`35
`
`40 45
`
`50 55 60 65 70
`
`*
`20
`
`Study day
`
`ALN-TTR02 dose groups (mg kg–1)
`
`*
`*
`*
`*
`
`5
`0
`siRNA
`dose
`
`100
`
`40
`
`20
`
`0
`
`−20
`
`−40
`
`−60
`
`−80
`
`−100
`
`Geometric mean percent
`
`TTR knockdown
`
`b
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`0
`siRNA
`dose
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Study day
`
`ALN-TTR01 dose groups (mg kg–1)
`
`80
`
`60
`
`40
`
`20
`
`0
`
`–20
`
`–40
`
`–60
`
`−80
`
`−100
`
`Geometric mean percent
`
`TTR knockdown
`
`a
`
`Placebo (N = 8)
`0.01 (N = 3)
`
`0.03 (N = 3)
`0.10 (N = 3)
`
`0.20 (N = 3)
`0.40 (N = 3)
`
`0.70 (N = 3)
`1.00 (N = 6)
`
`Placebo (N = 4)
`0.15 (N = 3)
`
`0.01 (N = 3)
`0.3 (N = 3)
`
`0.05 (N = 3)
`0.5 (N = 1)
`
`Control siRNA
`0.4 (N = 6)
`
`Fig. 3 | Phase I clinical trials of ALN-ttR01 and ALN-ttR02 (patisiran). a,b, mean percent serum transthyretin (TTR) knockdown at the indicated time points,
`as compared with the baseline in groups of patients receiving either placebo or increasing doses of ALN-TTR01 (a) and in healthy subjects receiving either
`placebo or increasing doses of ALN-TTR02 (patisiran) (b). The error bars indicate 95% confidence intervals. Data from ref. 13.
`
`to silence the FVII gene in hepatocytes,
`providing a convenient assay to optimize
`gene silencing potency in vivo. Initial work
`showed that LNP siRNA systems containing
`the ionizable lipid 1,2-dilinoleyl-N,N-
`dimethyl-3-aminopropane (DLinDMA)
`could silence genes in hepatocytes following
`intravenous (i.v.) administration8. However,
`the potency and tolerability of these LNP
`siRNA systems was not sufficient to warrant
`clinical development and a search for more
`active formulations commenced focusing
`primarily on the ionizable lipid component.
`A first breakthrough was reached with
`the development of the ionizable lipid
`DLinKC2DMA9, which substantially
`improved the potency and tolerability of
`the LNP, leading to an extensive research
`programme aimed at achieving ever
`more potent ionizable cationic lipids. The
`pharmacodynamics, pharmacokinetics
`and safety of promising formulations were
`evaluated in rodents, and lead candidates
`were then tested in non-human primates
`(NHPs). More than 300 ionizable lipids
`were designed and synthesized, leading
`to the identification of structure–activity
`relationships10. Notably, a remarkable
`dependence of LNP siRNA gene silencing
`potency on the acid dissociation constant
`(pKa) of the ionizable cationic lipid was
`found, with an optimum around pKa ≈ 6.4.
`Deviation from this pKa by as little as 0.5
`units could reduce potency by 100-fold or
`more10. This pKa optimum likely reflects
`the required balance between a low LNP
`
`surface charge to avoid rapid clearance in
`the circulation and a positive charge on the
`ionizable lipids to enable escape out of the
`acidic endosome following endocytosis.
`Positively charged lipids interact with
`negatively charged lipids to disrupt
`bilayer membranes11, which is a probable
`requirement for breaking out of endosomes
`and thus, for cytoplasmic delivery of siRNA.
`The remarkable affinity of these LNPs
`for the liver, in particular for hepatocytes,
`was found to be facilitated by the adsorption
`of apolipoprotein E (ApoE) on the surface
`of the LNPs following i.v. administration.
`The particle-associated ApoE acts as a
`highly effective targeting ligand by binding
`to lipoprotein receptors on the surface of
`hepatocytes, thereby triggering uptake into
`hepatocytes by endocytosis12. An integrated
`working model of LNP-mediated delivery of
`siRNA was then developed to describe the
`key steps of the LNP journey, from the site
`of administration to the release of the siRNA
`payload into the cytoplasm of hepatocytes
`(Fig. 1). This improved mechanistic
`understanding and predictability of lipid
`activity enabled the discovery of increasingly
`potent ionizable lipids, among which
`heptatriaconta-6,9,28,31-tetraen-19-yl-4-
`(dimethylamino) butanoate, later termed
`DLinMC3DMA (or simply MC3), exhibited
`an improvement in potency of more than
`two orders of magnitude compared to the
`benchmark DLinDMA formulation (Fig. 2).
`After confirmation of potent TTR
`silencing in NHPs, the MC3 formulation
`
`containing a human TTR-targeting
`siRNA was transitioned into preclinical
`development as ALN-TTR02 (later known
`as patisiran). Repeat-dose toxicology in rats
`and NHPs demonstrated a substantially
`improved therapeutic index compared to the
`first generation LNP.
`
`clinical development
`The translation of LNP-enabled siRNA
`systems for the treatment of hATTR
`amyloidosis in humans proceeded in two
`stages. The first generation DLinDMA-
`based formulation was evaluated in
`a placebo-controlled phase 1 trial to
`determine safety and efficacy of ALN-
`TTR01 after administration of a single
`dose, ranging from 0.01 to 1 mg kg–1. The
`study provided a number of key insights:
`(1) NHP studies seemed to provide a
`reasonable prediction of human efficacy
`(approximately 50% mean TTR reduction
`observed in NHPs at 1 mg kg–1); (2) ALN-
`TTR01 showed an encouraging safety profile
`with no drug-related serious adverse events
`or discontinuations or mild-to-moderate
`infusion-related reactions in a subset of
`participants; and (3) a single dose of ALN-
`TTR01 at 1 mg kg–1 led to a mean reduction
`in serum TTR levels of 38% compared to the
`placebo (Fig. 3a), with one patient achieving
`a substantial TTR reduction of >80%.
`These results validated the RNAi approach,
`the siRNA and the LNP platform in a real
`patient setting for the first time. Based on
`these results, the MC3-based 2nd generation
`
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`NAtuRe NANotechNoLogy | VOL 14 | DECEmbER 2019 | 1084–1087 | www.nature.com/naturenanotechnology
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`
`

`

`LNP (ALN-TTR02, patisiran) was advanced
`to the clinic in a first-in-human phase
`1 trial13. As predicted by the preclinical
`studies, patisiran showed improved clinical
`activity compared to ALN-TTR01 with
`rapid, robust and durable suppression of
`TTR levels of >80%, compared to the
`placebo at doses between 0.15 and
`0.5 mg kg–1 (Fig. 3b).
`The encouraging efficacy and safety
`profile observed in the phase 1 study paved
`the way for further clinical development,
`culminating in the randomized, double-
`blinded, placebo-controlled phase 3
`APOLLO study3 and finally, in the
`regulatory approval of Onpattro in the US
`and EU, with the potential for approval in
`other jurisdictions.
`
`Future prospects
`The clinical development pathway followed
`by Onpattro paves the way for the clinical
`translation of LNP nanomedicines
`containing nucleic acid-based drugs to
`enable many novel therapeutics based
`on silencing or expressing target genes.
`The ethanol-dilution rapid mixing
`manufacturing process employing ionizable
`cationic lipids can be readily extended to
`encapsulate much larger negatively charged
`molecules such as mRNA14,15 and effective
`transfection has been achieved in a variety
`of tissues in addition to the liver16. LNP
`systems containing mRNA show promise to
`target and use the liver as a bioreactor for
`the production of therapeutic proteins, such
`as monoclonal antibodies17 and hormones18
`following i.v. administration. Alternatively,
`when administered by intradermal or
`intramuscular routes, LNP mRNA systems
`provide highly effective vaccines for
`infectious diseases such as the Zika virus19
`or influenza virus20. Finally, LNPs containing
`
`mRNA coding for programmable nucleases
`show considerable potential for gene editing
`in vivo21,22. Challenges remain, including
`achieving improved site-specific transfection
`as well as improving our ability to transfect
`extrahepatic tissues. However, the rapid
`advances of recent years suggest that it is just
`a matter of time before these challenges are
`overcome.
`❐
`
`Akin Akinc1, Martin A. Maier1,
`Muthiah Manoharan1, Kevin Fitzgerald1,
`Muthusamy Jayaraman1, Scott Barros1,
` Steven Ansell2, Xinyao Du2, Michael J. Hope2,
`Thomas D. Madden2, Barbara L. Mui2,
`Sean C. Semple2, Ying K. Tam2,
`Marco Ciufolini3, Dominik Witzigmann 
`Jayesh A. Kulkarni3, Roy van der Meel 
` 3,4*
`and Pieter R. Cullis 
`1Alnylam Pharmaceuticals, Cambridge, MA, USA.
`2Acuitas Therapeutics, Vancouver, BC, Canada.
`3University of British Columbia, Vancouver, BC,
`Canada. 4NanoMedicines Innovation Network,
`University of British Columbia, Vancouver, BC,
`Canada.
`*e-mail: pieterc@mail.ubc.ca
`
` 3,
`
` 3
`
`Published online: 4 December 2019
`https://doi.org/10.1038/s41565-019-0591-y
`
`References
` 1. Allen, T. M. & Cullis, P. R. Liposomal drug delivery systems:
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` 18. Thess, A. et al. Sequence-engineered mRNA without chemical
`nucleoside modifications enables an effective protein therapy in
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` 19. Pardi, N. et al. Zika virus protection by a single low-dose
`nucleoside-modified mRNA vaccination. Nature 543,
`248–251 (2017).
` 20. Pardi, N. et al. Nucleoside-modified mRNA immunization
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`
`Competing interests
`A.A., M.A.M., M.M., K.F., M.J. and S.B. are employees
`of Alnylam Pharmaceuticals. S.A., X.D., M.J.H., T.D.M.,
`B.L.M., S.C.S. and Y.K.T. are employees of Acuitas. P.R.C.
`has financial holdings in Acuitas.
`
`NAtuRe NANotechNoLogy | VOL 14 | DECEmbER 2019 | 1084–1087 | www.nature.com/naturenanotechnology
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`1087
`
`comment
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