l e t t e r s
`
`rational design of cationic lipids for sirNA delivery
`Sean C Semple1,6, Akin Akinc2,6, Jianxin Chen1,5, Ammen P Sandhu1, Barbara L Mui1,5, Connie K Cho1,
`Dinah W Y Sah2, Derrick Stebbing1, Erin J Crosley1, Ed Yaworski1, Ismail M Hafez3, J Robert Dorkin2, June Qin2,
`Kieu Lam1, Kallanthottathil G Rajeev2, Kim F Wong3, Lloyd B Jeffs1, Lubomir Nechev2, Merete L Eisenhardt1,
`Muthusamy Jayaraman2, Mikameh Kazem3, Martin A Maier2, Masuna Srinivasulu4, Michael J Weinstein2,
`Qingmin Chen2, Rene Alvarez2, Scott A Barros2, Soma De2, Sandra K Klimuk1, Todd Borland2,
`Verbena Kosovrasti2, William L Cantley2, Ying K Tam1,5, Muthiah Manoharan2, Marco A Ciufolini4,
`Mark A Tracy2, Antonin de Fougerolles2, Ian MacLachlan1, Pieter R Cullis3, Thomas D Madden1,5 & Michael J Hope1,5
`
`We adopted a rational approach to design cationic lipids for
`use in formulations to deliver small interfering RNA (siRNA).
`Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-
`dimethylaminopropane (DLinDMA), a key lipid component of
`stable nucleic acid lipid particles (SNALP) as a benchmark,
`we used the proposed in vivo mechanism of action of ionizable
`cationic lipids to guide the design of DLinDMA-based lipids
`with superior delivery capacity. The best-performing lipid
`recovered after screening (DLin-KC2-DMA) was formulated
`and characterized in SNALP and demonstrated to have
`in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents
`and 0.1 mg/kg in nonhuman primates. To our knowledge, this
`represents a substantial improvement over previous reports of
`in vivo endogenous hepatic gene silencing.
`
`A key challenge in realizing the full potential of RNA interference
`(RNAi) therapeutics is the efficient delivery of siRNA, the molecules
`that mediate RNAi. The physicochemical characteristics of siRNA—
`high molecular weight, anionic charge and hydrophilicity—prevent
`passive diffusion across the plasma membrane of most cell types.
`Therefore, delivery mechanisms are required that allow siRNA to
`enter cells, avoid endolysosomal compartmentalization and localize
`in the cytoplasm where it can be loaded into the RNA-induced
`
` silencing complex. To date, formulation in lipid nanoparticles (LNPs)
` represents one of the most widely used strategies for in vivo delivery
`of siRNA1,2. LNPs represent a class of particles comprised of different
`lipid compositions and ratios as well as different sizes and structures
`formed by different methods. A family of LNPs, SNALP3–6, is charac-
`terized by very high siRNA-encapsulation efficiency and small,
`uniformly sized particles, enabled by a controlled step-wise dilution
`methodology. LNPs, including SNALP, have been successfully used to
`silence therapeutically relevant genes in nonhuman primates6–8 and
`are currently being evaluated in several clinical trials.
`An empirical, combinatorial chemistry–based approach recently
`identified novel materials for use in LNP systems7. A key feature of
`this approach was the development of a one-step synthetic strategy
`that allowed the rapid generation of a diverse library of ~1,200 com-
`pounds. This library was then screened for novel materials capable
`of mediating efficient delivery of siRNA in vitro and in vivo. Here, we
`instead used a medicinal chemistry (that is, structure-activity relation-
`ship) approach, guided by the putative in vivo mechanism of action
`of ionizable cationic lipids, for rational lipid design. Specifically,
`we hypothesized that after endocytosis, the cationic lipid interacts
`with naturally occurring anionic phospholipids in the endosomal
`membrane, forming ion pairs that adopt nonbilayer structures and
`disrupt membranes (Fig. 1)9–12. We previously advanced the concept
`
`Figure 1 Proposed mechanism of action for membrane disruptive effects of
`cationic lipids and structural diagram of DLinDMA divided into headgroup,
`linker and hydrocarbon chain domains. In isolation, cationic lipids and
`endosomal membrane anionic lipids such as phosphatidylserine adopt a
`cylindrical molecular shape, which is compatible with packing in a bilayer
`configuration. However, when cationic and anionic lipids are mixed together,
`they combine to form ion pairs where the cross-sectional area of the combined
`headgroup is less than that of the sum of individual headgroup areas in
`isolation. The ion pair therefore adopts a molecular ‘cone’ shape, which
`promotes the formation of inverted, nonbilayer phases such as the hexagonal
`HII phase illustrated. Inverted phases do not support bilayer structure and are
`associated with membrane fusion and membrane disruption9,21.
`
`+
`
`–
`
`+ –
`
`Cylindrical shape supports
`bilayer structure
`
`Cone shape disrupts
`bilayer structure
`
`Headgroup
`
`Linker
`
`Hydrocarbon
`chains
`
`Bilayer
`
`Hexagonal HII
`
`DLinDMA
`
`1Tekmira Pharmaceuticals, Burnaby, British Columbia, Canada. 2Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA. 3Department of Biochemistry and
`Molecular Biology and 4Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada. 5Present address: Alcana Technologies,
`Vancouver, British Columbia, Canada. 6These authors contributed equally to this work. Correspondence should be addressed to S.C.S. (ssemple@tekmirapharm.com)
`or A.A. (aakinc@alnylam.com).
`
`Received 16 September 2009; accepted 17 December 2009; published online 17 January 2010; doi:10.1038/nbt.1602
`
`172
`
`VOLUME 28 NUMBER 2 FEBRUARY 2010 nature biotechnology
`
`© 2010 Nature America, Inc. All rights reserved.
`
`ARBUTUS - EXHIBIT 2021
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation - IPR2019-00554
`
`

`

`l e t t e r s
`
`Figure 2 In vivo evaluation of novel cationic lipids. (a) Silencing
`activity of DLinDAP (), DLinDMA (), DLin-K-DMA () and DLin-
`KC2-DMA (•) screening formulations in the mouse Factor VII model.
`All LNP-siRNA systems were prepared using the preformed vesicle
`(PFV) method and were composed of ionizable cationic lipid, DSPC,
`cholesterol and PEG-lipid (40:10:40:10 mol/mol) with a Factor
`VII siRNA/total lipid ratio of ~0.05 (wt/wt). Data points are expressed
`as a percentage of PBS control animals and represent group mean
`(n = 5) ± s.d., and all formulations were compared within the same
`study. (b) Influence of headgroup extensions on the activity of
`DLin-K-DMA. DLin-K-DMA () had additional methylene groups added
`between the DMA headgroup and the ketal ring linker to generate
`DLin-KC2-DMA (•), DLin-KC3-DMA () and DLin-KC4-DMA (). The
`activity of PFV formulations of each lipid was assessed in the mouse
`Factor VII model. Data points are expressed as a percentage of PBS
`control animals and represent group mean (n = 4) ± s.d. (c) Chemical
`structures of novel cationic lipids.
`
`120
`100
`80
`60
`40
`20
`
`00
`
`b
`
`protein (%)
`
`Relative serum factor VII
`
`0.1
`.01
`1
`10
`Factor VII siRNA dose (mg/kg)
`
`120
`100
`80
`60
`40
`20
`
`00
`
`protein (%)
`
`a
`
`Relative serum factor VII
`
`0.1
`.01
`1
`10
`100
`Factor VII siRNA dose (mg/kg)
`
`O
`
`R
`
`Me2N
`
`O
`R
`
`O
`
`O
`DLinDAP
`
`RR
`
`O O
`
`Me2N
`
`R R
`
`O O
`
`Me2N
`
`R
`R
`
`O O
`
`Me2N
`
`O R
`R
`
`O
`
`DLinDMA
`
`Me2N
`
`R
`R
`
`O O
`
`c
`
`Me2N
`
`DLin-K-DMA
`
`DLin-KC2-DMA
`
`DLin-KC3-DMA
`
`DLin-KC4-DMA
`
`R =
`
`of using ionizable cationic lipids with pKas < pH 7.0 to efficiently
`formulate nucleic acids at low pH and maintaining a neutral or low
`cationic surface charge density at pH 7.4 (ref. 13). This strategy should
`provide better control of the circulation properties of these systems
`and reduce nonspecific disruption of plasma membranes. As posi-
`tive charge density is minimal in the blood but increases substan-
`tially in the acidic environment of the endosome, this should activate
`the membrane-destabilizing property of the LNP. Although these
`attributes may account for the activity of these systems upon inter-
`nalization by hepatocytes, they do not necessarily explain the high
`levels of hepatic biodistribution observed for many LNPs, including
`SNALP. Although these LNPs do not specifically include a targeting
`ligand to direct them to hepatocytes after systemic administration,
`it is possible that these LNPs associate with one or more proteins in
`plasma that may promote hepatocyte endocytosis.
`The ionizable cationic lipid DLinDMA has proven to be highly
`effective in SNALP, has been extensively tested in rodents and non-
`human primates, and is now being evaluated in human clinical
`trials. Therefore, we selected it as the starting point for the design
`and synthesis of novel lipids. We chose the mouse Factor VII model7,
`as the primary in vivo screening system to assess functional LNP-
`mediated delivery to hepatocytes. Briefly, C57BL/6 mice received
`a single dose of LNP-formulated Factor VII siRNA through bolus
`tail vein injection and serum was collected from animals 24 h after
`administration to analyze Factor VII protein level. The initial screen-
`ing of LNP-siRNA systems was conducted using LNPs prepared by
`a preformed vesicle method14 and composed of ionizable cationic
`lipid, distearoylphosphatidylcholine (DSPC), cholesterol and PEG-
`lipid (40:10:40:10 mol/mol), with a Factor VII siRNA/total lipid ratio
`of ~0.05 (wt/wt). Although not a bilayer-destabilizing lipid, a small
`amount of phosphatidylcholine was incorporated into the LNP to
`help stabilize the LNP both during formulation and while it was in
`circulation. A short acyl chain PEG-lipid was incorporated into the
`LNP to control particle size during formulation, but is designed to
`leave the LNP rapidly upon intravenous injection. As our goal was
`to identify novel ionizable cationic lipids for use in LNPs, we aimed
`to minimize other effects by using a single robust composition and
`set of formulation conditions suitable for all novel lipids tested. The
`preformed vesicle method employing the composition listed above
`provides a convenient platform for such testing, but uses a different
`formulation process, a different lipid composition and a different
`siRNA/lipid ratio than SNALP. The structure of DLinDMA can be
`divided into three main regions: the hydrocarbon chains, the linker
`and the headgroup (Fig. 1). A detailed structure-function study to
`
`investigate the impact of increasing the number of cis double bonds
`in the hydrocarbon chains found the linoleyl lipid containing two
`double bonds per hydrocarbon chain (DLinDMA) to be optimal15.
`We therefore maintained the linoleyl hydrocarbon chains present in
`DLinDMA as an element in our lipid design, and focused on optimiz-
`ing the linker and headgroup moieties.
`The linker region in a bilayer structure resides at the membrane
`interface, an area of transition between the hydrophobic membrane
`core and hydrophilic headgroup surface. Our approach to linker
`modification of DLinDMA involved introducing groups expected to
`exhibit different rates of chemical or enzymatic stability and to span
`a range of hydrophilicity. A variety of these rationally designed lipids
`were made, characterized and tested (Supplementary Syntheses 1 and
`Supplementary Table 1). LNPs based on the ester-containing lipid
`DLinDAP showed substantially reduced in vivo activity compared
`to LNPs based on the alkoxy-containing lipid DLinDMA (Fig. 2).
`Further, LNPs based on DLin-2-DMAP, a lipid with one alkoxy link-
`age and one ester linkage, yielded activity intermediate between
`DLinDAP- and DLinDMA-based LNPs (Supplementary Table 1).
`Although it is uncertain why the ester-containing lipids are consid-
`erably less active in vivo, we speculate that the diester lipid (DLinDAP)
`is relatively inactive because it is more readily hydrolyzed in vivo
`than the alkoxy analog (DLinDMA), and therefore, unable to either
`protect the siRNA adequately before release from the endosome
`and/or survive long enough in the endosome to disrupt the mem-
`brane. These hypotheses are being investigated. LNPs based on lipids
` containing carbamate or thioether linkages also resulted in dramati-
`cally reduced in vivo activity. Interestingly, the introduction of a ketal
`ring linker into DLinDMA resulted in LNPs that were ~2.5-fold more
`potent in reducing serum Factor VII protein levels relative to the
`DLinDMA benchmark, with an ED50 (that is, dose to achieve 50%
`gene silencing) of ~0.4 mg/kg versus 1 mg/kg, respectively (Fig. 2).
`Given the importance of positive charge in the mechanism-
`of-action hypothesis guiding the lipid design, the effects of structural
`changes in the amine-based headgroup were investigated in the con-
`text of DLin-K-DMA as the new benchmark lipid. A series of head-
`group modifications were made, characterized and tested to explore
`the effects of size, acid-dissociation constant and number of ionizable
`groups (Supplementary Syntheses 2 and Supplementary Table 2).
`Piperazino, morpholino, trimethylamino or bis-dimethylamino modifi-
`cations tested were not better than the benchmark dimethylamino
`headgroup of DLin-K-DMA. As an additional parameter, the distance
`between the dimethylamino group and the dioxolane linker was varied
`by introducing additional methylene groups. This parameter can
`
`nature biotechnology VOLUME 28 NUMBER 2 FEBRUARY 2010
`
`173
`
`© 2010 Nature America, Inc. All rights reserved.
`
`

`

`l e t t e r s
`
`Cationic lipid
`
`In vivo ED50
`(mg/kg)
`
`Table 1 Biophysical parameters and in vivo activities of
`LNPs containing novel lipids
`LII to HII phase transition
`Apparent
`temperature (°C)b
`a
`lipid pKa
`6.8 ± 0.10
`~1
`27
`DLinDMA
`6.2 ± 0.05
`40–50
`26
`DLinDAP
`5.9 ± 0.03
`~0.4
`19
`DLin-K-DMA
`6.7 ± 0.08
`~0.1
`20
`DLin-KC2-DMA
`7.2 ± 0.05
`~0.6
`18
`DLin-KC3-DMA
`7.3 ± 0.07
`>3
`18
`DLin-KC4-DMA
`apKa values ± s.d. (n = 3 to 9). bLII to HII phase transition was measured at pH 4.8 in equi-
`molar mixtures with DSPS, using differential scanning calorimetric, repeat scans reproducible
`to within 0.1 °C.
`
`affect both the pKa of the amine headgroup as well as the distance
`and flexibility of the charge presentation relative to the lipid bilayer
`interface. Inserting a single additional methylene group into the head-
`group (DLin-KC2-DMA) produced a dramatic increase in potency
`relative to DLin-K-DMA. The ED50 for this lipid was ~0.1 mg/kg,
`making it fourfold more potent than DLin-K-DMA and tenfold more
`potent than the DLinDMA benchmark when compared head-to-head
`in the Factor VII model (Fig. 2a). Further extension of the tether with
`additional methylene groups, however, substantially decreased activ-
`ity, with an ED50 of ~0.6 mg/kg for DLin-KC3-DMA and >3 mg/kg
`for DLin-KC4-DMA (Fig. 2b).
`As changes in lipid design and chemistry may affect the pharmaco-
`kinetics, target tissue accumulation and intracellular delivery of LNP
`formulations, we investigated the relative importance of these para-
`meters on LNP activity at an early stage in this research program.
`Several of the novel lipids were incorporated into LNP-siRNA for-
`mulations containing cyanine dye (Cy3)-labeled siRNA. Plasma,
`liver and spleen levels of siRNA were determined at 0.5 and 3 h after
`injection at siRNA doses of 5 mg/kg, and the results are presented in
`Supplementary Table 3. In general, formulations that were the most
`active in the mouse Factor VII screens achieved the highest liver levels
`of siRNA at 0.5 h; however, delivery of siRNA to the target tissue was
`not the primary factor responsible for activity. This is supported by
`the observations that most formulations accumulated in the liver and
`spleen quite quickly and that some formulations with similar liver lev-
`els of siRNA had large differences in activity. Moreover, plasma phar-
`macokinetics alone did not predict activity. For example, although
`DLin-KC2-DMA and DLinDMA had virtually indistinguishable
`blood pharmacokinetic profiles in mice (data not shown), the activity
`of DLin-KC2-DMA in LNPs is approximately tenfold greater than the
`
`same formulation with DLinDMA. Taken together, these results led
`us to conclude that rapid target tissue accumulation was important,
`but not sufficient, for activity. Moreover, other parameters were more
`critical for maximizing the activity of LNP-siRNA formulations.
`Two important parameters underlying lipid design for SNALP-
`mediated delivery are the pKa of the ionizable cationic lipid and the
`abilities of these lipids, when protonated, to induce a nonbilayer
`(hexagonal HII) phase structure when mixed with anionic lipids.
`The pKa of the ionizable cationic lipid determines the surface
`charge on the LNP under different pH conditions. The charge state
`at physiologic pH (e.g., in circulation) can influence plasma pro-
`tein adsorption, blood clearance and tissue distribution behavior16,
`whereas the charge state at acidic pH (e.g., in endosomes) can influ-
`ence the ability of the LNP to combine with endogenous anionic
`lipids to form endosomolytic nonbilayer structures9. Consequently,
`the ability of these lipids to induce HII phase structure in mixtures
`with anionic lipids is a measure of their bilayer-destabilizing capacity
`and relative endosomolytic potential.
`The fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic
`acid (TNS), which exhibits increased fluorescence in a hydrophobic
`environment, can be used to assess surface charge on lipid bilayers.
`Titrations of surface charge as a function of pH can then be used
`to determine the apparent pKa of the lipid in the bilayer (hereafter
`referred to as pKa) of constituent lipids17. Using this approach, the
`pKa values for LNPs containing DLinDAP, DLinDMA, DLin-K-DMA,
`DLin-KC2-DMA, DLin-KC3-DMA and DLin-KC4-DMA were deter-
`mined (Table 1). The relative ability of the protonated form of the
`ionizable cationic lipids to induce HII phase structure in anionic lipids
`was ascertained by measuring the bilayer-to-hexagonal HII transition
`temperature (TBH) in equimolar mixtures with distearoylphosphati-
`dylserine (DSPS) at pH 4.8, using 31P NMR18 and differential scan-
`ning calorimetric analyses19. Both techniques gave similar results.
`The data presented in Table 1 indicate that the highly active lipid
`DLin-KC2-DMA has pKa and TBH values that are theoretically favo-
`rable for use in siRNA delivery systems. The pKa of 6.4 indicates
`that LNPs based on DLin-KC2-DMA have limited surface charge in
`circulation, but will become positively charged in endosomes. Further,
`the TBH for DLin-KC2-DMA is 7 °C lower than that for DLinDMA,
`suggesting that this lipid has improved capacity for destabilizing
` bilayers. However, the data also demonstrate that pKa and TBH do
`not fully account for the in vivo activity of lipids used in LNPs. For
` example, although DLin-KC3-DMA and DLin-KC4-DMA have
`identical pKa and TBH values, DLin-KC4-DMA requires a more than
`fivefold higher dose to achieve the same activity in vivo. Moreover,
`
`*
`
`*
`
`**
`
`1
`mg/kg
`siApoB
`
`0.03
`mg/kg
`
`PBS
`
`0.3
`0.1
`mg/kg
`mg/kg
`siTTR
`
`1
`mg/kg
`
`1.4
`
`1.2
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0
`
`b
`
`Relative liver TTR/GAPDH mRNA levels
`
`Figure 3 Efficacy of KC2-SNALP in rodents
`and nonhuman primates. (a) Improved
`efficacy of KC2-SNALP relative to the
`initial screening formulation tested in
`mice. The in vivo efficacy of KC2-SNALP
`() was compared to that of the unoptimized
`DLin-KC2-DMA screening (that is, PFV)
`formulation (•) in the mouse Factor VII model.
`Data points are expressed as a percentage
`of PBS control animals and represent group
`mean (n = 5) ± s.d. (b) Efficacy of KC2-SNALP
`in nonhuman primates. Cynomolgus monkeys
`(n = 3 per group) received a total dose of either
`0.03, 0.1, 0.3 or 1 mg/kg siTTR, or 1 mg/kg
`siApoB formulated in KC2-SNALP or PBS
`as 15-min intravenous infusions (5 ml/kg)
`through the cephalic vein. Animals were euthanized 48 h after administration. TTR mRNA levels relative to GAPDH mRNA levels were determined in
`liver samples. Data points represent group mean ± s.d. *, P < 0.05; **, P < 0.005.
`
`0.1
`0.01
`1
`Factor VII siRNA dose (mg/kg)
`
`10
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`00
`
`.001
`
`a
`
`Relative serum factor VII protein (%)
`
`174
`
`VOLUME 28 NUMBER 2 FEBRUARY 2010 nature biotechnology
`
`© 2010 Nature America, Inc. All rights reserved.
`
`

`

`Table 2 Clinical chemistry and hematology parameters for KC2-SNALP–treated rats
`siRNA dose
`Total Bilirubin
`BUN
`(mg/kg)a
`(mg/dl)
`(mg/dl)
`
`Vehicle
`
`ALT (U/L)
`
`AST (U/L)
`
`l e t t e r s
`
`RBC
`(× 106/µl)
`
`Hemoglobin
`(g/dl)
`
`WBC
`(× 103/µl)
`
`PLT (× 103/µl)
`
`1,166 ± 177
`11 ± 3
`11.3 ± 0.4
`5.5 ± 0.3
`4.8 ± 0.8
`2 ± 0
`109 ± 31
` 56 ± 16
`PBS
`1,000 ± 272
`13 ± 2
`11.6 ± 0.6
`5.6 ± 0.2
`4.4 ± 0.6
`2 ± 0
`100 ± 14
` 58 ± 22
`1
`KC2-SNALP
`1,271 ± 269
`13 ± 4
`11.6 ± 0.3
`5.9 ± 0.3
`4.3 ± 0.6
`2.2 ± 0.4
` 81 ± 10
`73 ± 9
`2
`KC2-SNALP
` 958 ± 241
`15 ± 2
`11.9 ± 0.4
`6.0 ± 0.2
`5.0 ± 0.8
`2 ± 0
`100 ± 30
` 87 ± 19
`3
`KC2-SNALP
`aNontargeting, luciferase siRNA. Sprague-Dawley rats (n = 5) received 15-min intravenous infusions of KC2-SNALP formulated siRNA at different dose levels. Blood samples were taken 24 h
`after administration. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; RBC, red blood cells; WBC, white blood cells; PLT, platelets.
`
`DLin-KC2-DMA and DLin-KC4-DMA, which have very similar pKa
`and TBH values, exhibit a >30-fold difference in in vivo activity. This
`result suggests that other parameters, such as the distance and flex-
`ibility of the charged group relative to the lipid bilayer interface, may
`also be important. Thus, although the biophysical parameters of pKa
`and TBH are useful for guiding lipid design, the results presented in
`Table 1 support the strategy of testing variants of lead lipids, even
`ones with very similar pKa and TBH values.
`The lipid composition chosen for the initial formulation and
`screening of novel ionizable cationic lipids (cationic lipid/DSPC/
`cholesterol/PEG-lipid = 40:10:40:10 mol/mol, siRNA/total lipid
`~ 0.05 wt/wt) was useful for determining the rank-order potency
`of novel lipids, but is not necessarily optimal for in vivo delivery. In
`addition, the in vivo activity of resultant LNP-siRNA formulations
`is affected by the formulation process employed and the resulting
`particle structure. Improvements in activity were possible with the
`preformed vesicle process by modifying and optimizing lipid ratios
`and formulation conditions (results not shown). However, we chose
`to further validate DLin-KC2-DMA activity specifically in the context
`of the SNALP platform, currently the most advanced LNP formula-
`tion for delivery of siRNA in vivo. We therefore tested in vivo a ver-
`sion of SNALP (termed KC2-SNALP), which uses less PEG lipid than
`reported previously6 and in which DLinDMA was replaced with DLin-
`KC2-DMA. The incorporation of DLin-KC2-DMA into SNALP led
`to a marked improvement in potency in the mouse Factor VII model;
`the measured ED50 decreased from ~0.1 mg/kg for the unoptimized
`screening formulation to ~0.02 mg/kg for the KC2-SNALP formu-
`lation (Fig. 3a). KC2-SNALP also exhibited similar potency in rats
`(data not shown). Furthermore, after a single administration in rats,
`KC2-SNALP–mediated gene silencing was found to persist for over
`10 d (Supplementary Fig. 1).
`In addition to efficacy, tolerability is another critical attribute of
`a suitable LNP-siRNA delivery system for human use. We therefore
`studied the single-dose tolerability of KC2-SNALP in rats—a popular
`rodent model for assessing the toxicology of siRNA and nucleic acid–
`based therapeutics. As doses near the efficacious dose level were found
`to be very well tolerated (data not shown), single-dose escalation
`studies were conducted starting at doses ~50-fold higher (1 mg/kg)
`than the observed ED50 of the formulation. To understand formula-
`tion toxicity in the absence of any toxicity or pharmacologic effects
`resulting from target silencing, we conducted the experiments using
`a nontargeting control siRNA sequence directed against luciferase.
`KC2-SNALP containing luciferase siRNA was prepared in the exact
`same manner as that containing Factor VII siRNA, and the resultant
`size, lipid composition and entrapped siRNA/lipid ratio were similar.
`Clinical signs were observed daily and body weights, serum chem-
`istry and hematology parameters were measured 72 h after dosing.
`KC2-SNALP was very well tolerated at the high dose levels exam-
`ined (relative to the observed ED50 dose) with no dose-dependent,
`clinically significant changes in key serum chemistry or hematology
`parameters (Table 2).
`
`Given the promising activity and safety profile observed in rodents,
`studies were initiated in nonhuman primates to investigate the trans-
`lation of DLin-KC2-DMA activity in higher species. For these stud-
`ies, we chose to target transthyretin (TTR), a hepatic gene of high
`therapeutic interest20. TTR is a serum protein synthesized primarily
`in the liver, and although amyloidogenic TTR mutations are rare,
`they are endemic to certain populations and can affect the periph-
`eral nerves, leading to familial amyloidotic polyneuropathy, and the
`heart, leading to familial amyloid cardiomyopathy. Currently, the
`only disease-modifying therapy is liver transplantation. We treated
`cynomolgus monkeys with a single 15-min intravenous infusion of
`KC2-SNALP–formulated siTTR at siRNA doses of 0.03, 0.1, 0.3 and
`1 mg/kg. Control animals received a single 15-min intravenous infu-
`sion of PBS or KC2-SNALP–formulated ApoB siRNA at a dose of
`1 mg/kg. Tissues were harvested 48 h after administration and
`liver mRNA levels of TTR were determined. A clear dose response
`was obtained with an apparent ED50 of ~0.3 mg/kg (Fig. 3b).
`A toxicological analysis indicated that the treatment was well tolerated
`at the dose levels tested, with no treatment-related changes in animal
`appearance or behavior. No dose-dependent, clinically significant
`alterations in key clinical chemistry or hematological parameters
`were observed (Supplementary Table 4).
`In summary, we applied a rational approach to the design of novel
`cationic lipids, which were screened for use in LNP-based siRNA
`delivery systems. Lipid structure was divided into three main func-
`tional elements: alkyl chain, linker and headgroup. With DLinDMA
`as a starting point, the effect of each of these elements was inves-
`tigated in a systematic fashion, by holding the other two constant.
`First, the alkyl chains were established, then linker was varied and,
`finally, different headgroup structures were explored. Using this
`approach, important structure-activity considerations for ioniz-
`able cationic lipids were described and lipids with improved activ-
`ity relative to the DLinDMA benchmark were identified. A SNALP
`formulation of the best-performing lipid (DLin-KC2-DMA) was
`well-tolerated in both rodent and nonhuman primates and exhib-
`ited in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents,
`as well as silencing of a therapeutically significant gene (TTR) in
`nonhuman primates. Although the scope of the current work has
`been limited to hepatic delivery in vivo, the TTR silencing achieved
`in this work (ED50 ~ 0.3 mg/kg) represents a substantial improve-
`ment in activity relative to previous reports of LNP-siRNA mediated
`silencing in nonhuman primates.
`
`MeThoDS
`Methods and any associated references are available in the online
`version of the paper at http://www.nature.com/naturebiotechnology/.
`
`Note: Supplementary information is available on the Nature Biotechnology website.
`
`ACKNoWLEDGMENTS
`The authors thank K. McClintock for assistance with animal studies. The authors
`also thank the Centre for Drug Research and Development at the University
`
`nature biotechnology VOLUME 28 NUMBER 2 FEBRUARY 2010
`
`175
`
`© 2010 Nature America, Inc. All rights reserved.
`
`

`

`l e t t e r s
`
`of British Columbia for use of the NMR facilities and M. Heller for his expert
`assistance in setting up the 31P-NMR experiments.
`
`AUTHoR CoNTRIBUTIoNS
`J.C., M.A.C., P.R.C., T.D.M., M.J.H. and K.F.W. designed and advised on novel
`lipids. J.C., K.F.W. and M.S. synthesized novel lipids. M.J.H., T.D.M., J.C., K.F.W.,
`M.M., K.G.R., M.A.M., M.T. and M.J. analyzed and interpreted lipid data. T.D.M.,
`M.J.H. and M.A.T. co-directed novel lipid synthesis and screening program. S.C.S.
`designed and directed rodent in vivo studies. S.C.S., S.K.K., B.L.M., K.L., M.L.E.,
`M.K., A.P.S., Y.K.T., S.A.B., W.L.C., M.J.W. and E.J.C. generated rodent in vivo
`data, including Factor VII and tolerability analyses. L.N., V.K., T.B., R.A., Q.C.
`and D.W.Y.S. developed novel siRNAs targeting TTR. R.A. and A.A. designed and
`directed NHP in vivo studies. S.C.S., S.K.K., A.A., B.L.M., I.M., A.P.S., Y.K.T., R.A.,
`T.B., D.W. Y. S., S.A.B., J.Q., J.R.D. and A.d.F. analyzed and interpreted in vivo data.
`B.L.M., K.L., A.P.S., S.K.K., S.C.S. and E.J.C. generated and characterized preformed
`vesicle formulations with novel lipids. D.S. and C.K.C. developed methods and
`designed and conducted HPLC lipid analyses of preformed vesicle formulations.
`E.Y. and L.B.J. prepared SNALP formulations. P.R.C. directed biophysical studies
`and advised on methods. A.P.S., I.M.H., S.D. and K.W. performed biophysical
`characterization studies (pKa, NMR, differential scanning calorimetric) of novel
`lipids and formulations. M.J.H., P.R.C., T.D.M., A.P.S., I.M.H. and K.F.W. analyzed
`biophysical data. S.C.S., M.J.H., A.A. and P.R.C. co-wrote the manuscript. T.D.M.,
`M.M., M.A.M., M.A.T. and A.D.F. reviewed and edited the manuscript. S.C.S.,
`M.J.H., A.A., P.R.C., I.M. and A.D.F. were responsible for approval of the final draft.
`
`CoMPETING INTERESTS STATEMENT
`The authors declare competing financial interests: details accompany the full-text
`HTML version of the paper at http://www.nature.com/naturebiotechnology/.
`
`
`Published online at http://www.nature.com/naturebiotechnology/.
`reprints and permissions information is available online at http://npg.nature.com/
`reprintsandpermissions/.
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