Screening, Synthesis, and Applications of "Lipidoids",
a Novel Class of Molecules Developed for the Delivery of
RNAi Therapeutics
by
Michael Solomon Goldberg
Hon. B.Sc. Biological Chemistry
University of Toronto, 2003
M.Phil. BioScience Enteprise
University of Cambridge, 2004
Submitted to the Department of Chemistry in Partial
Fulfillment of the Requirements for the Degree of
MASSACHU •
8 INS
OF TEOHNOLOGY
DOCTOR OF PHILOSOPHY IN CHEMISTRY
JUN 10 2008
at the
LIBRARIES
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
ARCHNIEs
June 2008
) 2008 Massachusetts Institute of Technology. All rights reserved.
Signature of Author
Y
Uep ill.
t of C emnistry
April 14, 2008
Certified by
_
Robert Langer
Institute Professor
Thesis Supervisor
Accepted by
Robert W. Field
Chairman
Departmental Committee on Graduate Students
THESIS COMMITTEE
Certified by
Alexander Klibanov
Professor
Department of Chemistry
Committee Chair
Certified by
Robert Langer
Institute Professor
Thesis upervisor
Certified by
C t i b-
Stu a rt L ic ht
Assistant Professor
Department of Chemistry
Committee Member
TABLE OF CONTENTS
I. D edication ..........................................................................................
5
II. Acknowledgements............................................................6
III. Abstract............................................................................................ 7
IV. Chapter 1 (Introduction): Novel materials for delivery of RNAi therapeutics...........8
1.1 Introduction ............................................................... .............
a. Nucleic acid delivery .........
.........................
......
............ 8
b. RNA interference (RNAi) ............................... ....................8
c. Carriers for delivery of RNAi therapeutics ......................... 9........
1.2 siRNA design and modification......................................................10
1.3 Classes of materials (carriers/vectors) .............................................. 11
a. Viral shRNA delivery.. ....................
..
............. 11
b. Non-viral siRNA delivery ........................................................12
i. Polymers................................................. 13
ii. Liposomes and lipoplexes ............................................ 13
iii. "Lipidoids".........................................14
iv. Peptides......................................... .......
....... 15
v. Aptamers............................................... 16
1.4 Chemically-dynamic vectors .........
................
.......................... 17
1.5 Conclusion and Future Directions ....................................................18
a. Targeting considerations.............................
....
.............. 18
b. Combinatorial RNAi ........................................................... 19
c. MicroRNA..............................................................
.......... 19
d. Final thoughts ............................................................
......20
1.6 References ........................................
....................................... 22
V. Chapter 2: A combinatorial library of lipid-like materials for delivery of RNAi
therapeutics........ .............................................................................. 34
2.1 Abstract ...............................................................
........ 34
2.2 Introduction ......................................................................... 34
2.3 Results and Discussion ............................................................... 35
a. Library synthesis and screening .............................................. 35
b. In vitro screening confirmed the ability of lipidoids to transfect several
cell types................
.......................
.........................35
c. Lipidoid-formulated siRNA mediates target-specific knockdown in
vivo.......................................................36
d. Lipidoids are efficacious and well tolerated in rats.......... ............ 37
e. Lipidoids can be employed for local delivery to non-hepatic cell types
and for delivery of RNA therapeutics other than siRNA .................. 38
f. Lipidoids enable potent, specific, and durable knockdown in nonhuman primates ....................................................................... 38
2.4 Conclusion ..................................... .....................................39
2.5 Materials and Methods ................................................................ 40
46
2.6 Figures ............................................................. ......
2.7 References.......................................................57
VI. Chapter 3: Formulation optimization of a highly-effective lipidoid carrier for the
systemic delivery of RNAi therapeutics ....................................
59
3.1 Abstract .................................................................................. 59
3.2 Introduction............................
.......................... 59
3.3 Results and Discussion .............................................................. 60
a. Preliminary characterization................................................60
b. Formulation optimization ....................................
................. 61
c. The possibility of coding region-targeted siRNA-mediated
transcriptional gene silencing ....................................
. .......
.... 62
d. Dosing scheme influences knockdown ................................. 63
e. LNP01-formulated siRNA remains stable and active for several
m onths............................................................................63
3.4 Conclusion .............................................................................. 64
3.5 Materials and Methods .......................................................
.......
65
3.6 Figures ............................................................................
......67
3.7 R eferences .............................................................................. 77
VII. Chapter 4: Identification of a convergence of structure among highly-efficient
carriers of siRNA for the systemic delivery of RNAi therapeutics ....................... 80
4.1 A bstract ................................................................................. 80
4.2 Introduction ...
........................................................................ 80
4.3 Results and Discussion................................................................. 82
a. High-throughput lipidoid library screening: in vitro siRNA
transfection ........ ................................................................ 82
b. In vivo administration of top-performing lipidoids induces potent
knockdown
.............................................
83
c. Structure-activity relationships reveal a convergence of structure among
the top-performing lipidoids ......................................
.....83
4.4 Conclusion.........................................
........................... 85
4.5 Materials and Methods................................................................86
4.6 Figures.............. .............................................................................. 89
4.7 References............................................................................95
VIII. Chapter 5: Conclusion.........................................................................99
IX. Curriculum Vitae ............................................................................... 100
DEDICATION
I dedicate my thesis to my grandfather and best friend, Dave: my hero.
ACKNOWLEDGEMENTS
I would like to thank my family for their unending love and support: Karla, Dave, Zelda,
Gary, Linda, Orli, Shira, Jonny, Adam, Eden, Ari, Carmel, and Judah. In particular, I
would like to express my gratitude to my father for his passion and wisdom.
I would like to thank my friends - in Toronto, London, and Boston - for being such
wonderful people whom I admire and respect greatly.
I would like to thank the entire Langer Lab for being so special. I have been very
fortunate to interact with such capable, funny, intelligent, and sociable individuals. It is a
real treat to come to lab everyday.
I would like to thank all of my collaborators, but especially Elizaveta Leshchiner and
Valentina Busini - two superlatively-talented visiting students - as well as Dr. Andreas
Zumbuehl, chemist extraordinaire and esteemed officemate, and Dr. Akin Akinc, an
extremely-talented alumnus of the lab and valued collaborator who now works at
Alnylam Pharmaceuticals.
Finally, I would like to thank my lab mentor Dr. Daniel Anderson and my supervisor
Institute Professor Robert Langer. Dan demonstrated great confidence in me, and I am
very grateful for his advice, guidance, and tutelage. Bob encouraged me to address
important problems and to overcome the fear of failure, always offering words of
encouragement and wisdom along the way. It has been such a pleasure and privilege to
train under him, and I am extremely appreciative of the opportunities that he has
graciously provided to me.
Synthesis, Screening, and Applications of "Lipidoids",
a Novel Class of Molecules Developed for the Delivery
of RNAi Therapeutics
by
Michael Solomon Goldberg
Submitted to the Department of Chemistry
on April 14, 2008 in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy in
Chemistry
ABSTRACT
RNAi therapeutics represent a fundamentally new way of treating disease,
permitting the selective abrogation of protein production at the genetic level. As nucleic
acids do not readily enter cells, carriers are utilized to enhance uptake. While viruses are
extremely efficient, concerns about their safety has led to the desire to engineer novel
synthetic vectors. We have developed chemical methods using a combinatorial approach
that has resulted in the synthesis of a large library of structurally-diverse compounds
(1,200 in total), termed "lipidoids", without the need to use solvents, catalysts, or
protection/deprotection steps. The members of this library were screened for the ability to
transfect mammalian cells in vitro.
The top-performing compounds were formulated for in vivo administration and
resulted in durable, potent, and specific knockdown of reporter and therapeuticallyrelevant genes in four distinct species, including non-human primates. Local and
systemic administration effectively silenced target genes in three different tissue types. In
addition to reducing protein expression by directing cleavage of target mRNA through
delivery of siRNA, lipidoids were shown to mediate upregulation of proteins by
derpressing miRNA targets through delivery of anti-miR. Finally, inspection of the
chemical functional groups common to the top-performing lipidoids from the first library
informed the synthesis of a second-generation library. The dramatic increase in
percentage of effective materials from this second library (52% as opposed to 3%)
confirmed that a convergence of structure has been identified.
Thesis Supervisor: Robert Langer
Title: Institute Professor
CHAPTER 1:
NOVEL MATERIALS FOR DELIVERY OF RNAI THERAPEUTICS
INTRODUCTION
Nucleic acid delivery
Nucleic acid therapeutics afford the opportunity to treat disease at the genetic
level. Unlike antibodies, which are typically restricted to extracellular targets, and small
molecules, which often have off-target effects because their binding is based simply on
the three-dimensional interactions between the drug and an active or allosteric site on a
target enzyme or receptor, nucleic acids are inherently specific owing to the
complementarity of Watson-Crick base pairing.
Unfortunately, nucleic acids do not readily cross the cell membrane as the lipid
bilayer is hydrophobic and the cell exterior has a negative charge, which repels the likecharged polyphosphate backbone of nucleic acids 1. Nucleic acid delivery can be
facilitated by both viral and non-viral vectors. While extremely efficient, virus-mediated
delivery poses safety concerns - including acute toxicity, the induction of cellular and
humoral immune responses 2,oncogenicity due to insertional mutagenesis 3,and the risk
of viral presence in germ-line cells 4 - which have led to the desire to engineer novel
synthetic carriers. Some groups have attempted to engineer viruses to be safer, while
others have elected to engineer safe materials to be more efficacious.
In addition to being much safer and less expensive, non-viral vectors can
generally be synthesized in large quantities, are amenable to rational design and diverse
chemical modification, and afford much greater nucleic acid carrying capacity 5
Examples of non-viral nucleic acid delivery include physical manipulation of naked
ilasmid DNA, using techniques such as electroporation, magnetofection, and ultrasound
. Synthetic vectors have been created to improve both biostability and cellular uptake.
Polycationic lipids 7,8 and polycationic polymers 9,10 electrostatically bind and condense
nucleic acids to form nanometer-sized complexes, whose small size facilitate uptake 11
Delivery itself is insufficient to achieve therapeutic results. If the complex is
taken up by the target cell via endocytosis then it should promote endosomolysis by
means of a pH-responsive functionality in the carrier backbone, such as a "proton
sponge" 12, 13, and the payload must dissociate from its carrier. In the case of DNA, the
prodrug must further cross the nuclear membrane and be transcribed by host machinery.
Nucleic acids can be used not only for gain-of-function, as in gene therapy, upon
delivery of DNA but also for loss-of-function, as in RNA interference (RNAi), upon
delivery of RNA. The field of RNA delivery, however, is much less mature than the field
of DNA delivery. As a result, less is known about the pathways through which the uptake
of RNA/carrier complexes is mediated. In theory, functional RNA delivery is a simpler
endeavor than functional DNA delivery, as RNAi does not require nuclear translocation
or transcription since the necessary cellular machinery resides in the cytosol.
RNA interference (RNAi)
The 2006 Nobel Prize in Physiology or Medicine was awarded to Dr. Andrew
Fire and Dr. Craig Mello "for their discovery of RNA interference (RNAi) - gene
silencing by double-stranded RNA." RNAi is a conserved defense mechanism evolved by
eukaryotic cells against double-stranded RNA (dsRNA) introduced by invading viruses,
transposons, and transgenes 14, 15. Specifically, RNAi refers to the entire process of posttranscriptional dsRNA-dependent gene silencing 16. Upon recognition, long dsRNAs are
processed by the ribonuclease III-like enzyme Dicer into siRNAs 17, 18. These short
dsRNA fragments - typically 21- to 23-base pairs in length, possessing two-nucleotide 3'
overhangs as well as 5' phosphate and 3' hydroxyl termini 19, 20 - direct the sequencespecific degradation of single-stranded target mRNA via integration into the RNAinduced silencing complex (RISC) 21, 22. Once incorporated into RISC, the sense
("passenger") strand - determined by the decreased thermodynamic stability at its 3' end
relative to its complementary strand 23 - is cleaved by Argonaute 2 ("Slicer") 24, priming
Argonaute 2 to cleave target mRNA possessing the same sequence
RNAi's utility as a potent tool for reverse genetic analysis - providing a link
between sequence data and biological function 26, 27 - is surpassed only by its potential
for use in gene-specific therapeutics 28-31. Chemically-synthesized siRNAs have been
shown to induce post-transcriptional gene silencing in vitro in human cells, which are
usually killed by longer dsRNA 32. Accordingly, such siRNAs might provide a means to
regulate both exogenous and endogenous gene expression, particularly "undruggable"
targets that are not amenable to conventional therapeutics 33. Specifically, small
molecules are typically limited to the following molecular targets: G protein-coupled
receptors, ion channels, nuclear hormone receptors, kinases, and proteases 34. RNAi, in
contrast, can modulate the expression of any protein - by degrading the encoding mRNA
- thereby affecting transcription factors, protein-protein interactions, and other proteinnucleic acid interactions.
RNAi has the potential to become a new major class of drugs, as it represents a
fundamentally new way of treating disease, which is generally caused by the
inappropriate behavior of proteins, allowing for selectively-abrogated protein production
31 It can be used to address a broad range of unmet medical needs, as the pathway
involved is present in all cell types and is indiscriminate to target mRNA sequence. The
pathway is inherently potent owing to the fact that it is catalytic, whereas small molecules
act in a stoichiometric manner. Furthermore, rather than antagonize aberrantlyfunctioning proteins once they have been produced, RNAi instructs the cell to block the
production of disease-causing proteins before they are translated. Finally, RNAi
therapeutics afford a greatly-simplified process of drug discovery and development.
Rather than screen millions of compounds and optimize scale-up chemistries - which
must be performed afresh for each candidate - scientists can determine appropriate
siRNA sequences using computer algorithms by reverse translating a protein into its
encoding primary sequence.
Carriers for delivery of RNAi therapeutics
The foremost obstacle to the widespread use of RNAi therapeutics is safe and
efficient nucleic acid delivery 35 36. Standard in vitro siRNA transfection reagents are
often ineffective or toxic in vivo, hence, alternative strategies and novel structures are
being explored. Because siRNAs are large, charged, and susceptible to nuclease
degradation, chemical modifications are often employed to enhance their lipophilicity
and stability. Lipophilic moieties have also been conjugated to the ends of siRNAs in
order to enhance their uptake. Still, the majority of efforts have focused on developing
vectors to protect the payload, enhance its uptake, and improve its pharmacokinetic
properties. Because viral vectors have significant drawbacks, which will be discussed,
non-viral vectors are considered to be a promising alternative. These synthetic carriers
are produced from a variety of materials, including polymers, liposomes, lipidoids,
peptides, and aptamers. Excitingly, progress is beginning to be made in the development
of chemically-dynamic and targeted vectors. This chapter will discuss the maturation of
the field of siRNA delivery, placing emphasis on the materials used to achieve this feat.
SIRNA DESIGN AND MODIFICATION
Because many siRNAs can be utilized as templates to direct the cleavage of a
given mRNA, design criteria are employed to maximize potency, specificity, and
nuclease stability 37, 38. Positions 2-8 (the "seed region") on the antisense ("guide")
strand are responsible for minimizing off-target silencing 39, 40, which can arise as a result
of partial homology shared between the target and other genes. Interestingly, modifying
position 2 alone is generally sufficient to stem the majority of off-targeting silencing 41
Positions 10 and 16 are also important for cleavage specificity, possibly owing to their
contribution to helical structure . Computer algorithms have been developed to assist in
the design of sequences that will minimize off-target effects 43, 44. In silico design is
augmented by in vitro screening of the resultant lead candidates to maximize the
specificity of sequences prior to in vivo testing.
siRNAs have in vivo half-lives on the order of minutes 45, owing to the action of
serum nucleases 46. To increase their biostability and pharmacokinetic properties,
chemical modifications to the oligonucleotide bases, sugar, and backbone have been
performed and evaluated 38. 2,4-difluorotoluyl ribonucleoside can be used as a
noncanonical nucleoside mimetic, as it does not affect siRNA-mediated target mRNA
cleavage despite increasing nuclease resistance 47. By enforcing the C3'-endo pucker
conformation 48, the introduction of 4'-thioribose increases the stability of siRNA by 600
times 49. Protection against endonuclease-mediated and exonuclease-mediated
degradation are conferred by 2' sugar modifications (2'-OMe or 2'-F) and the
introduction of a phosphorothioate backbone linkage at the 3' end of the guide strand,
respectively 50. The activity and stability of the resultant oligonucleotides depends on the
number and position of the modifications and can be tuned through the judicious
combination of these modifications. Chemical modifications can also be used to reduce
stimulation of the innate immune system via activation of Toll-like receptor 7 in antigenpresenting cells 51, 52
An siRNA can be modified not only within its coding sequence but also at its
termini. The first systematic study of lipophile-siRNA conjugates involved conjugating
derivatives of cholesterol, lithocholic acid, or lauric acid to the 5' end of the guide strand,
the passenger strand, or both using phosphoramidite chemistry 53. This work was
extended to in vivo studies, wherein the conjugation of cholesterol to the 3' end of the
passenger strand via a pyrrolidine linker was shown to result in enhanced cellular uptake
and pharmacokinetic properties - presumably attributable to increased binding to serum
proteins, such as albumin 46. While this work represented the first example of silencing of
an endogenous target by systemic administration of modified siRNA using a
conventional technique (low-pressure, intravenous injection), the dose required was very
high (50 mg/kg, three administrations) and unsuitable for clinical applications.
More recently, a collection of nine lipophiles - including cholesterol, bile acids,
and long-chain fatty acids - were systemically conjugated to siRNAs at the 3' end of the
passenger strand to demonstrate that efficient and selective uptake of these conjugates are
governed by interactions with lipoprotein particles 54. Successful delivery requires the
appropriate lipoprotein receptors to be expressed on the cells in the target organ as well
as the mammalian homolog of the C. elegans transmembrane protein Sidl. This
understanding of the uptake mechanism will likely facilitate the design of future
lipophilic siRNA conjugates to maximize the efficiency and specificity of delivery.
CLASSES OF MATERIALS (CARRIERS/VECTORS)
In addition to direct modification of the nucleic acid, various approaches have
been taken to address the unmet need of siRNA delivery, primarily in the form of novel
vector design. Some groups are working to remove the virulence of viruses, while others
prefer synthetic carriers. Component materials include polymers, liposomes, lipidoids,
peptides, and aptamers. Such carriers can protect the payload from nucleases and
facilitate siRNA delivery into target cells. The resulting increase in delivery efficiency
can allow for lower doses, less frequent dosing, or both. Biocompatibility must, however,
be considered when selecting these materials. While naturally-occuring biomolecules
(small molecules, peptides, and antibodies) may also be used as conjugates to enhance
delivery - particularly as a means of targeting - care must be taken to ensure that they do
not perturb the normal physiological function of their endogenous targets. The inclusion
of non-siRNA components renders large-scale manufacturing more complex 38; still, this
increased complexity is highly acceptable if the carrier material potentiates siRNA
delivery in vivo at levels and sites that would not be otherwise achievable.
VIRAL SHRNA DELIVERY
Viruses can deliver constructs that encode short hairpin RNAs (shRNAs) - noncoding RNA transcripts that contain the two complementary strands of an siRNA
(separated by a non-complementary oligonucleotide linker that forms a short hairpin) that
can be processed by Dicer upon entry into the cytoplasm, resulting in mature siRNAs 55
enabling regulated, sustained expression of RNAi therapeutics. This is advantageous for
chronic diseases, such as cancer and neurodenegenerative diseases, that would otherwise
require frequent dosing 56 Viral vectors must be selected to match their desired
application. A major consideration is the choice between payload integration and
episomal persistance within expression cassettes. Promoter strength and type constitutively and ubiquitously active versus drug inducible and spatiotemporally
controlled 57- are other facets of great importance.
Adenoviruses deliver double-stranded DNA and afford highly-robust shRNA
expression. They are especially well suited for oncology treatments, owing to the ability
to generate tumor-restricted variants that conditionally replicate in and subsequently lyse
transformed cells 58. The disadvantages of adenoviruses include strong immunogenicity
and short duration of shRNA expression 56. Lentiviruses are retroviruses that can infect
dividing as well as non-dividing cells, including refractory cells such as human
hematopoietic and embryonic stem cells 59. They are also capable of stable integration,
rendering them desirable for the treatment of chronic diseases; however, integration into
inappropriate sites results in insertional mutagenesis, and the use of lentiviruses is thus
limited by the vector's inadequate safety profile. Adeno-associated viruses (AAV)
mediate strong and stable shRNA expression 60 but integrate at much lower frequencies
than the other two commonly-used classes of virus. Moreoever, because they can deliver
a much greater shRNA copy number per infected cell than lentiviruses, they afford an
expanded therapeutic window 61. While safer, the low integration efficiency of AAVs
increases the dosing regimen, arguably forfeiting the advantage of viral vectors, whose
inherent immunogenicity is already a concern.
The most highly-explored applications of viral shRNA delivery have been local
delivery to treat neurological and oncological diseases and systemic delivery to treat
infectious diseases. Viral delivery to the nervous system has been reported using all three
classes of virus. Examples include normalization of disease phenotypes, both behavioral
and neuropathological, in animal models of Alzheimer's disease , amyotrophic lateral
sclerosis 63 , and Huntington's disease 64. The selected shRNAs may not be well tolerated
in humans, as the sequences studied have not distinguished between the mutant and wildtype genes 56; moreover, repeated intraspinal injections may not be tractable in patients.
Viral delivery of shRNAs to xenografts via intratumoral injection have
demonstrated efficacy in mouse models of adenocarcinoma 65, glioma 66, and prostate
cancer 67, as evidenced by decreased tumor size. shRNAs can target cancers not only
directly by silencing oncogenes, such as bcl-2 68, but also indirectly by reducing the
expression of angiogenic factors, such as VEGF 69. Perhaps the most exciting target for
RNAi therapeutics are viral infections themselves, as the viral nucleic acids represent
sequences that are entirely exogenous and less likely to have endogenous homologous
counterparts that might lead to off-target effects 29. The greatest successes thus far have
been observed for the treatment of hepatitis B (using an AAV vector) 70 and HIV (using a
lentiviral vector) 71
Unfortunately, while they afford the potentials to decrease dosing dramatically
and target specific cell types through incorporation of appropriate glycoproteins, viral
vectors are assoicated with several safety concerns, most notably immunogenicity and
insertional mutagenesis. Of great significance was the finding that shRNA expression
levels must be tightly controlled because excessive amounts of shRNA saturate the
endogenous microRNA pathway, resulting in profound morbidity and mortality in mice
72. Viral delivery of RNAi therapeutics profoundly reduces the ability to control drug
exposure, in terms of dose and timing, which is a critical aspect of medical treatment
from the perspectives of both safety and efficacy 38
NON-VIRAL SIRNA DELIVERY
While delivery of RNAi therapeutics can be facilitated by both viral and non-viral
vectors, concerns about the safety of viruses have led to the desire to engineer novel
synthetic carriers. Additionally, delivery of chemically-synthesized siRNAs offers the
advantage of bypassing the transport and processing steps of shRNAs. This ability to
harness the natural pathway in a consistent and predictable fashion renders siRNA
particularly attractive as a therapeutic molecule. For this reason, siRNAs are the class of
RNAi therapeutics that is most advanced in preclinical and clinical studies 33. Several
materials have been investigated for their ability to protect and deliver siRNA in a safe
manner. This section will focus on the most robust systems reported to date.
Polymers
Polymers afford access to tremendous chemical diversity and are amenable to
extensive modification. This property enables this class of materials to be tuned to
specific applications 73. Cationic polymers can electrostatically condense the
polyphosphate backbone of nucleic acids, yielding neutral or slightly-positively-charged
nanoparticles that can be more-readily taken up by cells 74. Commonly used cationic
polymers include poly(ethyleneimine) (PEI), poly(L-lysine), and imidazole-containing
polymers 75, 76. In addition to having the highest charge density, PEI greatly enhances
endosomal escape through its "proton sponge" capability 13,77
PEI has been used to deliver siRNA locally to the nervous system to reduce pain
78 and systemically to the lungs to reduce influenza infection 79. PEI-siRNA
complexes
have also been utilized in the treatment of cancer, inhibiting tumor growth in xenograft
models of glioblastoma upon local administration 80 and of ovarian carcinoma upon
systemic administration 8'. PEI can also be targeted to tumor vasculature by conjugating
it to PEG-RGD, inhibiting angiogenesis and tumor growth rate 82. Unfortunately, PEI is
extremely toxic at high doses, as it is not readily biodegradable 83, 84. Cytotoxicity has
been shown to correlate with molecular weight and degree of branching 85. Groups are
working to improve the safety profile of PEI by optimizing its structure 86, 7 and
imparting biodegradable fiinctionalities to shorter analogs 88'89
Two naturally-derivecf:biopolymers have also 'demonstrated effective siRNA
delivery. Atelocollagen (protease-treated collagen), a liquid at 40 C that gels at 370 C, has
been shown to mediate-aiititumor effects in xenografts 90, 91 as well as in bone metastases
92. Chitosan-siRNA complexes has been successfully delivered to the lungs via intranasal
administration 93 and to xenografts via intravenous administration 94. Efforts to employ
poly(lactic-co-glycolic acid) and polyamidoamine as siRNA carriers in vivo are ongoing.
Perhaps the most versatile polymeric siRNA delivery system is that of
cyclodextrin-containing polycations, which can be readily functionalized to include
targeting moieties, such as transferrin. This robust system was demonstrated to inhibit
tumor growth in a murine model of metastatic Ewing's sarcoma, delivering siRNA
effectively to transferrin receptor-expressing tumor cells .This delivery system was also
shown to be well tolerated by non-human primates at lower doses, though no evidence of
in vivo therapeutic silencing was shown 96
Liposomes and Lipoplexes
Liposomes, discrete lipid bilayers that surround an aqueous core, improve the
pharmacokinetic properties of drugs and decrease their toxicity 97. Nucleic acids can also
be combined with lipids to forn amorphous particles known as lipoplexes 98. These
systems generally contain multiple components, including cationic lipids to facilitate
nucleic acid entrapment, polyethylene glycol to shield surface charges, cholesterol to
increase particle stability, and fusogenic lipids to enhance endosomal release 33, 99
Local administration of siRNA-containing liposomes and lipoplexes have led to
positive outcomes in the eye 00, 101, in the nervous system
10,
and in tumors 104
Lipoplexed siRNA can also induce gene silencing following direct application to mucosal
surfaces, such as the vagina 105, 106 and colon 106. This effective delivery route is very
exciting because mucosa are entry sites for many viruses and afford easy access for
subsequent treatment 33
Stable nucleic acid lipid particles (SNALPs) 51 have been shown to be effective
for hepatic siRNA delivery in animal models of infection by HBV 107 and Ebola virus 108
In addition to the treatment of viral infections, SNALPs were used to silence an
endogenous hepatocyte-expressed gene upon systemic delivery in rodents as well as nonhuman primates, resulting in a physiologically-relevant outcome (serum cholesterol
reduction) 109. The treatment resulted in significant target gene silencing after a single
administration, though transient increases in liver enzymes (an indication of mild
toxicity) were noted. The duration of effect was observed for less than two weeks,
however, so the utility of this platform for the treatment of chronic diseases remains
unclear, as does the ability to use this formulation to deliver to tissues other than the liver.
Systemic administration of other liposomal formulations has resulted in
efficacious siRNA delivery in mice to the lungs 110 and kidney "1,conferring physiologic
effects. Systemic administration of siRNA-containing liposomes has also led to improved
outcomes for arthritis in knee joints 112 and for cancer in mouse models of solid tumors
113-115 and metastases 116. Biodistribution must be taken
into account, though, as the
structure of each lipid, particlarly as it relates to charge, affects cellular uptake patterns,
tissue specificity, and toxicity
117, 118
"Lipidoids"
While lipid-based formulations for systemic siRNA delivery constitute one of the
most promising short-term opportunities for the development of RNAi therapeutics 33, the
present collection of delivery materials and their diversity remain limited, in part due to
their slow, multi-step syntheses. The yield is often poor, and the throughput is low, so
alternative chemistries are attractive. Recently, chemical methods were developed based
on a combinatorial approach that permit the simple and rapid synthesis of a large library
containing over 1,200 structurally-diverse lipid-like materials (termed "lipidoids") 119.
Whereas current chemistries used to synthesize cationic lipids involve several onerous
steps - including protection, deprotection, solvent exchanges, and serial purification this innovative route enables high-throughput synthesis in a single step, increasing the
number of lipofection materials studied to date by nearly two orders of magnitude.
The conjugate addition of oligoamines to acrylates or acrylamides was employed
to generate a large library of compounds that chemically resemble lipids and thus protect
and enhance the uptake of their siRNA payload. The resultant unique, novel class of
materials manifests as a hybrid structure between cationic lipids and first-generation
dendrimers. Notably, unlike cationic lipids, which are typically composed of a discrete
cationic head group and a hydrophobic tail, lipidoids are cationic owing to the presence
of reversibly-protonatable backbone amines. Another interesting chemical property that
renders lipidoids a distinct class of biomaterials is the inversion of its ester linkage with
respect to the aliphatic chain when compared to natural lipids, such as triglycerides.
The library members were screened for the ability to transfect mammalian cells
both in vitro and in vivo, and the top-performing material was tested in four species,
including non-human primates. Therapeutic efficacy was observed in liver, lung, and
macrophages. Lipidoids were shown to be effective vectors in both local and systemic
applications, as they facilitate durable, potent, and specific silencing of endogenous and
exogenous gene transcripts when formulated with either double-stranded siRNA or
single-stranded antisense oligoribonucleotides targeting microRNA 120. Importantly, the
knockdown resulted in physiologically-relevant outcomes in disease models related to
obesity and infectious disease.
Lipidoids were also used recently to confirm that siRNA-mediated gene silencing
does not interrupt the endogenous microRNA pathway - affecting neither the production
of mature microRNAs nor their function - confirming the safety of siRNAs as a
therapeutic molecule 121
Peptides
Like cationic polymers and liposomes, cationic peptides can electrostatically bind
to and condense siRNA to yield stable nanoparticles 22, facilitating the delivery of
oligonucleotides into cells 123. For example, VEGF-targeting siRNA complexed with
cholesteryl oligo-D-arginine (Chol-R9) resulted in tumour regression in a xenograft
mouse model upon local administration 124. In addition to acting as a cationic entity that
complexes siRNA, peptides can transport their cargo across cell membranes. Such cellpenetrating peptides (CPPs), or protein transduction domains (PTDs), have received
notable attention 125
MPG, a short amphipathic peptide, forms non-covalent complexes with siRNA
that, unlike most CPPs, enter cells independently of the endosomal pathway 126. It is the
sole CPP demonstrated to induce siRNA-mediated gene silencing both in vitro and in
vivo 127. Most CPPs include a nuclear localization sequence, causing them to transport
their cargo into the nucleus. This feature allowed MPG to deliver promoter-targeted
siRNA, resulting in transcriptional silencing via DNA methylation 128. MPG was shown
to deliver siRNA upon injection into the inner cell mass of mouse blastocysts, silencing
an early transcription factor and impairing cardiogenesis 129. An MPG variant has been
isolated that delivers siRNA to the cytosol, where the RISC complex and target mRNA
are situated, allowing for robust post-transcriptional gene silencing 130. Variants of other
CPPs must be identified to restrict their delivery to the cytoplasm, as post-transcriptional
silencing does not occur in the nucleus. This may prove challenging, as these basic
residues essential for nuclear localization, which seem undesirable for this application,
contribute to nucleic acid condensation and cell surface binding 131
While CPPs can condense siRNA through non-covalent interactions, they can also
be conjugated to siRNA directly 132 or, more generally, via a disulfide bond that liberates
the siRNA in the reducing environment of the cell 131. Unfortunately, while the advent of
a soluble, monomeric siRNA-CPP conjugate would be desirable, only three reports
involving this approach (all involving in vitro delivery) have been published, using TAT
133 and penetratin 134, 135. All three were published in 2004, and none has been
reproduced. Toxicity was not evaluated, and the conjugates were not characterized
sufficiently well to rule out the possibility that the actual mediators of delivery were
noncovalent complexes. Membrane integrity is compromised when cultured cells
experience prolonged exposure to CPP-siRNA complexes, and siRNA uptake was likely
attributable to this membrane infidelity 131
Important considerations in the development of such well-defined conjugates which are difficult to synthesize because of the natural tendency of the opposite charges
to interact electrostatically to yield complexes - are safety and purity. Neutralization of
the CPP's positive charge abolishes its ability to affect cellular uptake 131. Though
potentially exciting, CPPs face several challenges. Systemic administration of siRNACPP complexes or conjugates has yet to be reported, and cytotoxicity due to membrane
disruption is a valid concern.
Peptides can be used not only to enhance uptake into cells but also to target
siRNA delivery to desired cell types when combined with other delivery systems 136
Arg-Gly-Asp (RGD) has been used to target siRNA delivery to tumor cells and their
neovasculature 82, 137. Antibody-protamine fusions afford the ability to condense siRNA
and to target specific cellular antigens. This platform was shown to deliver siRNA
discriminately to HIV-infected primary CD4 T cells in vitro and to HIV-envelopeexpressing melanoma xenografts in vivo upon intratumoral or intravenous administration
to mice, causing inhibition of tumor proliferation 38. It was also used to deliver siRNA
selectively to breast cancer cells expressing the ErbB2 (HER2) receptor via a single chain
antibody fragment-protamine fusion. A single chain antibody fragment-protamine fusion
protein that targets the human integrin lymphocyte function-associated antigen-i has
been demonstrated to induce siRNA-mediated silencing in leukocytes; notably, gene
silencing can be limited to activated leukocytes when the construct is comprised of an
antibody variant that preferentially recognizes activation-dependent conformational
changes in the antigen 139. Production costs will be a major consideration in the
commercialization of this technology.
Peptides derived from parasitic organisms have also been explored to confer
tissue targeting and facilitate cellular uptake. A 19-amino acid peptide from the
Plasmodium protozoan that is responsible for malaria was attached to the distal end of a
lipid-PEG conjugate to target the liver 140. More impressively, siRNA was transported
transvascularly to the brain following complexation with a chimeric peptide comprising
nine arginines and a 29-amino acid sequence from the rabies virus glycoprotein 14. Four
intravenous injections of the formulation protected the majority of mice from fatal viral
encephalitis for one month. The glycoprotein binds to the acetylcholine receptor
expressed by microglia, astrocytes, endothelial cells, and neurons, so it is unclear which
neuronal cells are transfected with siRNA. Furthermore, the peptide may not cross the
blood-brain barrier to enter cells in the functional regions of the brain 142. Still, this
approach represents an exciting step forward in the treatment of neuronal disease by
systemically-administered RNAi therapeutics.
Aptamers
Aptamers are oligonucleotides (ssDNA or ssRNA, 20-40 base pairs in length) that
bind to their targets with high specificity owing to their three-dimensional shapes that
result from intramolecular base pairing 143. Huge libraries (>1014 sequences) can be
screened by the systematic evolution of ligands by exponential enrichment (SELEX) 144
145 to isolate aptamers with high affinity and selectivity to virtually any molecule 146
These nucleic acid binding species are non-protein-based alternatives to antibodies 147
affording the advantages of nonimmunogenicity, simplified discovery, and abiological
synthesis. They can be used not only as direct agonists or antagonists but also as targeting
moieties 148. Notably, aptamers can be selected for based on the ability to bind both to
known antigens and to a given cell type without a prioriknowledge of the molecular
target 149
Two particularly-interesting accounts of aptamer-mediated siRNA delivery have
been reported. In one study, the quadravalency of steptavidin was exploited to
incorporate (on average) two biotin-conjugated PSMA-targeting aptamers and two biotinconjugated siRNA molecules per carrier 147. The resulting drug delivery system was
readily taken up by PSMA-expressing cells, producing efficient gene silencing. The
utility of this system for in vivo siRNA delivery, however, remains to be demonstrated.
In the second study, an aptamer-siRNA chimeric RNA was utilized to deliver
siRNA in vivo, eliciting tumor regression in a xenograft model of prostate cancer 150. The
siRNA was annealed prior to conjugation, but one might envision synthesizing a single
strand of RNA that, through Watson-Crick base pairing, spontaneously folds into an
aptamer (targeting) segment and an shRNA (effector) segment. The shRNA could be
cleaved by Dicer upon entry into the targeted cell to generate a functional siRNA. While
the notion of a therapeutic comprised entirely of RNA is enticing, the commercial-scale
synthesis of longer constructs (>50 nucleotides) may prove challenging. Furthermore,
while shorter aptamer-siRNA conjugates are effective for local delivery in vivo, they can
be cleared rapidly by the kidney and possess a half-life that is prohibitive for systemic
administration unless they are incorporated into larger particles 33
CHEMICALLY-DYNAMIC VECTORS
While efficient, safe, and targeted siRNA delivery are all major advances, the
ability to design "smart" delivery systems is still highly desirable. Chemically-dynamic
vectors contain bonds that are cleaved in the presence of physiological conditions,
resulting in delivery specifically when exposed to particular stimuli 151. A common
example of such a system is the use of masked endosomolytic agents, which trigger the
vector's endosomolytic functionality - thereby facilitating endosomal escape - only upon
degradation of a pH-labile bond by the acidic environment of the endosome 1 2. The
ability to escape the endosome is of paramount importance because endocytosis is the
main method by which cells uptake materials, and nucleic acids are membrane
impermeable 151
The use of PEG as a pH-labile masking agent has garnered the most interest. PEG
deshielding from amphiphilic methacrylate-containing solycations yields endosomolysis
and intracellular oligonucleotide delivery in vitro . In addition to unmasking an
endosomolytic agent, the liberation of PEG can also increase the transfection efficiency
of polycationic polymers in vivo 154. The preferred linkages for these systems are acetal
155 and hydrazone 156 bonds. Notably, PEG can be reversibly linked directly to the siRNA
payload - rather than to the polycationic carrier - and subsequently complexed with a
polycationic carrier to enhance gene silencing in vitro 157
pH can trigger not only bond cleavage but also conformational changes that
confer functional attributes. In particular, peptides that contain protonatable groups
become more amphipathic and membrane active in acidic environments in a manner
similar to antimicrobial agents and viruses 158, 159. Limiting the membrane activity of
these materials to acidic vesicles minimizes effects on the plasma membrane and thereby
cellular toxicity. The GALA peptide, for example, adopts the shape of a pore-forming
transmembrane helix under acidic conditions to facilitate endosomal escape 160
Fusogenic peptides, derived from the influenza virus, similarly enhance the release of
cargo into the cytosol and have been shown to improve siRNA-mediated silencing in
vitro 161
The development of Dynamic PolyConjugates confirmed that chemicallydynamic vectors could be efficacious in vivo 162. Perhaps the most interesting chemicallydynamic vector for siRNA delivery reported to date, this multicomponent system
includes a membrane-active polymer to which siRNA is conjugated via a reducible
disulfide bond and PEG and galactose are conjugated via a maleamate-derived acid-labile
bond. As the endosome acidifies, the PEG and galactose - which serve beneficial
functions in the extracellular environment, namely shielding and targeting, respectively are detached. A single intravenous injection in mice resulted in the silencing of
hepatocyte-expressed genes, yielding a physiological response. The modular design of
these particles may enable other polymers, linkages, and targeting ligands to be
incorporated, though this potential has not yet been realized. This system merits further
investigation in non-human primates.
Disulfide bonds, which are reduced within the cell to release cargo from its
carrier, can also be used to trigger intracellular delivery. Examples employing this
strategy include the return of function to a membrane pore-forming protein when
separated from protamine 163, the enhanced transfection efficacy of disulfide-crosslinked
PEI 164, and the separation of PEG from oligonucleotides 165. To increase transfection at
specific tissue sites, PEG can be deshielded from vectors by incorporating a peptide
linkage that is recognized by specific proteases. Such a system was demonstrated to
enhance nucleic acid uptake in tumors expressing signature matrix metalloproteinases
following systemic administration 166. While the great majority of these systems have
focused on the delivery of plasmid DNA or antisense oligonucleotides, chemicallydynamic vectors represent a potentially exciting development in the advancement of
"smart" siRNA delivery vehicles.
CONCLUSION AND FUTURE DIRECTIONS
Targeting considerations
Targeted nucleic acid delivery can be achieved transcriptionally or
transductionally. Transcriptional targeting refers to the use of genetic regulatory elements
to restrict gene expression to specific cells or to delivery of an siRNA that interacts with
an mRNA that is expressed specifically by a given subset of cells. Transductional
targeting refers to the delivery of nucleic acid to specific cells using a targeting moiety
that is attached to the vector but is unrelated to the nucleic acid sequence, such as small
molecules, aptamers, peptides, or antibodies. Notably, the ability to identify materials
from large libraries that inherently confer cell type-specific delivery owing to their
structure would obviate the need to add such targeting moieties.
The selection of appropriate target sequences can render transcriptional targeting
intrinsic to siRNA delivery. In such cases, transductional targeting is only required to
minimize the dose, thereby decreasing the cost of goods and the likelihood of side effects.
siRNAs exhibit very high specificity for their intended molecular targets, so exposure of
nontargeted tissues is only an issue if the target mRNA is expressed in these tissues and
has an important role in normal cellular function within these tissues 38. In these cases,
local delivery might circumvent undesired side effects caused by systemic delivery, and
transductional targeting can be used if local delivery is impractical. By increasing the
specificity of delivery, transductional targeting can enhance both efficacy and safety 136
Interestingly, compartmental modeling of trasferrin-mediated tumor targeting revealed
that the efficacy of these targeted nanoparticles was attributable to increased cellular
uptake by tumor cells rather than to increased overall tumor localization relative to nontargeted particles 167. This led the scientists to conclude that the optimization of
internalization may be the critical step in the development of highly-effective,
nanoparticle-based targeted therapeutics.
Combinatorial RNAi
Cellular processes are networked in a manner that incorporates redundancy and
multifunctionality 168. For this reason, RNAi monotherapies may be limited. Perhaps
most famously, the field of oncology has taken advantage of combination therapies to
achieve great success 169. A second impetus for administering RNAi therapeutics against
multiple targets is related to the regulation of exogenous genes in the treatment of
infectious disease. Specifically, combinatorial RNAi (coRNAi) can be harnessed to
address the natural diversity and rapid evolution of viruses.
There is great genetic diversity within a given class of viruses, so the
identification of a single conserved target sequence is nearly impossible. Furthermore,
because of the specificity of RNAi, viruses need only accrue a single point mutation to
develop resistance to an siRNA or shRNA 70. Several accounts of such behavior have
already been reported 171, 172. The likelihood of a virus developing resistance is related to
the number of coincidental mutations across the number of 21-mer sequences targeted,
either genomic or transcribed RNA, so coRNAi offers an important means by which to
maximize selection pressure and minimize viral escape 173. Additionally, it is becoming
apparent that viruses interact with the endogenous RNAi pathway, having developed
suppressors of RNAi silencing (SRSs) 174, a finding that might render RNAi
monotherapies less effective.
The majority of work in this young field has focused on the coexpression of
multiple microRNAs or shRNAs either from individual promoters or in tandem from a
single promoter. The hairpins can be designed to target various sequences within a single
target or multiple targets (viral and/or host). coRNAi can also involve the delivery of
RNAi therapeutics in concert with other RNA-based gene silencers, such as antisense
oligonucleotides 17s or ribozymes 176, or with plasmids that encode therapeutic proteins
177. Though it has yet to be demonstrated in animal
models of disease, coRNAi clearly
represents an exciting advancement in the development of next-generation RNAi
therapeutics. siRNA cocktails will likely receive enhanced interest in coming years, as
viral delivery is associated with safety concerns and control over multiple promoters may
prove challenging.
MicroRNA
In addition to siRNA, another class of small non-coding RNAs, termed
microRNAs (miRNAs), has received considerable attention of-late. miRNAs are singlestranded RNAs of -22 nucleotides in length that are generated from endogenous hairpin-
shaped transcripts 178. They function as guide molecules in post-transcriptional gene
silencing by base pairing with target mRNAs, resulting in translational repression or
mRNA cleavage ' . miRNAs are believed to constitute 1-4% of human genes and to
regulate 30% of protein-encoding genes 180
As a large set of master regulators of gene expression, miRNAs represent a
particularly interesting class of therapeutic targets 181. They are pleiotropic and
effectively mediate systems biology at the molecular level, repressing the expression of
multiple transcripts that encode proteins that interact in common biochemical pathways.
miRNA misregulation has been implicated in several diseases, including cancer 182, heart
failure 183, and metabolic disorders 184. The loss or gain of miRNA function can be
generated by a single point mutation in either the miRNA or its target mRNA's 3'
untranslated region or via epigenetic silencing of primary miRNA transcription units 185
miRNA function can theoretically be restored by administering miRNA mimics or
precursors, while miRNA function can be inhibited by delivering antisense
oligonucleotides (ASOs) 186, 187 "Antagomirs", single-stranded cholesterol- and 2'-Omethyl-modified ASOs, were shown to bind to complementary miRNAs, thereby
derepressing their mRNA targets and yielding the expected physiological outcome in
mice 188. 2'-methoxyethyl-modified ASOs directed to the same miRNA demonstrated
similar cellular and physiologic effects even in the absence of cholesterol conjugation,
which facilitates hepatic targeting 189. Most recently, in vitro studies revealed the
significant contribution of secondary structural elements - highly-structured, doublestranded flanking regions around the reverse complement core - to the potency of antimiRNAs 190
miRNA mimics and inhibitors are likely to become important molecules as the
field of RNAi therapeutics matures. The delivery of these molecules will surely benefit
from the efforts invested in siRNA delivery.
Final thoughts
RNAi, which has great potential as a therapeutic modality, is a platform
technology. Delivery remains the principal obstacle to the realization of the broadest
applications of RNAi therapeutics. An appropriate carrier must protect the payload,
confer a good pharmacokinetic profile, enhance cellular uptake, and release its cargo into
the cytosol. To this end, many different classes of materials have been used. Targeted and
chemically-dynamic vectors represent some of the more-exciting and innovative recent
developments in the field. It is unclear whether one vector may be applied to sundry
diseases - as the carrier is presumably indiscriminate to the siRNA sequence - with the
modular exchange of targeting moieties to direct the vector to the desired tissue or
whether the entire delivery vehicle will have to be customized for each target cell type
and disease state.
While viruses have evolved over millions of years to deliver nucleic acids into
cells efficiently, non-viral vectors have been developed over the short course of the past
ten years. The amount of progress that has been made in this short period of time is
astounding, combining the principles of rational design and the ability to learn from
experiments in an incremental, empirical fashion. Ultimately, an improved understanding
of the mechanisms of uptake and determining the critical physical properties that
facilitate this uptake - to be derived from insightful structure-activity relationships - will
hopefully enable the judicious design of next-generation vectors that display maximal
efficacy and minimal toxicity.
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CHAPTER 2:
A COMBINATORIAL LIBRARY OF LIPID-LIKE MATERIALS FOR
DELIVERY OF RNAI THERAPEUTICS
ABSTRACT
The safe and effective delivery of RNAi therapeutics remains the central
challenge to its clinical development. The current collection of delivery materials and
their diversity remains limited, in part due to their slow, multi-step syntheses. Here, we
describe a new class of lipid-like delivery molecules, which we term "lipidoids", as
delivery agents for RNAi therapeutics. Chemical methods were developed to allow the
rapid synthesis of a large library of over 1,200 structurally-diverse lipidoids. From this
library, lipidoids were identified that facilitate high levels of specific silencing of
endogenous gene transcripts when formulated with either double-stranded small
interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe)
oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids
were studied in three animal models - mice, rats, and non-human primates. The studies
reported herein suggest these new materials could potentially have broad utility for both
local and systemic delivery of RNA therapeutics.
INTRODUCTION
The specific reduction, or "silencing", of gene expression through RNAi has
tremendous potential to create a new class of therapeutics that address previouslyuntreatable diseases 1,2. High doses of cholesterol-modified siRNA. 3 and cholesterolmodified 2'-OMe oligoribonucleotides ("antagomirs") 4 have been demonstrated to
reduce gene expression specifically in vivo in liver and other tissues. Certain viruses,
such as Respiratory Syncytial Virus (RSV) can be inhibited locally following
administration of naked siRNA 5,6. Recently, systemic delivery of siRNA to the liver of
mice and non-human primates was demonstrated using a lipid formulation 7,8. Despite
these advances, to our knowledge there are few ublished reports demonstrating efficacy
of systemically delivered siRNA in primates 9. While significant efforts have been
dedicated to cationic lipid delivery systems, the development of safe and effective
methods both in vitro and in vivo has proven challenging.
To date, the delivery of siRNA has been mediated by direct conjugation of
delivery agents 3,10-12; formulation using lipid- 7,, 13-16, polymer- 9,17-20, or peptide-based
delivery systems 21-23; and, more recently, complexation with antibody fusion proteins 24,
25. A key barrier to the larger exploration of delivery
material space is the difficulty of
new material synthesis. Conventional lipid synthesis typically requires individuallyoptimized, multiple-step synthesis, including time-intensive procedures such as chemical
protection and deprotection, the use of and removal of catalysts, solvent exchanges, and
purification 26 The customization of each synthetic reaction and the multiple steps
required limit throughput and, correspondingly, ability to generate significant library size
and diversity. To address these problems, we have developed, to our knowledge, the first
chemical methods capable of rapid, parallel production of lipid-like molecules (Fig. 1).
RESULTS AND DISCUSSION
Library synthesis
The synthetic scheme employed is based upon the conjugate addition of alkylacrylates or alkyl-acrylamides to primary or secondary amines (Fig. lb). This particular
chemistry - unlike many traditional lipid synthesis chemistries - allows for reactions in
the absence of solvent or catalysts, results only in lipidoid product, and thereby
eliminates the need for protection/deprotection steps, purification, or concentration steps.
Using these methods, we first synthesized a pilot library of more than 700 lipidoid
members, in which a number of parameters were systematically varied, including 1) alkyl
chain length from C10 to C18, 2) linkage between the alkyl chain and the amine through
the degradable ester bond or the more stable amide bond, 3) primary R group on the
amine, and 4) the post-synthetic introduction of a constitutive positive charge to the
acrylate-derived lipidoids by quaternization of the amine with the alkylating agent methyl
iodide.
Characterization of representative lipidoids demonstrated nearly-complete
conjugation for acrylate-based lipidoids under conditions used (Materials and
Methods). Acrylamide conjugation was slower, but the majority of material was
conjugated after one week at elevated temperature. This combinatorial approach has the
advantages of both simple and rapid synthesis as well as the potential to generate a large
and diverse collection of materials.
In vitro screening confirmed the ability of h'pidoids to transfect several cell types
Once synthesized, this first library was screened for the ability to deliver siRNA
to the human cervical cancer cell line HeLa (Fig. 2a). A HeLa cell line was created that
stably expresses both Firefly luciferase and Renilla luciferase. Efficacy of siRNA
delivery by lipidoids was determined by treating these cells with siRNA-lipidoid
complexes, prepared using a Firefly luciferase-targeting siRNA (siLuc), and then
measuring the ratio of Firefly to Renilla luciferase expression. In this assay, toxic or other
non-specific effects result in reduction of expression of both luciferase proteins, while
non-cytotoxic, specific silencing results in reduction of only Firefly luciferase. To
facilitate screening throughput, siRNA-lipid complexes were formed by simple mixing of
siRNA-lipidoid solutions in microtiter plates.
Analysis of these results revealed several trends in lipidoids capable of delivering
siRNA to HeLa cells. Overall, enhanced delivery performance was achieved using
lipidoids containing more than two amines per head unit (e.g. monomers 61-64, 95-103).
Furthermore, effective materials often had either two long amide tails (e.g. 64N16) or
several smaller amide tails (e.g. 98N 12). Based on this data set, we synthesized a second
generation library of 500 lipidoids, expanding the structures tested based on these trends.
In particular, we hypothesized that a number of these design features could enrich the
library with effective materials. Specifically, we expanded the library to include lipidoids
with even shorter amide tails (e.g N9, Ni1 ) and more head units with two or more amines
(e.g. monomers 109-117). Since many of these head units have the potential for more
than two tails, lipidoids were resynthesized with varied reaction stoichiometry to generate
lipidoids with varying numbers of tails.
The ratio of delivery material to nucleic acid is known to affect delivery potential
of formulations. Therefore, this second generation library of 500 lipidoids was screened
in quadruplicate, as above, for delivery to HeLa cells at four different lipidoid:siRNA
ratios. Using these screening methods, we identified a total of 56 lipidoids capable of
inducing gene silencing at levels similar to or better than the commercially-available in
vitro transfection agent LipofectamineTM 2000 (Fig. 2b). Notably, the top performing
lipidoids in this second library contained several structural similarities: 1) amide linkages,
2) more than two alkyl tails, 3) tail length in the range of 8-12 carbons, and 4) one tail
short of total substitution of the amine reactants, therefore containing one secondary
amine. These relatively short tails are surprising in comparison to typical gene deliver
cationic lipids such as DOTAP and DOTMA, which contain 18 aliphatic carbon atoms
Interestingly, many of the effective lipidoids (e.g. 100N 9) are structurally unlike both
conventional lipids and cationic polymers. This novel set of effective siRNA delivery
materials significantly expands the collection and chemical diversity of materials known
to facilitate siRNA delivery to cells.
To examine in vitro performance more closely, a collection of purified lipidoids
selected from those showing efficacy in HeLa cells, was tested at different doses, with
three cell types: HeLa, human hepatocellular liver carcinoma cell line HepG2, and
primary bone marrow-derived murine macrophages (Fig. 2c-e). In vitro analysis
confirmed that, in general, both lipidoids and commercial lipids are relatively noncytotoxic at the concentration ranges in which they are efficacious (data not shown).
Notably, these lipidoids performed differently in the different cell types. While all
materials were able to facilitate good silencing at the highest siRNA levels, the
commercial reagents LipofectamineTM RNAiMAX and LipofectamineTM 2000 were able
to facilitate greater silencing at the lowest siRNA levels in HeLa and HepG2 cells. In
contrast, with primary bone marrow-derived macrophages, lipidoids were more effective
delivery vehicles at low siRNA concentrations. Dose-dependant silencing of GFP
expression was observed for 6 of the 7 lipidoids tested with 50% silencing observed even
at a 1 nM siRNA concentration (Fig. 2e). In contrast, LipofectamineTM 2000 and
Lipofectamine TM RNAiMAX were not effective at silencing GFP expression even at the
highest siRNA concentration used (Fig. 2e).
Lipidoid-formulated siRNA mediates target-specific knockdown in vivo
To examine the utility of this library, we first attempted to mine the lipidoids as
novel materials for the in vivo systemic delivery of siRNA to liver. The liver represents
an attractive organ for therapeutic intervention both because of the number of potential
hepatic targets as well as the highly-perfused nature of the organ, which may render it
more amenable to delivery of exogenous siRNAs. Using a liver-directed in vivo screen of
the lipidoid library, we identified a series of compounds that facilitate high levels of
siRNA-mediated gene silencing in hepatocytes, the cells comprising the liver
parenchyma. Factor VII, a blood clotting factor, is an ideal target gene for assaying
functional siRNA delivery to liver. It is produced specifically in hepatocytes; therefore,
gene silencing indicates successful delivery to parenchyma, as opposed to delivery solely
to the cells of the reticuloendothelial system (e.g. Kupffer cells). Furthermore, Factor VII
is a secreted protein that can be readily measured in serum, obviating the need to sacrifice
animals. Finally, owing to its short half-life (2-5 hours), silencing at the mRNA level is
manifest as silencing at the protein level with minimal lag.
A number of top performing lipidoids, identified through the in vitro screens,
were screened in vivo, and several lipidoid formulations were identified that mediated
significant reduction of serum Factor VII protein levels, with the largest reduction
observed for 98N 12 (>90%) (Fig. 3a). Variants of 98N 12 with different tail numbers were
purified and tested individually (data not shown). 98N 12-5 (i.e. 5-tailed) was found to be
optimal for in vivo delivery. To verify the specificity of gene silencing, liver mRNA
levels were measured for both Factor VII and another hepatocyte-expressed gene,
apolipoprotein B (Apob) (Fig. 3b). In animals treated with formulations containing Factor
VII-specific siRNA (siFVII), only silencing of the Factor VII mRNA was observed.
Conversely, in animals treated with formulations containing Apob-specific siRNA
(siApoB), only silencing of the Apob mRNA was observed. Further, administration of a
lipidoid-formulated 1:1 (wt:wt) mixture of the two siRNAs resulted in silencing of both
Factor VII and Apob genes with no detectable loss in potency or competition between
siRNAs. No silencing of Factor VII was observed when a mismatch Factor VII siRNA
was used (data not shown). These data indicate that the observed gene silencing is a
direct result of the specific effects of lipidoid-siRNA on mRNA levels in the liver and
that these effects are applicable to multiple hepatocyte-expressed genes.
Lipidoids are efficacious and well tolerated in rats
To explore further the in vivo activity of 98N 12-5 observed in mice, we conducted
efficacy and tolerability studies in rats. Rats were given a single i.v. injection of siFVII
formulated in 98N 12-5 at doses of 1.25, 2.5, 5, and 10 mg/kg. Significant, dose-dependent
reductions in liver Factor VII mRNA levels were observed, with 40%, 80%, and greater
than 90% silencing at 1.25, 2.5, and 5 mg/kg, respectively (Fig. 3c). No silencing was
observed using a formulated control siRNA (siCont), demonstrating specificity of
silencing. The reduction in liver Factor VII mRNA levels produced a concomitant dosedependent reduction in serum Factor VII protein levels, with nearly complete silencing at
the highest dose levels (Fig. 3d).
As would be expected, significantly-reduced serum Factor VII levels produced a
phenotypic effect in the treated animals. Because Factor VII is part of the extrinsic
coagulation pathway, treated animals had impaired clotting through this pathway, as
measured by prolongation in prothrombin time (PT) (Fig. 3e). The phenotypic effect was
found to be specific and not attributable to the delivery vehicle, with the formulated
control group demonstrating no perturbations in PT. The resultant gene silencing was
highly durable. Single injections of 98N 12-5-formulated siFVII were capable of mediating
silencing persisting for nearly 4 weeks (Fig. 30).
Next, the tolerability of the lipidoid formulation was investigated in rats. Animals
received 4 once-per-week i.v. bolus injections of a formulated siRNA at doses as high as
10 mg/kg/week. The non-physiological siRNA siCont was used in order to control for
any potential target silencing-related toxicities. The formulation was generally welltolerated at the dose levels tested, as determined by cage-side observations and measures
of clinical chemistry and hematology parameters (data not shown). However,
enlargement of the spleen--a major clearance organ for nanoparticles--was observed at
the highest doses. The appearance and weights of all other organs were normal.
Lipidoids can be employed for local delivery to non-hepatic cell types and for delivery
of RNA therapeutics other than siRNA
To explore the versatility of the lipidoid approach, an additional disease model
was examined next. Specifically, local siRNA delivery to lung after intranasal
administration in a mouse model of Respiratory Syncytial Virus infection 5 was tested
(Fig. 4a). In separate experiments, the absence of gene silencing in the liver and kidney
after local pulmonary administration of the formulation was confirmed (data not shown).
While "naked" siRNA provided roughly one log reduction in viral plaques, 98N 12-5formulated siRNA at the same dose provided greater than two log reduction in viral
plaques. Importantly, these data demonstrate that lipidoids can be used in non-systemic
applications of RNAi technology and are capable of delivering siRNA to non-hepatic cell
types.
The macrophage is a cell type frequently implicated in the pathology of
inflammatory diseases. Following our success in transfecting siRNAs into primary
macrophages in cultures in vitro (Fig 2e.), we tested whether lipidoid-formulated siRNAs
could mediate silencing in macrophages in vivo. Mice were injected intraperitoneally
with thioglycollate as a sterile inflammation stimulus, followed by injection of 98N12 -5formulated siCD45. A 65% reduction of CD45 protein expression was observed in the
peritoneal macrophage population (Fig. 4b). While the intraperitoneal administration
circumvents some of the challenges associated with systemic delivery to macrophages,
these results indicate that lipidoid formulations can potentially be employed for the
delivery of siRNA to macrophages in vivo.
To examine the utility of lipidoid materials in the delivery of other nucleic acid
therapeutic drugs, we tested the potential of 98N 12-5 to facilitate the delivery of singlestranded 2'-O-Me oligoribonucleotides targeting miRNAs ("anti-miRs"). In vivo delivery
of anti-miR results in specific target miRNA silencing, and, consequently, the specific
upregulation of genes regulated by the target miRNA 4. 98N 12-5-formulated anti-miR122
dosed at 5 mg/kg on three consecutive days in mice resulted in greater miR-122
repression than the cholesterol-conjugated version of the same oligoribonucleotide
(antagomirl22) dosed at 80 mg/kg on three consecutive days (Fig. 4c). Further, this
effect was shown to be specific, as mismatched control anti-miR122 (mm-anti-miR122)
did not result in appreciable effects on miR-122. Consistent with miR-122 downmodulation, gene targets of miR-122 were shown to be derepressed in anti-miR122treated mice relative to mismatched controls (Fig. 4d).
Lipidoids enable potent, specific, and durable knockdown in non-human primates
To determine the effects of 98N 12-5 lipidoid in a third animal species, we initiated
studies in non-human primates. Cynomolgus monkeys were treated with a single i.v.
injection of lipidoid-formulated siApoB at siRNA doses of 2.5 and 6.25 mg/kg. Separate
control animals were treated with either saline or lipidoid-formulated siCont. Serum
samples were taken from animals for up to 30 days post-administration to determine both
extent and duration of serum ApoB protein silencing. Silencing of ApoB was observed in
a dose-dependant manner, with maximal serum ApoB reduction of up to 75% relative to
pre-dose levels (Fig. 5a). In contrast, no significant silencing was observed in the saline
or formulated control groups, indicating specific activity of the formulated ApoB siRNA.
Silencing was remarkably persistent for animals in the high dose group after a single i.v.
bolus injection, with nadir levels of ApoB achieved rapidly by day 3, >50% silencing still
maintained at 2 weeks, and full recovery achieved only a full month later. The data herein
regarding durability for ApoB silencing thereby extends the previous reports for systemic
RNAi in non-human primates reported by Zimmermann et al. 8, in which the study was
terminated at 11 days. Consistent with ApoB silencing, therapeutic efficacy, as measured
by specific and persistent reductions in LDL-C of up to 60%, was also observed (data not
shown).
A subsequent primate study was performed with a next-generation 98N 12-5
lipidoid-based formulation in cynomolgus monkeys. This newer formulation was
optimized in part by maximizing the siRNA loading in the lipidoid formulation.
Interestingly, the total mass of delivery material relative to siRNA in this formulation is
roughly 1/3 that of the previously published SNALP delivery system 8. Also, unlike the
previously-published SNALP system, this formulation contains fewer total components
(three different agents plus siRNA instead of four). Animals were treated with formulated
ApoB siRNA at siRNA doses of 2.5 and 6.25 mg/kg or formulated control siRNA at 2.5
mg/kg administered via single i.v. injection. Tissues were harvested 48 h postadministration for liver mRNA determination. Silencing of Apob liver mRNA of up to
85% was observed (Fig. 5b), corresponding to a maximal reduction in serum ApoB
protein of up to 74% (Fig. 5c). As early as 2 days post administration, LDL-C was
reduced by greater than 50% (Fig. 5d). Toxicological analysis indicated that the
formulations were well tolerated at the dose levels tested, with no observed treatmentrelated changes in appearance or behavior. No clinically-relevant changes in coagulation
or hematological parameters were observed other than a mild reduction in platelets at the
highest dose. No significant changes in clinical chemistry parameters were observed,
except for a slight increase in ALT and AST. These elevations were less than those
observed with the previously-published SNALP formulations .
CONCLUSION
RNAi technology is poised to form the basis for the next major class of
pharmaceutical drugs. However, effective delivery of RNAi therapeutics remains the key
hurdle for advancement of this technology. We believe the development of lipidoids
could extend the scale and diversity of available delivery solutions. Lipidoid formulations
of siRNA and anti-miR demonstrated potent, specific, and durable effects on gene
expression in three distinct species, including non-human primates. Therapeutic efficacy
was observed in vivo in liver, lung, and peritoneal macrophages. Further studies are
warranted to investigate lipidoid formulations of siRNA for delivery to additional tissues
and to extend this technology for the broadest applications of RNAi therapy and drug
delivery.
MATERIALS AND METHODS
Library synthesis. Lipidoids were synthesized by addition of acrylamides or acrylates to
amines. Amines were purchased from Sigma-Aldrich (St. Louis, MO) and TCI America
(Portland, OR). Acrylates were purchased from Sigma-Aldrich (St. Louis, MO), Dajac
Monomer-Polymer (Feasterville, PA), Hampford Research (Stratford, CT), Scientific
Polymer (Ontario, NY), and TCI America (Portland, OR). Acrylamides were synthesized
by the drop-wise addition of acryloyl chloride to the appropriate alkylamine (see below
for details). The ester portion of the lipidoid library was synthesized at a ratio of 2:1
acrylate:amine, with no solvent, unless otherwise specified. The amide portion of the
lipidoid library was synthesized at the maximal ratio of acylamide:amine for each amine
(e.g. 6 acrylamide:amine for amine monomer 98). All library reactions were carried out
in 5 mL Teflon-lined glass screw-top vials. 200 mg of amine was added to the
corresponding amount of acrylate or acrylamide. The mixture was stirred at 90 OC for 1
or 7 days for acrylate or acrylamide monomers, respectively. After cooling, the lipid
mixtures were used without purification unless otherwise specified.
Acrylamide synthesis. Acrylamides were synthesized by the drop-wise addition of
acryloyl chloride to the appropriate 1-aminoalkane in dry tetrahydrofuran at -150 C with
stirring for several hours. Excess triethylamine was added to neutralize the hydrochloric
acid that was generated. The product was extracted in ethyl acetate, the organic phase
was dried over Na2SO4, and the solvent was evaporated under reduced pressure. The
product was resuspended in petroleum ether, and the solution was filtered over charcoal
to remove impurities. The crude material was purified using silica gel column
chromatography, running a solvent gradient (petrol ether:ethyl acetate = 1:0; 3:1; 1:1;
0:1).
Library purification and characterization. Representative library members were
purified and characterized. Chromatographic purification was performed by flash
chromatography using Merck Silica Gel 60. Mixtures of dichloromethane (75%),
methanol (22%), and ammonium hydroxide (3%) with varying amounts of additional
dichloromethane (depending on the lipid) yielded pure products. Solvent removal was
performed by evaporation on a Biichi rotavapor, with heating to 40 OC. TLC was
performed on Merck Silica Gel 60 F254 TLC glass plates and visualized with ninhydrin
stain or UV 254. IR spectra were recorded on a Nicolet Magna-IR 550 spectrometer
using polyethylene sheets or ATR technology. NMR spectra were recorded on Varian
Mercury-300 or Varian Inova-500 spectrometers with the internal signal of the deuterated
solvent as standard. Fast Atom Bombardment analysis and Positive-ion Electrospray
analysis was carried out by M-Scan (West Chester, PA) on a VG Analytical ZAB 2-SE
high field mass spectrometer and a Micromass Q-Tof API US hybrid quadrupole/time of
flight mass spectrometer.
Synthesis of PEG-DMG lipid. PEG-DMG was synthesized from (S)-1,2-di-Otetradecyl-snglycerol and 1-methoxy-PEG2000-amine (mPEG2000-NH2). Treatment of
(S)-1,2-di-Otetradecyl-sn-glycerol with N,N'-disuccinimidyl carbonate in the presence of
triethylamine in dichloromethane under argon atmosphere and subsequent reaction of the
intermediate formed with mPEG2000-NH2 in the presence of pyridine in
dichloromethane afforded the desired compound (R)-3-[(1-methoxy-PEG2000carbamoyl)]-1,2-di-O-tetradecyl-sn-glyceride (PEGDMG) in 90 % yield.
Nucleic acids. All siRNAs and 2'-O-Me oligoribonucleotides were synthesized by
Alnylam. Oligonucleotides were characterized by ESMS and anion-exchange HPLC.
The sequences for the sense and antisense strands of siRNAs are as follows:
siLuc sense: 5'-CUUACGCUGAGUACUUCGATT-3',
antisense: 5'-UCGAAGUACUCAGCGUAAGTT-3'.
siFVII sense: 5'-GGAucAucucAAGucuuAcT*T-3',
antisense: 5'-GuAAGAcuuGAGAuGAuccT*T-3'.
siApoB sense: 5'-GGAAUCuuAuAuuuGAUCcA*A-3',
antisense: 5'-uuGGAUcAAAuAuAAGAuUCc*c*U-3'.
siCont sense: 5'-cuuAcGcuGAGuAcuucGAT*T-3',
antisense: 5'-UCGAAGuACUcAGCGuAAGT*T-3'.
siGFP sense: 5'-CcAcAuGAAGcAGcACGACu*U-3'
antisense: 5'-AAGUCGUGCUGCUUCAUGUGg*u*C-3'.
siCD45 sense: 5'-cuGGcuGAAuuucAGAGcAT*T-3',
antisense: 5'-UGCUCUGAAAUUcAGCcAGT*T-3'.
mm-siRSV sense: 5'-CGAUUAUAUUACAGGAUGAT*T-3',
antisense: 5'- UCAUCCUGUAAUAUAAUCGT*T-3'.
siRSV sense: 5'- UCCUAGAAUCAAUAAAGGGTT-3',
antisense: 5'- CCCUUUAUUGAUUCUAGGATT-3'.
2'-O-Me oligoribonucleotides:
antagomirl22: 5'-a*c*aaacaccauugucacacu*c*c*a-Cholesterol-3'
mm-antagomirl22: 5'- a*c*acacaacacugucacauu*c*c*a-Cholesterol-3'
anti-miR122: 5'-a*c*aaacaccauugucacacu*c*c*a-3'
mm-anti-miR122: 5'- a*c*acacaacacugucacauu*c*c*a-3'
2'-O-Me modified nucleotides are in lower case, 2'-Fluoro modified nucleotides are in
bold lower case, and phosphorothioate linkages are represented by asterisks. siRNAs
were generated by annealing equimolar amounts of complementary sense and antisense
strands.
In vitro siRNA transfection assay. The protocol was adapted from Anderson, D.G., et
aL 27. HeLa cells, stably expressing Firefly luciferase and Renilla luciferase were seeded
(14,000 cells/well) into each well of an opaque white 96-well plate (Coming-Costar,
Kennebunk, ME) and allowed to attach overnight in growth medium. Growth medium
was composed of 90% phenol red-free DMEM, 10% fetal bovine serum, 100 units/mL
penicillin, 100 .tg/mL streptomycin (Invitrogen, Carlsbad, CA). Cells were transfected
with 50 ng of Firefly-specific siLuc complexed with lipidoid at lipidoid:siRNA ratios of
2.5:1, 5:1, 10:1, and 15:1 (wt:wt) to determine the optimum for transfection efficiency.
Transfections were performed in quadruplicate.
Working dilutions of each lipid were prepared (at concentrations necessary to
yield the different lipid/siRNA weight ratios) in 25 mM sodium acetate buffer (pH 5.2).
25 gIL of the diluted lipid was added to 25 gL of 60 jtg/mL siRNA in a well of a 96-well
plate. The mixtures were incubated for 20 min to allow for complex formation, and then
30 jiL of each of the lipidoid/siRNA solutions was added to 200 tL of media in 96-well
polystyrene plates. The growth medium was removed from the cells using a 12-channel
aspirating wand (V&P Scientific, San Diego, CA) after which 150 pLL of the
DMEM/lipidoid/siRNA solution was immediately added. Cells were allowed to grow for
1 day at 37 oC, 5% CO 2 and were then analyzed for luciferase expression. Control
experiments were performed with Lipofectamine TM 2000, as described by the vendor
(Invitrogen, Carlsbad, CA).
Firefly and Renilla luciferase expression was analyzed using Dual-Glo assay kits
(Promega, Madison, WI). Luminescence was measured using a Victor3TM luminometer
(Perkin Elmer, Wellesley, MA). A standard curve for luciferase was generated by
titration of luciferase enzyme (Promega, Madison, WI) into growth medium in an opaque
white 96-well plate.
Bone marrow-derived macrophage transfection. Murine bone marrow-derived
macrophages were cultures according to standard protocol 28. C57B1/6 mice expressing
GFP under the control of the RAGE locus promoter where used as a source of bone
marrow 29. Cells were cultured in 12-well dishes for 5 days in the presence of 8 ng/mL of
M-CSF. The optimal siRNA to lipidoid ratio was determined for each lipidoid (a ratio of
either 5 or 10 wt:wt was used). Mixtures siGFP or control siCD45 with lipidoids were
prepared as described above. LipofectamineTM 2000 and Lipofectamine TM RNAiMAX
(Invitrogen, Carlsbad, CA) were complexed with siRNA according to manufacturer's
instruction. siRNA-lipidoid mixtures were added to macrophage cultures at the desired
concentrations for 6 hr. Media was exchanged and GFP expression was analyzed by flow
cytometry 5 days later.
Lipidoid-siRNA Formulation. Lipidoid-based siRNA formulations comprised lipidoid,
cholesterol, poly(ethylene glycol)-lipid (PEG-lipid), and siRNA. Formulations were
prepared using a protocol similar to that described by Semple and colleagues 30, 31. Stock
solutions of 98N12-5(1)-4HCI MW 1489, mPEG2000-Ceramide C16 (Avanti Polar
Lipids, Alabaster, AL) MW 2634 or mPEG2000-DMG MW 2660 (synthesized by
Alnylam, see Supplementary Information), and cholesterol MW 387 (Sigma-Aldrich,
St. Louis, MO) were prepared in ethanol and mixed to yield a molar ratio of 42:10:48.
Mixed lipids were added to 125 mM sodium acetate buffer (pH 5.2) to yield a solution
containing 35% ethanol, resulting in spontaneous formation of empty lipidoid
nanoparticles. The resultant nanoparticles were extruded twice through a 0.08 pmn
membrane (Sterlitech, Kent, WA) using a LIPEXTM Extruder (Northern Lipids, Burnaby,
BC, Canada). siRNA 50 mM sodium acetate (pH 5.2) and 35% ethanol was added to the
nanoparticles at 1:7.5 (wt:wt) siRNA:total lipids and incubated at 37 'C for 30 min.
Ethanol removal and buffer exchange of siRNA-containing lipidoid nanoparticles was
achieved by tangential flow filtration against phosphate-buffered saline (PBS) using a
100,000 MWCO membrane. Finally, the formulation was filtered through a 0.2 pm sterile
filter (Pall, Ann Arbor, MI). Particle size was determined using a Malvern Zetasizer
NanoZS (Malvern, UK). siRNA content was determined by UV absorption at 260 nm,
and siRNA entrapment efficiency was determined by the Quant-iTTM RiboGreen@ RNA
assay (Invitrogen, Carlsbad, CA) 32. Resulting particles had a mean particle diameter of
approximately 50 nm, with peak width of 20 nm, and siRNA entrapment efficiency of
>95%.
Formulation of lyophilized materials for use in vivo. For acrylate-based lipidoids, 15
mg of lipidoid, 0.8 mg of cholesterol, and 7 mg of mPEG2000-ceramideCl6 (Avanti
Polar Lipids, Alabaster, AL) were dissolved in 2 mL of 25 mM sodium acetate in a 15
mL conical tube with vortexing for 5-10 minutes. For acrylamide based lipidoids, 15 mg
of lipidoid and 0.8 mg of cholesterol were first dissolved in 0.8 mL of ethanol. 7 mg of
mPEG2000-ceramideC16 and 1.2 mL of 25 mM sodium acetate were added subsequently
with continued vortexing. 20 mg of sucrose was added with vortexing. 0.1 mL of 10
mg/mL siRNA solution was added to 1.9 mL of 25 mM sodium acetate. This solution
was added to the solution containing the complexes and vortexed for an additional 20
min. The solution was extruded 11 times through 400 nm polycarbonate membranes
(Whatman, Florham Park, NJ) and .11 times through 200 nm polycarbonate membranes.
An additional 10 mg/mL of sucrose was added to the extruded samples. The samiples
were then frozen at -80 "C for 2 h and lyophilized overnight.
Determination of entrapment efficiency. A modified RiboGreen@ (Invitrogen,
Carlsbad, CA) assay was used to quantify entrapment of siRNA. Samples were diluted
1:200 in TE buffer and mixed with Quant-iTTM RiboGreen® RNA reagent (1:200 in TE
buffer) at 37 "C for 30 minutes in the presence or absence of 0.5 % final w/v Triton X100'. Entrapment efficiency of siRNA in lipidoid complexes was determined by
comparing the fluorescent signal of the lipidoid-siRNA sample in the presence (total
siRNA) and absence (free siRNA) of Triton X-100 detergent. Signal in the absence of
detergent yields the "free" or accessible siRNA fraction, while signal in the presence of
detergent yields the total siRNA content.
siRNA quantification by UV absorption. Formulated siRNA samples were diluted 1:10
in PBS (200 pL). 800 gtL of a 2.5:1 mixture of methanol:chloroform was added to the
diluted siRNA sample and mixed, resulting in a single clear phase. The absorbance at 260
nm was measured on a UV. spectrophotometer. siRNA was quantified based on an
experimentally-determined extinction coefficient for the duplex siRNA.
In vivo rodent Factor VII and Apob silencing experiments. All procedures used in
animal studies conducted at Alnylam Pharmaceuticals were approved by the Institutional
Animal Care and Use Committee (IACUC) and were consistent with local, state, and
federal regulations as applicable. C57BL/6 mice (Charles River Labs, Wilmington, MA)
and Sprague-Dawley rats (Charles River Labs, Wilmington, MA) received either saline
or siRNA in lipidoid formulations via tail-vein injection at a volume of 0.01 mL/g.' Serum
levels of Factor VII protein were determined in samples collected by retroorbital bleed
using a chromogenic assay (Coaset Factor VII, DiaPharma Group, West Chester, OH or
Biophen FVII, Aniara Corporation, Mason, OH). Liver mRNA levels of Factor VII and
Apob were determined using a branched-DNA assay (QuantiGene Assay, Panomics,
Fremont, CA) 8
In vivo mouse RSV silencing experiments. BALB/c mice (Harlan Sprague-Dawley
Laboratories, Indianapolis, IN) were anesthetized by intraperitoneal (i.p.) administration
of 2,2,2-tribromoethanol (Avertin) and instilled intranasally (i.n.) with lipidoid-siRNA
formulations in a total volume of 50 pL. At 4 h post-siRNA instillation, the mice were
anesthetized and infected i.n. with 106 PFU of RSV/A2 or RSV/B1. Prior to removal of
lungs at day 4 post-infection, anesthetized mice were exsanguinated by severing the right
caudal artery. Lung tissue was collected on ice in PBS to determine virus titers. RSV
titers from lungs were measured by immunostaining plaque assay. Lungs were
homogenized with a hand-held Tissumiser homogenizer (Fisher Scientific, Pittsburgh,
PA). The lung homogenates were placed on ice for 5-10 minutes to allow debris to settle.
Clarified lung lysates were diluted 10-fold in serum-free DMEM, added to 95% confluent
Vero E6 cells cultured in DMEM in 24-well plates, and incubated for 1 h at 37 'C,
followed by 2% methylcellulose overlay. At 5 days post-infection, the media was
removed, and the cells were fixed with acetone:methanol (60:40) and immunostained.
Plaques were counted and log (10) pfu/g lung versus PBS or siRNA mismatch control
was determined.
Silencing in peritoneal macrophages. C57B1/6J mice (Jackson Labs, Bar Harbor, ME)
were injected intraperitoneally with 1 mL of 4% Brewers Thioglycollate medium (Difco,
Franklin Lakes, NJ) 3 days prior to injecting 10 mg/kg of siCD45 or siGFP i.p (4 mice
per group). Peritoneal lavage was collected 4 days later and stained with fluorophore
conjugated antibodies to CD lb, Grl, and CD45 (BD Biosciences, Franklin Lakes, NJ).
Flow cytometry samples were run on a LSRII flowcytometer (BD Biosciences, Franklin
Lakes, NJ), and FlowJo software (Tree Star, Ashland, OR) was used to identify the
CD1 lbhighGrllow macrophage population and quantify CD45 expression.
In vivo miRNA silencing experiments. C57BL/6NCRL mice (Charles River Labs,
Sulzfeld, Germany) received lipidoid formulations of antagomir or anti-miR via tail-vein
injection at 5 mg/kg (0.5 mg/mL) on 3 consecutive days. Livers were taken at day 4, and
expression levels of miR-122 were determined. Liver tissue was dissolved in Proteinase
K-containing Tissue and Cell Lysis Buffer (Epicentre, Madison, WI) and subjected to
sonication. Total RNA was extracted with TE-saturated phenol (Roth, Karlsruhe,
Germany) and subsequently precipitated in ethanol. Synthetic DNA probes
complementary to mouse miR-122 as well as mouse U6 RNA were 5'-end labeled using
polynucleotide kinase (New England Biolabs, Ipswich, MA) and y-32P ATP (GE
Healthcare,
Munich, Germany).
Probe sequences
were: miR-122,
5'AAACACCATTGTCACACTCCACAGCTCTCTCTTCT
-3';
U6,
5'
CACGAATTTGCGTGTCATCCTTGCGCAGGGGCCATGTTCTTCTTCTTCTTC- 3.
Total liver RNA was simultaneously hybridized in solution to a miR-122-specific probe
and the U6 probe. The hybridization conditions allowed detection of U6 RNA and mature
miRNA but not pre-miRNA. Following treatment with S1 nuclease, samples were loaded
on denaturing 10% acrylamide gels. Gels were exposed to a phosphoimager screen and
analyzed on a Typhoon 9200 instrument (GE Healthcare, Munich, Germany). Relative
signal intensities of miR-122 versus U6 were calculated for each sample.
Expression level analysis of miR-122 target genes by branched DNA assay. The assay
was performed as described previously 4. Briefly, 30-50 mg of frozen liver tissue was
lysed in 1 mL Tissue and Cell Lysis Buffer (Epicentre, Madison, WI) by sonication. 1040 jiL lysate was used for the branched-DNA assay, depending on signal strength of the
target gene. Probe sets were designed using QuantiGene ProbeDesigner software. Target
gene expression was assayed according to QuantiGene Assay (Panomics, Fremont, CA)
recommendations and normalized to corresponding GAPDH housekeeper expression
from the same liver tissue lysate.
In vivo non-human primate experiments. All procedures using cynomolgus monkeys
were conducted by a certified contract research organization using protocols consistent
with local, state, and federal regulations as applicable and approved by the Institutional
Animal Care and Use Committee (IACUC). Cynomolgus monkeys (6 animals per group)
received either 5 mL/kg phosphate-buffered saline, 2.5 mg/kg formulated siCont (1.25
mL/kg), 2.5 mg/kg (1.25 mL/kg) formulated siApoB, or 6.25 mg/kg (3.125 mL/kg)
formulated siApoB as bolus i.v. injections via the brachial vein. For apoB-100 protein
measurements, serum was collected pre-dose and at 0.5, 1, 2, 3, 4, 6, 8, 11, 14, 17, 20, 23,
26, and 30 days post-administration. In a subsequent experiment, cynomolgus monkeys
(3 animals per group) received either 2.5 mg/kg formulated siCont or 2.5 or 6.25 mg/kg
of formulated siApoB as bolus i.v. injections via the saphenous vein. For apoB-100
protein measurements, serum was collected pre-dose and at 12, 24, and 48 h postadministration. ApoB-100 protein was determined using an ELISA assay as previously
described 8. Clinical chemistries were analyzed at pre-dose and 24 and 48 h postadministration. Hematology and coagulation parameters were analyzed at pre-dose and
48 h post-administration. Animals were sacrificed at 48 h. Liver Apob mRNA levels were
determined in liver samples using a branched-DNA assay (QuantiGene Assay, Panomics,
Fremont, CA) 8
FIGURES
Figure 1. Synthesis of Lipidoids. a) Alkyl-acrylate, alkyl-acrylamide, and amino
molecules were used to synthesize a combinatorial library of lipidoids. b) Synthesis
occurs through the conjugate addition of amines to an acrylate or acrylamide. Depending
on the number of addition sites in the amino monomer, lipidoids can be formed with
anywhere from 1 to 7 tails. Amino groups in the lipidoid can be quaternized by treatment
with methyl iodide. For ease of nomenclature, lipidoids are named as follows: (amine
number)(acrylate or acrylamide name)-(number of tails)("+" if quaternized).
Figure 2. In vitro screening of siRNA delivery, a) HeLa cells expressing both Firefly and
Renilla luciferase were treated with Firefly luciferase-targeting siRNA-lipidoid
complexes. The average % reduction in Firefly luciferase activity following treatment
with siRNA-lipidoid complexes at a 5:1 w/w ratio in quadruplicate is shown. For ease of
analysis, data is presented grouped as follows: (no test), 0-20% knockdown, 20-40%, 4060%, 60-80%, 80-100%. b) Optimized in vitro knockdown by lipidoids in HeLa cells.
Lipidoids were optimized for delivery using four lipidoid:siRNA ratios, 2.5:1, 5:1, 10:1,
and 15:1. Materials were tested in quadruplicate. Data are presented for the optimal
siRNA/lipidoid w/w ratio for each lipidoid, including standard deviation. This data set
includes only lipidoids with no significant cytoxicity, as assessed by no significant
change in Renilla luciferase expression relative to untreated cells. c) Dose response of
silencing in HeLa, d) HepG2, and e) primary macrophage cultures. Data were generated
in quadruplicate, as a function of siRNA molarity, at a lipidoid:siRNA ratio of 5:1 w/w,
including standard deviation. Day 5 GFP-expressing bone marrow-derived macrophage
cultures were incubated with siRNA-lipidoid complexes composed of the indicated
lipidoids or commercial transfection reagents (LipofectamineTM 2000 and
LipofectamineTM RNAiMAX) and siGFP or siCD45 for 6 hours. GFP expression was
quantified by flow cytometry. Silencing is expressed as the % of untreated cultures
performed in parallel.
Figure 3. In vivo delivery in rodents. a) Mice (n = 3) received 2 daily i.v. injections of
different lipidoid formulations of siRNA at a dose of 2.5 mg/kg. Factor VII protein levels
were quantified 24 h after the second administration. b) Simultaneous silencing of 2
genes in vivo in mice. Mice (n = 3) received a single i.v. bolus injection of a 98N 12-5(1)formulated 1:1 wt:wt mixture of siFVII and siApoB at 10, 6, or 4 mg/kg (5, 3, or 2 mg/kg
of each siRNA). For comparison, control animals received PBS or lipidoid-formulated
siFVII alone or lipidoid-formulated siApoB alone at 5, 3, or 2 mg/kg. 48 h after
administration, animals were sacrificed, and livers were harvested. Liver mRNA levels of
Factor VII or ApoB (normalized to GAPDH) were determined by branched-DNA assay.
c-e) Rats (n = 4) were injected with lipidoid-formulated siRNA at 1.25, 2.5, 5, and 10
mg/kg. Animals were bled and sacrificed 48 h after administration. c) Liver mRNA
levels. d) Serum Factor VII protein levels. e) Prothrombin time. f)Durability of silencing
in rats. Rats (n = 5) received a single i.v. administration of lipidoid-formulated siRNA at
5 mg/kg. Animals were bled at various time points post-administration, and serum Factor
VII protein levels were quantified. Data points represent group mean ± s.d. Data points
marked with asterisks are statistically significant relative to control-treated groups (* p <
0.05, ** p < 0.005, ***p < 0.001; t-test, single-tailed).
Figure 4. a) Inhibition of RSV/A2 in Balb/c mouse lungs. Mice (n = 5) were intranasally
administered "naked" siRNA or lipidoid-siRNA (siRSV or mm-siRSV) formulation at a
dose of 2 mg/kg. Lungs were harvested at day 4 post-infection and assayed by
immunostaining plaque assay. b) Inhibition of CD45 protein in thioglycollate-elicited
mouse peritoneal macrophages. Mice (n = 4) were injected with thioglycollate (i.p.) 3
days prior to treatment with 10 mg/kg of lipodoid-formulated siCD45 or siGFP
administered via i.p. injection. 4 days post-administration, CD45 expression on
macrophages was analyzed by flow cytometry. Macrophage cells were gated, and median
fluorescence intensity of the CD45 staining is plotted. c) Lipidoid delivery of antimiR122 in vivo. Nuclease protection assay and miRNA detection. Liver RNA samples of
3 representative animals per treatment group are shown. The U6 signal serves as RNA
input control. Animals treated with antagomirl22 show a marked decrease of miR-122
signal (lanes 7-9). Even further reduction of miR-122 signal is observed for lipidoidformulated anti-miR122 treated animals (lanes 10-12). The mismatched control
antagomir and anti-miR had little effect on miR-122 signals (lanes 4-6 and 13-15). d)
Derepression of miR-122 target genes following miR-122 inhibition in mice. Mice (n =
6) received i.v. injections of either lipidoid-anti-miR122 (black bars) or lipidoid-mm-antimiR122 (gray bars) at a daily dose of 5 mg/kg for 3 consecutive days. 24 h after the last
injection, expression levels of 7 reported miR-122 target genes were analyzed. Data
points represent group mean ± s.d. Data points marked with asterisks are statistically
significant relative to control-treated groups (* p < 0.05, ** p < 0.005, *** p < 0.001; ttest, single-tailed).
Figure 5. In vivo delivery in primates, a) Extent and duration of serum ApoB- 100 protein
reduction in cynomolgus monkeys following single bolus i.v. administration of lipidoidformulated siRNA. For all groups except saline control, n = 6 for data points up to and
including 2 d, and n = 3 for data points beyond 2 d. For saline control, n = 4 for data
points up to and including 2 d, and n = 2 for data points beyond 2 d. Data points represent
group mean ± s.d. No error bars are shown for saline group where n = 2. b-d) Animals (n
= 3) were treated with either formulated control siRNA at 2.5 mg/kg, formulated Apobtargeting siRNA at 2.5 mg/kg, or formulated Apob-targeting siRNA at 6.25 mg/kg. b)
Liver Apob mRNA levels normalized to GAPDH mRNA 48 h post-administration. c)
Serum ApoB-100 protein reduction at 12, 24, and 48 h post-administration as % of predose levels. d) LDL-C and HDL-C levels at 48 h post-administration, normalized to predose levels. Data points represent group mean ± s.d. Data points marked with asterisks
are statistically significant relative to control-treated groups (* p < 0.05, ** p < 0.005,
"** p < 0.001; t-test, single-tailed).
Disclosure: While the author performed synthesis, screening, purification,
characterization, and formulation, the animal studies and non-hepatocyte-targeted
delivery efforts were conducted by his collaborators at Alnylam.
Figure la.
O
1
6
0.-.NH2
33 HO-
NH2
.O•
34
J
HO
HO.NH2
36
HO
38
HO
7 >(OrNH
0
woc
10 ,O
11
013
•'NH 2
OvNHz
13
82
60
NH2
0
17
0
20
HO•'-
21
XNH
0NH
L-
25
HO
Ho
63
Sv
vvvv-N'
H
N
64
91
H
NH,
'-N
ON
NH,
N."
94HN-NH
H
2
N
111
H
H
N
H
H
113 H
114
,,N-NH
H2N
F
-N
96
"HN
98
H2N--- H
NH2
116
NH
NH2
99
26
H
28 HO
77
HO,->.K6H
NH
H2N,-NNH2
NH
,
2
79
N75
31
H
HO0
32 HO
N
O"NH2
-'ý
100 H
f '
NV
I NH2
.Nv-.
80 /N
NH2 81
N
NH
HON-_N-•-OH
H
117
NH,
103
H
H
109 H•-N•J-nH
I,·-
NH2
N H2
H
O
Figure lb.
0
1
o
Rj-O ký
+
H 2 N-R
AT
0
R1-O
2
N-R
2
Mel .
R 1-O
R-O O
O
O0
R 1 N-N
H
+
H2N
NH2 -
N-R,
H
R1-N
AT
,-
H
H
HR
R1-N
NH,
H2 N
NH2
2
76 CN -,NH2
2
NH
NH
H0
NH
NH,
N
115
95
H
N-'NNH2
75
HN
H 112 H
NH91 12H,
\
94N
H
N
90QON---NH2
-N
H
NH2
110 H2N
NH2
70 HN,__NH
O
N16
- /- -
HO
HO
2
NH 2
0
HO,, N
N
H
2
NH2
HOv ZNH
~~Nkl
87
93
.-
OH
22 ,,- NH
2
H
HO'N
NH2
H
SN,
N
H
61
o
H
86
-NH2
HO
NH2
H
15 -0O
•
CN -- NH,
90
2
-0 -NH
NH2
0N-R,
O
Figure 2a.
010 01013
01015 01N1214N145NsN1 10
Q Q13
NO TEST
0-20
20-40
40-60
10
60-80
11
4
18
1
6
7
80-100
13
15
17
20
21
22
24
25
26
28
31
32
33
34
36
38
60
61
62
63
64
70
75
77
79
80
81
82
8
87
91
93
94
95
96
98
103
109
Figure 2b.
100
90
80
70
60
50
40
30
20
10
0
~·,zt
~
Aa·~~~~t:P"~~~~~~~~~
5,·.ýNN umNA
NV
N"NN'Vj
NN~~3rZ
Figure 2c.
140
0 0.0 nM
M0.4nM
M1.1 nM
S3.3 nM
E10 nM
5 30 nM
090 nM
98N12
110N9
111N9
112N9
114N9
115N9 LF2000
RNAi
MAX
Figure 2d.
O 0.0nM
E 0.4 nM
N 1.1nM
* 3.3 nM
10 nM
1 30 nM
090 nM
98N12
110N9
111N9
112N9
114N9
115N9
LF2000
RNAi
MAX
Figure 2e.
120 1
10080-
* 0.4 nM
0 1.1 nM
E 3.3 nM
l0 nM
98N12
110N9
111N9
112N9
114N9
115N9
LF2000
RNAi
MAX
Figure 3a.
2.0
1.8
1.6
C
Z 1.4
1.2
S1.0
U.
0.8
Z 0.6
0.4
0.2
0.0
0.0
(o o
3b.aFigure
D, ~
(Z
o0 oC
~m0090~~0
'V
0
•
Z
0
0
,
0
0
0
o
I
"-
Figure 3b.
2.0
1.8
w 1.6
E
m 1.4
- 1.2
Q 1.0
0.8
S0.6
c
0.4
0.2
0.0
5 mg/kg
3 mg/kg
2 mg/kg
5 mg/kg
3 mg/kg
2 mg/kg
10 mg/kg
6 mg/kg
4 mg/kg
1.6
1.4
ES1.2
> 1.0
...J 0.8
0.6
**
S0.4
iC
* = <0.05
**= <0.005
0.2
0.0
5 mglkg
3mg/kg
2 mg/kg
5 mglkg
3mg/kg
2 mg/kg
10 mg/kg
6 mg/kg
4 mg/kg
Figure 3c.
1.4
<t
1.2
z
~
E 1.0
S
LL.
"Q)
0.8
>
:J
Q)
> 0.6
'';:;
~
Q)
~
0.4
** = <0.005
*** = <0.001
0.2
0.0
PBS
siCont
siFVII
siFVII
siFVII
siFVII
10mg/kg
10mg/kg
5mg/kg
2.5mg/kg
1.25mg/kg
Figure 3d.
1.4
1.2
-
1.0
S
LL.
0.8
t:
'ai
0
0-
"-
*
Q)
>
'';:;
~
0.6
Q)
~
0.4
0.2
* = <0.05
= <0.001
***
***
0.0
PBS
siCont
siFVII
10mg/kg
10mg/kg
siFVII
5mg/kg
siFVII
siFVII
2.5mg/kg
1.25mg/kg
52
Figure 3e.
40
35
- 30
E
S25
T
E 20
2
1r
1
15
1-
10
PBS
siCont
siF
VII
siFVII
siFVII
siFVII
**= <0.005
0
PBS
siCont
siFVII
siFVII
siFVII
siFVII
10mglkg
10mglkg
5mg/kg
2.5mglkg
1.25mglkg
Figure 3f.
I.0
1.4
1.2
0 1.0
> 0.8
0)
0.6
0.4
0.2
0.0
0
5
10
15
Time (d)
20
25
30
Figure 4a.
* *, *
I
6
I
__.
I
***
Cm
5,
c
I
7
40
U.
.3-
o1
0L
* = <0.001
v
Saline
mm-siRSV
siRSV
98N1 2 5-
mm-siRSV
98N 12 5siRSV
Figure 4b.
150-
,
100-
U,
O
10
1000
100
CD45
P9 4
Figure 4c.
t-I.Cli1
PBS
MM-Antagomirl22 AntagomirI22
2 3
4
5
6
7
8
9
98N12-5
Anti-miR122
98N12-5
MM-Anti-miR122
10 11 12 13 14 15
]U6
miRI22
S4
SS
SS
A4
A5
A86
4
B5
B6
C4 CS
CS
D4
D5
D6
animal #
Figure 4d.
3.5 -
SMM-Anti-mirl22
**
U
**
Anti-mirl 22
3.0 0
t-
2.5 -
I
C)
x 2.0
w
I-
T
I
-
1.5
1.0* = <0.05
** = <0.005
-- r-
Gpx7
Hfe2
-- "r--
---'r--
___
Lass6
Ir
"---r-
-r
Sic35a4
Tmed3
Tmem50Ob
AldoA
Figure 5a.
160
140
-0-- PBS
--
siCont 2.5 mglkg
-- U- siApoB 2.5 mg/kg
---
siApoB 6.25 mg/kg
120
100
80
60
40
0
5
10
15
20
Time Post-dose (Days)
25
Figure 5b.
1.2
z
LL
E
0.8
0.6
i
-J
0,
.s
(FI
*= <0.05
***= <0.001
4)
0ý
siCont
2.5 mg/kg
siApoB
6.25 mg/kg
siApoB
2.5 mg/kg
Figure 5c.
140120r--L-
100-
12 h
24 h
fT
48 h
8060T
*:
40-
I
20-
I
0siCont
2.5 mg/kg
I
siApoB
2.5 mg/kg
I I
* = <0.05
L
siApoB
6.25 mg/kg
Figure 5d.
1.4
m LDL m HDL
1.2
1.0
I
0.8-
I
0.60.40.20n."'
+-
siCont
2.5 mg/kg
siApoB
2.5 mg/kg
siApoB
6.25 mg/kg
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CHAPTER 3:
FORMULATION OPTIMIZATION OF A HIGHLY-EFFECTIVE LIPIDOID
CARRIER FOR THE SYSTEMIC DELIVERY OF RNAI THERAPEUTICS
ABSTRACT
RNAi therapeutics afford the potential to silence target gene expression
specifically, preventing the accumulation of disease-causing proteins. Safe and effective
systemic siRNA delivery in vivo remains an obstacle. Cationic liposomes are currently
the most widely-validated means for systemic delivery of siRNA to the liver, having been
demonstrated in several animal models, including non-human primates. Recently, we
described the synthesis and screening of a novel combinatorial lipidoid library and the
identification of novel highly-effective vector for siRNA delivery, 98N 12-5(1). Here, we
depict the process of formulation optimization for this lipidoid to maximize its efficacy
and minimize its toxicity in murine models. The relative amount of formulation
constituents as well as differences as little as one methylene group in PEG-lipid length
greatly affect extent of silencing, weight gain, and clearance time. Optimally-formulated
siRNA remains stable in solution at 4 OC, 25 OC, and 37 'C for over five months and
retains in vivo activity after six months of storage at 4 OC.
INTRODUCTION
The emergence of genetic therapies will potentially enable physicians to replace
defective genes and modulate their expression specifically '. The primary impediment to
the prevalent administration of RNAi therapeutics is efficient and safe nucleic acid
delivery into mammalian cells in vivo 2. Because virus-mediated delivery poses vast
safety concerns 3., non-viral nucleic acid delivery has attracted a great deal of interest s
In addition to the physical manipulation of naked nucleic acids 6, synthetic vectors have
been created to improve biostability 7. Specifically, polycationic lipids can
electrostatically bind and condense nucleic acids to form nanometer-sized complexes 8,
whose small size enhance their cellular uptake 9.
Identified by Fire and Mello 10, RNA interference offers a powerful platform for
functional genomics, in vivo target validation, and gene-specific medicines ". This
catalytic natural pathway, found in all eukaryotic cells, is inherently potent. Unlike small
molecules, which are generally limited to binding to and blocking ion channels,
receptors, and enzymes, siRNAs can conceivably be used to modulate the expression of
any gene transcript - endogenous or exogenous - in a highly-specific manner
Clearly, with only a limited number of examples of systemic siRNA
administration published to date 12-24, the development of additional carriers is desirable.
Many in vitro siRNA transfection reagents are ineffective or toxic in vivo. Cationic
liposomes are currently the most widely-validated means for systemic delivery of siRNA
to the liver, whose cells are accessible through fenestrated endothelium (100-150 nm) by
nanoparticles that are engineered to be less than 100nm in diameter. In addition to
protecting nucleic acids from degradation in serum, such particles inhibit renal clearance
and excretion. Liposomes are believed to enter cells by endocytosis 25, 26. thus, "proton
sponge"-mediated endosomal escape enhances payload delivery into the cytoplasm 27. In
addition to promoting delivery by lipid mixing (fusion) with endosomal membranes,
standard cationic lipids are thought to destabilize endosomes by interacting with anionic
lipids in the endosomal membrane 28. Excessive positive charge can, however, have
negative consequences in vivo, including non-specific interactions with biological
surfaces, increased protein binding, opsonization, rapid clearance by the
reticuloendothelial system, hemolysis, and cytotoxicity 29. For this reason, formulations
must be tailored to discover the optimal balance of components.
The synthesis and screening of a novel combinatorial cationic lipidoid library has
led to the identification of a new non-toxic, non-viral vector that is able to transfect
mammalian cells in vivo following systemic administration 30. Efficacy has been
demonstrated in multiple animal species, including non-human primates, and more than
10 genes have been silenced. To maximize efficacy and minimize toxicity, siRNA and
lipidoid were combined with varying amounts of cholesterol and PEG-lipid. Here, we
depict the formulation optimization for this top-performing lipidoid, 98N 12-5(1).
RESULTS AND DISCUSSION
A combinatorial lipidoid library was synthesized and screened for the ability to
transfect mammalian cells with siRNA in vitro 30. The top 20-performing lipidoids were
subsequently administered to mice systemically by intravenous injection. While several
lipidoids yielded respectable levels (>50%) of mRNA and protein knockdown in vivo,
98N 12 -5(1) conferred drastic (>99%) post-transcriptional gene silencing in the absence of
associated toxicity. Importantly, administration of 98NI 2-5(1) has resulted in delivery to
three tissue types in four distinct species following a single injection 30. Here, we describe
the process of formulation optimization for the in vivo systemic administration of 98N1 2 -
5(1).
Preliminarycharacterization
Introductory experiments examined the relative efficacies and toxicities of
laurylaminopropionate (acrylamide N12 ) and triethylenetetramine (amine 98) when
combined in various stoichiometric ratios. The sole product that induced appreciable
knockdown was the one synthesized at the maximal ratio of 6:1. Because
triethylenetetramine comprises four nitrogen atoms - two primary and two secondary
amines - there were several possible structures for the active component.
Extensive purification procedures based on flash chromatography resulted in the
isolation of the variously-substituted products. The pure products were tested in vivo and
further characterized. One product generated the greatest knockdown and least toxicity.
Matrix-assisted laser desorption/ionization mass spectrometry confirmed that the active
species contained five laurylaminopropionate units or tails 30. Nuclear magnetic
resonance studies distinguished that the isomer with one unsubstituted internal amine
(isomer 1) is more efficacious than the isomer with one unsubstituted terminal amine
(isomer 2), achieving >99% protein knockdown (Fig. 1).
Target gene silencing was determined by inspecting serum levels of Factor VII in
mice following a single tail-vein injection. Factor VII, a protease involved in the bloodclotting cascade ", was used as a reporter protein because it has the shortest plasma half-
life (2-5 hours) among the clotting factors 32. Thus, silencing at the mRNA level is
manifest as silencing at the protein level with minimal lag. Prior to injection,
nanoparticles containing Factor VII-specific siRNA were analyzed to determine their
size, percent siRNA encapsulation, and concentration.
Formulationoptimization
The first formulations comprised lipidoid, cholesterol, PEG-lipid, sucrose, and
siRNA in varying ratios. Initially, the formulations were extruded through 200 nm filters
11 times, but much material was lost on the membranes. The minimal solubility of
lipidoid and cholesterol in 25 mM sodium acetate buffer (pH 5.2) proved to be a great
hindrance. This buffer was used because it was the solvent used for the in vitro studies, as
the acidity promoted protonation of the amine backbone, which resulted in spontaneous
electrostatic condensation of the siRNA. The rre-dissolution of lipid in 100% ethanol
was a major breakthrough, decreasing
(comprising up to 35% of the total volume)
material loss and extrusion pass number. The ethanol was removed post-extrusion by
dialyzing against phosphate-buffered saline for 90 minutes at room temperature.
Originally, sucrose was subsequently added as a cryoprotectant, as the samples were
lyophilized for storage prior to injection. Once it was determined that the formulations
retained activity after months of storage in solution, lyophilization was discontinued.
Cholesterol was added to increase circulation time and drug retention 34. PEG is
known to solubilize the carrier, to prevent non-specific fusion of liposomes with cell
membranes, to shield electrostatic charge, to minimize immunogenicity, and to hinder
renal excretion 35, 36. The use of other co-lipids, such as dipalmitoylphosphatidyl-choline
(DPPC) and distearoylphosphatidylcholine (DSPC), resulted in decreased efficacy when
used in conjuction with 98N 12-5(1). Many PEG-lipid conjugates - with PEG molecular
weights ranging from 750 to 5000 and lipid components including phophoethanolamine
and sphingosine - purchased from Avanti Polar Lipids Inc. (Alabaster, AL), were used in
the formulation of the siRNA-containing nanoparticles. Ultimately, N-palmitoylsphingosine-l-[succinyl(methoxy polyethylene glycol) 2000] (mPEG2000-Ceramide
C16) and mPEG2000-Glyceride C14 (synthesized by Alnylam) were found to induce the
greatest knockdown and least toxicity.
Titrations were performed to determine the optimal composition of the
nanoparticles. For each of the four components, the amounts of three were held fixed
while the fourth was varied. Specifically, initial studies using a wide range of PEG-lipid
and cholesterol masses indicated that 7 mg of PEG and 5 mg of cholesterol granted the
best ratio of efficacy-to-toxicity ratio when added to 15 mg of 98N 12-5(1) in a total of 4
mL formulation volume (65% 200 mM sodium acetate, 35% ethanol). Adding fewer than
7 mg of PEG-lipid resulted in significant weight loss, as did adding more than 10 mg of
cholesterol. Weight loss is used as a surrogate for tolerability as it is sensitive to and
correlates well with observed toxicology parameters, including detectable changes in
hematology (PLT reduction) and clinical chemistry (ALT elevation). The percent
knockdown increased as the amount of cholesterol approached 5 mg. A cholesterol
titration up to 5 mg was performed to confirm that 5 mg was the optimal amount to
maximize the efficacy-to-toxicity ratio (Fig. 2). This titration was performed using 15 mg
of 98NI 2-5(1) and 7 mg of either mPEG2000-lipid or mPEG5000-lipid to determine the
effect of PEG chain length on efficacy. mPEG2000-lipid was shown to induce greater
knockdown.
The therapeutic index can be improved not only by optimizing the component
ratios but also by optimizing the structure of the PEG-lipid, while keeping all other
structures fixed. The lipid portion of the PEG-lipid conjugate enables PEG to be
physically, rather than covalently, incorporated into the liposomes, allowing the
deshielding rate to be controlled by varying the lipid anchor length. PEGylation has a
significant effect on particle properties, altering the particles' pharmacokinetics and
biodistribution. Additionally, it impacts liposomal interaction with cells, biological
matrices, and blood components, which, in turn, impact efficacy and toxicity.
Since PEG2000 was demonstrated to be most effective, the length of the lipid
component was subsequently investigated. A glycerol backbone was derivatized with one
PEG chain and two alkyl chains of various lengths, ranging from C10-C16. These PEGlipids were incorporated into the formulation, and efficacy and tolerability were
compared in mice (Fig. 3a). A serial dilution dose-response study was performed from 20
mg/kg to 1.25 mg/kg, and C14-C16 were confirmed to maximize the therapeutic index.
As mentioned, increasing the lipid anchor length is believed to control rate of
disassociation of PEG-lipid from liposome. The data confirm that increasing PEG
shielding improves tolerability but decreases efficacy. PEG deshielding can also be
achieved by reducing the molar percent of the PEG-lipid in the formulation; however,
this results in larger particles, which may explain why they are less efficacious in
delivery to hepatocytes. The improvement in tolerability and decrease in efficacy appear
more pronounced in rats as PEG-lipid anchor length increases (Fig. 3b).
Once the optimal amounts of cholesterol and PEG-lipid - as well as length and
type of PEG-lipid - were determined, an RNA titration was performed. Maintaining the
molar ratio of lipidoid:cholesterol:PEG-lipid at 42:48:10, the weight-to-weight ratio of
the lipids to siRNA was systematically varied (Fig. 4). The data suggest that formulation
tolerability is increased as the lipid:siRNA ratio is reduced and that a ratio of total lipids
to siRNA of -7.5:1 (w/w) maximizes the loading of siRNA to saturation, thereby
imparting the greatest efficacy-to-toxicity ratio. Accordingly, through sequential titrations
of the various formulation components, the optimal weight-to-weight ratios of
constituents was determined to be 98N 12-5(1):cholesterol:mPEG2000-Glyceride
C14:siRNA = 15:5:7:3.5.
Having determined its ability to induce potent knockdown in the absence of
toxicity, we elected to examine the biodistribution, dosing, stability, and size of this
optimized formulation, LNPO1. The formulation is cleared from the plasma very rapidly,
with a plasma half-life in cannulated rats of less than 15 minutes, and is almostimmediately accumulated in entirety in the liver (the maximal accumulation in the lung
or kidney is less than 0.1%). The signal of fluorophore-labeled siRNA attenuates in the
liver with a half-life of -1.5 hours (Fig. 5a).
The possibility of coding region-targeted siRNA-mediated transcriptional gene
silencing
Remarkably, silencing persists for more than three weeks after a single bolus
intravenous injection in non-human primates 30 and in mice at doses as low as 5 mg/kg
(Fig. 5b). Owing to protein turnover and siRNA degradation, Ago2-mediated cleavage of
target mRNA may no longer a factor at such later time points - although the half-life of
holo-RISC is unknown, RISC has been shown to reload after 9-12 h in the presence of
surplus siRNA 37 - SO this observation reveals the principal possibility of gene silencing
at the transcriptional level. While the role of RNAi in heterochromatin formation has
been studied primarily in A. thaliana,S. pombe, and D. melanogaster3 8, 39 , it is certainly
possible that siRNA mediates epigenetic changes in trans. Thus, while some antisense
("guide") strands remain in the cytoplasm as components of activated RISC, other
molecules may be trafficked to the nucleus to silence gene expression in a manner similar
to that recently reported for endogenous antisense transcripts o
Such migration of small RNAs that initiate RNAi in the cytoplasm into the
nucleus, where they induce methylation of homologous DNA, has been observed in plant
cells 41. Moreover, the ability of mammalian cells to translocate small RNAs from the
cytoplasm to the nucleus has been confirmed by the presence of mature miRNAs - which
are produced in the cytoplasm - in nuclear cellular fractions in addition to cytoplasmic
fractions 42. siRNAs may also interact with DNA when the nuclear envelope breaks down
during mitosis.
Transcriptional gene silencing (TGS) owing to DNA methylation 43 or histone
methylation 44 has been observed upon administration of promoter-targeted siRNA,
confirming that siRNA-directed transcriptional silencing is conserved in mammals. The
observation of RNA-directed DNA methylation in mammalian cells is consistent with the
fact that these cells possess homologs of most of the enzymes required by plants to
perform this operation 45. Excitingly, the histone (H3K9 and H3K27) methylation was
determined to be mediated by the siRNA guide strand 44, as suggested above. Notably,
TGS in human cells does not require both forms of epigenetic control; it has been
realized in the absence of DNA methylation 46 or histone modification 47. While the
mechanism by which siRNA-mediated TGS is achieved is poorly understood, the process
appears to involve communication between Ago2 and Agol 47 as well as Polycomb-based
silencing 48. Unlike all reports of siRNA-induced TGS to date, our study does not employ
promoter-targeted siRNA. Thus, further studies are required to confirm our hypothesis of
potential coding region-targeted siRNA-mediated TGS.
Dosing scheme influences knockdown
Interestingly, dosing scheme also has a dramatic effect on efficacy. Multiple
dosings were compared to a single administration, and the data indicate that dividing the
dosage over multiple days is not equivalent to a single bolus administration of the same
dose (Fig. 6a). Dose additivity in mice was further investigated. The results suggest a
clear time dependence on dose additivity. Specifically, doses are roughly additive up to a
30-minute interval between dosings, whereas two doses at 1.5 mg/kg are not significantly
more efficacious than a single 1.5 mg/kg dose for intervals greater than 30 minutes
between dosings (Fig. 6b).
LNPO1-formulated siRNA remains stable and active for several months
Finally, the physical properties of the formulated particles were observed to be
very important to their function. The formulation provides stability to siRNA in serum,
increasing the half-life of full-length product from -15 minutes for unformulated material
to over 24 hours (Fig. 7a). The particles remain stable, as determined by particle size and
percent siRNA entrapment, for over five months (Fig. 7b). They have a mean diameter of
-50 nm (Fig. 8a), and smaller particle size improves efficacy and tolerability (Fig. 8b).
The formulation has minimal positive charge (+2-4 mV, as measured by Zeta potential),
which facilitates interaction with the negative surfaces of cells but is insufficient to
induce non-specific toxicity.
CONCLUSION
RNAi has the potential to become a new major class of drugs, as it represents a
fundamentally new way of treating disease. Specifically, disease is generally caused by
the inappropriate behavior of proteins, and RNAi allows protein production to be
inhibited selectively. RNAi therapeutics can be used to address a broad range of unmet
medical needs. Thus, the introduction of a novel, effective, and safe vector for siRNA
delivery offers tremendous opportunities. Because RNAi is a platform technology, one
vector can potentially be applied to sundry diseases, as the lipidoid is presumably
indiscriminate to siRNA sequence. Potential therapeutic applications include Alzheimer's
disease, amyotrophic lateral sclerosis, cirrhosis, coronary heart disease, diabetes,
influenza, cancer, and pain. Hopefully, LNP01 will enable scientists and physicians to
address some of these diseases. This formulation has been used successfully to silence
multiple (>10) genes in multiple species (mouse, rat, hamster, monkey). Efficacy is
observed in the range of 2-5 mg/kg for a numerous hepatic targets. This study
demonstrates the importance of formulation optimization to maximize the therapeutic
index of novel treatments.
MATERIALS AND METHODS
98N 12-5(1) Synthesis, Purification, and Characterization. Chemicals were purchased
from Aldrich (Milwuakee, WI). Laurylaminopropionate (acrylamide N12) was
synthesized by the drop-wise addition of 3 molar equivalents of acryloyl chloride to
dodecylamine in dry THF at 0 oC in the presence of 4 molar equivalents of triethylamine
with stirring for 12 h. The reaction mixture was dissolved in ethyl acetate and washed
with 5% hydrocloric acid and water three times. The organic phase was dried using
Na2 SO 4 , and the solvent was evaporated under reduced pressure. The product was
recrystallized from hexanes. The crude material was then purified using silica gel column
chromatography, running a solvent gradient (hexane:ethyl acetate= 1:0; 3:1; 1:1; 1:3;
0:1). The structures of the pure products were confirmed by 1H NMR, FTIR, MALDITOF-MS. Six equivalents of laurylaminopropionate were added to one equivalent of
triethylenetetramine. The solvent-free reaction mixture was stirred for 7 days at 90 IC
under nitrogen.
The reaction does not go to completion under these conditions. Chromatographic
purification was performed by flash chromatography using Merck Silica Gel 60. A
mixture of dichloromethane (75%), methanol (22%), and ammonium hydroxide (3%) was
combined with a gradient of increasing additional dichloromethane to yield good
separation of the variously-substituted triethylenetetramine products. TLC was performed
on Merck Silica Gel 60 F254 TLC glass plates, and the isomers were visualized with
ninhydrin stain. Solvent removal was performed by evaporation on a Biichi rotavapor
with heating to 40 oC.
siRNA Synthesis. Synthesized by Alnylam, the siRNA was characterized by ESMS and
anion-exchange HPLC.
siFVII sense: 5'-GGAucAucucAAGucuuAcT*T-3',
antisense: 5'-GuAAGAcuuGAGAuGAuccT*T-3'.
2'-O-Me modified nucleotides are in lower case, 2'-Fluoro modified nucleotides are in
bold lower case, and phosphorothioate linkages are represented by asterisks. siRNAs
were generated by annealing equimolar amounts of complementary sense and antisense
strands.
Lipidoid-siRNA Formulation. Lipidoid-based siRNA formulations comprised lipidoid,
cholesterol, poly(ethylene glycol)-lipid (PEG-lipid), and siRNA. Formulations were
prepared using a protocol similar to that described by Semple and colleagues 30,31. Stock
solutions of 98N 12-5(1)-4HCl MW 1489, mPEG2000-Ceramide C16 (Avanti Polar
Lipids, Alabaster, AL) MW 2634 or mPEG 20oo-Glyceride C14 MW 2660 (Alnylam,
Cambridge, MA), and cholesterol MW 387 (Sigma-Aldrich, St. Louis, MO) were
prepared in ethanol and mixed to yield a molar ratio of' 42:10:48. Mixed lipids were
added to 125 mM sodium acetate buffer (pH 5.2) to yield a solution containing 35%
ethanol, resulting in spontaneous formation of empty lipidoid nanoparticles. The resultant
nanoparticles were extruded twice through a 0.08 inm membrane (Sterlitech, Kent, WA)
using a LIPEXTM Extruder (Northern Lipids, Burnaby, BC, Canada). siRNA in 50 mM
sodium acetate (pH 5.2) and 35% ethanol was added to the nanoparticles at 1:7.5 (wt:wt)
siRNA:total lipids and incubated at 37 'C for 30 min. Ethanol removal and buffer
exchange of siRNA-containing lipidoid nanoparticles was achieved by tangential flow
filtration against phosphate buffered saline using a 100,000 MWCO membrane. Finally,
the formulation was filtered through a 0.2 gIm sterile filter. Particle size was determined
using a Malvern Zetasizer NanoZS (Malvern, UK). siRNA content was determined by
UV absorption at 260 nm, and siRNA entrapment efficiency was determined by the
Quant-iTTM RiboGreen® RNA assay (Invitrogen, Carlsbad, CA). The particles had a
mean particle diameter of approximately 50 nm, with peak width of 20 nm, and siRNA
entrapment efficiency of>95%.
Determination of entrapment efficiency. A modified RiboGreen@ (Invitrogen,
Carlsbad, CA) assay was used to quantify entrapment of siRNA. Samples were diluted
1:200 in TE buffer and mixed with Quant-iTTM RiboGreen® RNA reagent (1:200 in TE
buffer) at 37 OC for 30 minutes in the presence or absence of 0.5 % final w/v Triton X100. Entrapment efficiency of siRNA in lipidoid complexes was determined by
comparing the fluorescent signal of the lipidoid-siRNA sample in the presence (total
siRNA) and absence (free siRNA) of Triton X-100 detergent. Signal in the absence of
detergent yields the "free" or accessible siRNA fraction, while signal in the presence of
detergent yields the total siRNA content.
In vivo rodent Factor VII silencing experiments. All procedures used in animal studies
conducted at Alnylam were approved by the Institutional Animal Care and Use
Committee (IACUC) and were consistent with local, state, and federal regulations, as
applicable. C57BL/6 mice (Charles River Labs, Wilmington, MA) and Sprague-Dawley
rats (Charles River Labs, Wilmington, MA) received either saline or siRNA in lipidoid
formulations via tail vein injection at a volume of 0.01 mL/g. Serum levels of Factor VII
protein were determined in samples collected by retroorbital bleed after 24 or 48 hours
using an activity-based chromogenic assay (Coaset Factor VII, DiaPharma Group, West
Chester, OH or Biophen FVII, Aniara Corporation, Mason, OH). All experiments were
performed in triplicate.
FIGURES
Figure 1. a) Five-tailed triethylenetetramine-laurylaminopropionate (98N12) with free
internal amine (isomer 1) and free terminal amine (isomer 2). b) Both five-tailed isomers
of 98N 12 were administered (3.0 mg/kg does) via a single tail-vein injection. Relative
plasma level protein is shown after 24 h. Each experiment was performed in triplicate.
Figure 2. A cholesterol titration using a) mPEG2000-Ceramide C16 and b) mPEG5000Ceramide C16. Relative plasma level protein, 24 h after a single tail-vein injection, is
shown by the bars with y-axis on the left-hand side. Average weight gain (g) is
represented by * with y-axis on the right-hand side. Cholesterol mass and dosing
concentration are indicated. The same effect is observed at different doses. Maximal
knockdown is achieved using 5 mg cholesterol per 15 mg of lipidoid. While the trend
continues with increasing amounts of cholesterol, toxicity is observed with these higher
amounts (data not shown). Each experiment was performed in triplicate.
Figure 3. The optimal alkyl chain length for the PEG-lipid was determined to be between
C14-C16. a) C57BL/6 mice (n = 3) were treated with a single bolus intravenous injection
of siRNA formulated with 98N 12-5(1), cholesterol, and PEG-glyceride with alkyl chain
lengths ranging from C10-C16 at either 20 mg/kg or 2.5 mg/kg. Relative serum Factor
VII levels and body weights were measured after 48 h. b) Based on these results,
Sprague-Dawley rats (n = 5) were treated with a single bolus intravenous injection of
siRNA formulated with 98N 12-5(1), cholesterol, and PEG-glyceride with alkyl chain
lengths ranging from C14-C16. Serum Factor VII levels relative to PBS control were
measured after 48 h.
Figure 4. a) The RiboGreen® RNA Assay (performed in quadruplicate) revealed that
total lipids:siRNA (w/w) of 5:1 contains excess siRNA. Excess siRNA was removed by
ultrafiltration through 100,000 MWCO hollow-fiber cartridge. Saturation is achieved at
total lipids:siRNA (w/w) -7.5:1. b) Lipid nanoparticles (LNP) prepared with an initial
excess of siRNA that is removed by ultrafiltration is efficacious. c) Animal weight gain
after a single 10 mg/kg injection. No appreciable weight changes were observed at 3
mg/kg, 2 mg/kg, or 1.5 mg/kg). Each experiment was performed in triplicate.
Figure 5. a) Kinetics of liver distribution of Cy3-labeled siRNA formulated in LNP01 in
C57BL/6 mice at a dose of 2.5 mg/kg. b) Silencing is potent and durable. C57BL/6 mice
(n = 5) were treated with a single bolus intravenous injection of LNP01-formulated
siRNA at a dose of 5 mg/kg.
Figure 6. Cmax is critical for efficacy. a) C57BL/6 mice received either a single injection
of 1.5 mg/kg, 3.0 mg/kg, or 4.5 mg/kg or a dose of 0.5 mg/kg, 1.0 mg/kg, or 1.5 mg/kg
for three consecutive days. Serum factor VII levels were measured 96 h after the first
injection. b) Formulated siRNA was administered to C57BL/6 mice as two consecutive
1.5 mg/kg doses with different times in between the two doses (15 min, 30 min, 1 h, 2 h,
and 4 h). Single doses of 3 mg/kg and 1.5 mg/kg were included for comparison. Serum
Factor VII levels were measured 48 h after the first injection. Each experiment was
performed in triplicate.
Figure 7. a) Unformulated and formulated siRNAs incubated in human serum at 37 oC.
The percent of full-length product was determined by HPLC. b) Formulations are stable
in solution. These particles maintain in vivo activity after 6 months of storage at 40 C.
Figure 8. Hepatocyte delivery requires particles with a mean diameter <100 nm. a) The
optimal formulation has a reproducible mean diameter -50 nm, as determined by
dynamic light scattering. b) Smaller particle size improves efficacy and tolerability.
Relative serum Factor VII levels and weight gains were determined following a single
bolus intravenous injection in C57BL/6 mice. Each experiment was performed in
triplicate.
Disclosure: While the principal formulation optimization was performed by the author,
the subsequent studies (determination of optimal PEG alkyl chain length, optimal dosing,
and optimal particle size) were conducted by his collaborators at Alnylam.
Figure la.
Isomer 1
H
H
H
H
N
H
Isomer 2
H
H
O
O
O
NNNH
N
H
0
NH
N0
H
Figure lb.
12..
"
-
1
0.8
0.6
-
0.4 0.2
-
0-
-I
PBS
98N12 5(1)
98N12 5(1+2)
,
'I
98N12 5(2)
I
Figure 2a.
r,
------
1.2
1
0.8
0.6
0.4
0.2
0
PBS
control
98N12
Omg
2mg/kg
98N12 98N12
1mg
2.5mg
2mg/kg 2mg/kg
98N12
5mg
2mg/kg
98N12 98N12
Omg
1mg
lmg/kg lmg/kg
98N12
2.5mg
lmg/kg
98N12
5mg
lmg/kg
Figure 2b.
1.4
1.2
0.8
0.6
0.4
0.2
0
PBS
control
98N12
98N12
98N12
98N12
98N12
98N12
98N12
98N12
0mg
1mg
2.5mg
5mg
Omg
1mg
2.5mg
5mg
1.5mg/kg 1.5mg/kg 1.5mg/kg 1.5mg/kg 0.5mg/kg 0.5mg/kg 0.5mg/kg 0.5mg/kg
Figure 3a.
Relative FVII Protein
Efficacy
Body Weight Gain-glerability
1.8
1.6
-
-
1.4 .. .
20 mg/kg
t-.-..
-2.5 mg/kg
1.2
10
0.8
0.60.4
0.2.
0.0
"
PBS PEG- PEG- PEG- PEG- PEG- PEG- PEG0
1C
C1
1
C12
C13
C14
C15
C16
36.
Figure
I.2
1.0
0.8
0.6
0.4
0.2
0.0
10
7.5
5
Dose mg/kg)
3.5
Figure 4a.
I1
I ''nI-------1
L.•. 0.8
4...
tw
ORi-____
0
* 0.4
U-
0.2
0.0
~~~1~
LNP 5:1
LNP 5:1 LNP10:1
post UF
LNP15:1
LNP20:1
LNP30:1
Figure 4b.
I.U
0.9
S0.8
0.7
S10 mg/kg
0.6
S3 m g/kg
0.5
0.4
m0.3
0.2
O 2 mg/kg
0 1.5 mg/kg
0.1
0.0
LNP 5:1
post UF
LNP10:1
LNP15:1
LNP20:1
LNP 30:1
Figure 4c.
L.UU
1.00
0.00
.-
-1.00
f= -2.00
-3.00
-4.00
-5.00
EDayl
1 Day2
I· a 2
Figure 5a.
30 mins
1.5 hours
--
7 hours
3 hours
24 hours
24 hours PBS
V - Central Vein
Figure 5b.
I .70
80%
60%
40%
20%
0%
5
10
15
Time post administration (d)
20
25
Figure 6a.
1.2
-
------------
r
1.0
= 0.8
> 0.6
I
0.4
-i
T
L.
---
F---~-----
-4
---
0.2
I
0.0
4.5 mg/kg
PBS
3 mg/kg
1.5 mglkg
3X 1.5 mg/kg
3X 1 mg/kg
3X 0.5 mg/kg
Figure 6b.
-------------~--II~
1.2
I
1.0
T-
0.8
L.
-----F-----
- -------------- -t----~-~-~--~
!
& 0.4
---
T
I
0.2
-I
0.0
PBS
1.5 mg/kg
3 mg/kg
]
1.5 mg/kg 2X 1.5 mg/kg 2X 1.5 mg/kg 2X 1.5 mglkg 2X 1.5 mg/kg 2X
15 min
30 min
1h
2h
4h
I
Figure 7a.
Unformulated
Formulated
ND-3110 in HSS
ND98-3110 inHSS
120
100
80
120
100
c 80
60
*
S 40
S20
0
60
S40
P
0
0.25
0.50
1
3
6
15
24
20
0
PBS-
0
0.25
0.50
1
3
6
24h
Timepoint [h]
Timepoint [h]
*lsense-FLP Iantisense FLP
ssense-FLP gantisense-FLP
T112 > 24 h
T112 ~ 15 min
Figure 7b.
4C
Time
(d)
(nm)
25 C
eak
idth
ntrap- d
ent (nm)
(nm) (%)
37 C
eak
ntrapidth ment (nm)
(nm) (%)
eak
idth
ntrapent
(nm) (%)
S
61
20
7
1
0
0
8
59
19
98
1
20
7
6
1
19
7
59
1
6
59
20
6
10
1
19
7
1
0
7
0
20
8
17
1
0
6
60
20
97
1
20
5
37
2
1
8
63
19
8
9
21
9
59
58
1
8
57
1
98
59
20
98
80
1
19
8
58
21
8
0
20
8
115
6
20
7
3
23
6
57
10
7
164
6
19
7
61
4
96
6
21
7
15
24
PBS24h
Figure 8a.
Figure 8a.
Size Distribution by Volume
251
20
E,
15 -
E
0
10
5
0
!
..-
.
0.1
P..
1
10
100
1000
10000
Size (d.nm)
Overlay of 3 independent runs
Record 5: LNFO1-1955 post filter
Record 7: LNP01-1955 post filter
Record 6: LNF01-1955 post filter
Figure 8b.
Efficacy at 3 mg/kg
Tolerability
1.0
1.50
1.0
1.00
5 080.8
.0.50
U> 0.6
M
TE
2
00.4
0.00
-0.50
0.2
n0
.
PBS
150 nm
85nm
60nm
50nm
nfl
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CHAPTER 4:
IDENTIFICATION OF A CONVERGENCE OF STRUCTURE AMONG
HIGHLY-EFFICIENT CARRIERS OF SIRNA FOR THE SYSTEMIC
DELIVERY OF RNAI THERAPEUTICS
ABSTRACT
A combinatorial library of lipid-like molecules, termed "lipidoids", was
synthesized using a Michael-type conjugate addition of amines to acrylates or
acrylamides to yield hundreds of degradable and non-degradable cationic lipids,
respectively. The library members were tested for their ability to transfect mammalian
cells with siRNA both in vitro and in vivo. Preliminary screening revealed nine lipidoids
that afforded greater siRNA transfection levels than the commercially-available
transfection reagent LipofectamineTM 2000, as determined by their ability to knockdown
Firefly luciferase protein expression; however, the top-100 performing compounds were
further screened at multiple lipidoid:siRNA weight-to-weight (w/w) ratios to yield 25
materials that outperformed LipofectamineTM 2000. The top-20 performing lipidoids
were subsequently formulated for systemic administration. siRNA delivery using a
member of the library, 98N 12-5(1), resulted in >95% target protein knockdown following
a single intravenous injection in mice. Finally, analysis of structures-activity relationships
revealed critical chemical functionalities that impart good siRNA transfection ability
upon lipidoids. A second-generation combinatorial lipidoid library was synthesized and
screened for the ability to transfect mammalian cells with siRNA in vitro, and a
convergence of structure was observed. Whereas only 3% (25/761) of the first-generation
library was able to induce greater knockdown than LipofectamineTM 2000 in vitro, more
than 50% (25/48) of the second-generation library was able to achieve this feat.
Furthermore, a member of the second-generation library, 110N9-5, was shown to induce
>99% target protein knockdown following a single intravenous injection of formulated
siRNA in mice.
INTRODUCTION
The advent of RNAi therapeutics, which confer highly-specific gene silencing,
has the potential to revolutionize the field of medicine. Efficient and safe nucleic acid
delivery into mammalian cells in vivo remains a critical obstacle to the widespread use of
RNAi therapeutics '. Nucleic acid delivery can be achieved using viral or non-viral
vectors. Viruses are able to induce foreign gene expression with high proficiency and
specificity; hence, viruses would be theoretically-ideal vectors if their pathogenic genes
were to be replaced by therapeutic nucleic acids. However, while extremely efficient,
virus-mediated delivery poses safety concerns, including acute toxicity, the induction of
cellular and humoral immune responses 2, oncogenicity due to insertional mutagenesis 3,
and the risk of viral presence in germ-line cells 4.
In addition to being generally safer and less expensive to produce, non-viral
vectors can typically be synthesized reproducibly in large quantities, are amenable to
rational design and diverse chemical modification, and afford much greater nucleic acid
carrying capacity 5.Synthetic vectors have been created to improve bio-stability and
cellular uptake. Polycationic lipids 6, 7and polycationic polymers 8,9 can electrostatically
bind and condense nucleic acids to form nanometer-sized complexes, whose small size
facilitate uptake 1o
Identified by Fire, Mello et al. ", RNA interference offers a powerful platform for
functional genomics, in vivo target validation, and gene-specific medicines. RNAi is a
defense mechanism evolved by eukaryotic cells against double-stranded RNA (dsRNA)
introduced by invading viruses, transposons, and transgenes 1,12. Specifically, RNAi
refers to the entire process of post-transcriptional dsRNA-dependent gene silencing 13
Upon recognition, long dsRNAs are processed by the ribonuclease III-like enzyme Dicer
into siRNAs 14, 15. These short dsRNA fragments - typically 21- to 23-base pairs in
length, possessing two-nucleotide 3' overhangs as well as 5' phosphate and 3' hydroxyl
termini 16,17 - direct the sequence-specific degradation of single-stranded target mRNA
via integration into the RNA-induced silencing complex (RISC) 18, 19. RNAi's utility as a
potent tool for reverse genetic analysis - providing a link between sequence data and
biological function 20, 21 - is bested only by its potential for use in gene-specific
therapeutics 22-24
Chemically-synthesized siRNAs have been shown to induce post-transcriptional
gene silencing in vitro in human cells, which stimulate interferon synthesis in response to
the presence of longer dsRNA 25, 26. Accordingly, such siRNAs might provide a means to
regulate both exogenous and endogenous gene expression, particularly "non-druggable"
targets that are not amenable to conventional therapeutics 27. siRNAs have been
effectively utilized to inhibit virus propagation via local - vaginal 28 and intranasal 29-31 _
and systemic 32, 33 delivery in mice, with one example of a therapy targeted to infected
cells via cell-surface receptors 34.. The expression of endogenous genes has also been
moderated. Notably, RNAi has been employed to treat murine models of cancer 35,36 and
hypercholesterolemia 27 via systemic siRNA administration. RNAi has also been realized
in vivo in non-human primates 37. The successful use of siRNA to treat chronic myeloid
leukaemia in a human patient has been reported 38, but long-term safety has yet to be
demonstrated. The use of RNAi therapeutics as a potential new class of pharmaceutical
drugs is reviewed particularly well by de Fougerolles et al. 39, Kim et al. 40 ,Bumcrot et
al.4 ,and Dykxhoorn et al. 4
Clearly, given the limited number of examples of systemic siRNA administration
published to date, the development of additional carriers is desirable. Many in vitro
siRNA transfection agents are ineffective or toxic in vivo. By synthesizing and screening
a novel combinatorial cationic lipidoid library, a new non-toxic, non-viral vector that is
able to transfect mammalian cells, for use in systemic siRNA delivery, has been
identified 43. This article depicts the identification of the critical chemical functionalities
that impart good transfection ability upon cationic lipidoids. A second-generation library
was synthesized and screened for the ability to transfect mammalian cells in vitro to
support the authors' hypothesis that a convergence of structure had been discovered, and
the data confirmed that oligoamines separated by ethyl or. propyl groups in the lipidoid
backbone confer maximal transfection.
RESULTS AND DISCUSSION
Cationic liposomes provide protection against chemical degradation and facilitate
cellular uptake of negatively-charged nucleic acids 44. Despite the clear benefits of a
lipid-based carrier for nucleic acid delivery, the rational design of such a system is an
onerous task 45. This work was designed to generate a broad library from which a robust
candidate could be identified. In addition to its simplicity, the reaction scheme - the
conjugate addition of a nucleophile to an a,3-unsaturated system - offers several benefits:
commercially-available starting reagents, no by-products, and potential for highthroughput synthesis.
The library's starting reagents provide extensive chemical diversity (Fig. la),
covering much of the available space of commercially-available amine reagents. Also,
many of the amines were shown to impart good transfection ability upon members of a
poly(P-amino ester) library that was synthesized previously for applications in nucleic
acid delivery 46. The reaction scheme is depicted in Fig. lb. Amines were conjugated to
acrylates or acrylamides to yield a degradable and non-degradable series of lipidoids,
respectively. Conjugation of amines to acrylates reaches quantitative conversion
overnight; acrylamides are less reactive, and these reaction mixtures were stirred at 90 OC
for one week.
Lipidoids exhibit several interesting chemical properties that render them a unique
class of biomaterials. The ester linkage has an inverted orientation with respect to the
aliphatic chain when compared to natural lipids, such as triglycerides. Additionally,
whereas standard cationic lipids are typically composed of a constitutively-cationic head
group, owing to a quartemized amine, lipidoids are transiently cationic due to the
presence of environmentally-dependent, reversibly-protonatable amines situated in the
backbone of the molecules. Furthermore, cationic lipids are amphipathic, with welldefined cationic heads and hydrophobic tails; in contrast, lipidoids have hydrophobic tails
emanating from a central backbone and, thus, structurally resemble first-generation
dendrimers as much as they resemble lipids.
The effect of charge on the efficacy of a lipidoid was investigated by synthesizing
quaternized analogues of degradable and non-degradable lipids. For this purpose, methyl
iodide was used, and quaternization gave quantitative yields. In total, 230 non-degradable
lipidoids, 267 degradable lipidoids, and 264 of their quaternized analogues were
synthesized, yielding 761 compounds in the first generation library.
High-throughput lipidoid library screening: in vitro siRNA transfection
The entire library was assayed for siRNA transfection ability at a lipidoid:siRNA
w/w ratio of 5:1, yielding only nine hits. The top-100 performing lipidoids were further
screened at lipidoid:siRNA. w/w ratios of 2.5, 5, 10, and 15 (Fig. 2a). 25 lipidoids
achieved superior transfection levels to the commercially-available transfection reagent
LipofectamineTM 2000 (Fig. 2b), with no apparent toxicity, as determined by specific
knockdown of the reporter protein Firefly luciferase in the absence of off-target Renilla
luciferase knockdown. The data obtained are extremely important, as only a modest
number of siRNA delivery systems have been developed to date, and many of them are
toxic in vivo. This work demonstrates the possibility of synthesizing a number of efficient
siRNA delivery vectors of comparable magnitude to all reported examples in the
literature of this field.
The chemical functionalities present in a lipidoid's backbone correlate strongly with
the material's transfection ability. Lipidoids derived from oligoamines separated by ethyl and
propyl groups were the most efficient subset of structures. Ether-containing lipidoids did not
exhibit high efficacy, with few exceptions (those derived from amine 31). Similarly,
lipidoids derived from monoamines without hydroxyl groups or from cyclic amines
displayed poor transfection ability. Surprisingly, only a few of the lipidoids containing
hydroxyl groups - a functionality that is common to effective DNA transfection reagents 47were found among the top-performing siRNA delivery agents. Quaternized lipidoids were
generally not efficient siRNA vectors, likely owing to their inability to act as "proton
sponges" 48, a feat that would facilitate endosomal escape.
The type and length of their hydrophobic tails also influenced the effectiveness of
lipidoid-mediated siRNA transfection. Specifically, lipidoids derived from the shortest
acrylamides, N 12 and N 14, were most effective; in contrast, lipidoids derived from longer
acrylates, especially O16, were the most effective. Surprisingly, this optimal length of 12-16
carbons is less than the average tail of commonly-used lipids, which typically comprise 18
carbons. Interestingly, only two of the top-25 lipidoids possessed an odd-numbered alkyl
chain. The number of degradable lipidoids roughly equaled the number of non-degradable
lipidoids among the most efficient materials.
In vivo administration of top-performing lipidoids induces potent knockdown
Because the ultimate aim of this work was to identify a carrier that will enable the
treatment of human disease, the top-20 performing lipidoids from the first-generation
library were tested for siRNA delivery ability in vivo. The formulations comprised
lipidoid, cholesterol, poly(ethylene glycol) (PEG), and siRNA, and were administered to
female C57BL/6 mice systemically by intravenous injection at doses ranging from 0.1-5
mg of siRNA per kg of mouse mass (Materials and Methods).
Factor VII was used as a reporter protein because it has the shortest plasma halflife (2-5 hours) among the clotting factors 49. Thus, silencing at the mRNA level is
manifest as silencing at the protein level with minimal lag time. Prior to injection,
liposomes containing Factor VII-specific siRNA were analyzed to determine percent
siRNA encapsulation and sample concentration.
Several lipidoids enabled mRNA and protein knockdown levels of greater than
50% 43. The greatest efficacy was achieved using 98N 12-5(1) - of the two possible fivetailed isomers of 98N1 2 , this molecule has an internal secondary amine (Fig. 3a) - as an
siRNA carrier, affording >95% knockdown without apparent toxicity (weight loss). As
shown in Fig. 3b, the knockdown levels were highly dose dependent, which is an
important feature for therapeutics.
Structure-activity relationships reveal a convergence of structure among the topperforming lipidoids
Analysis of the most efficacious materials from the original lipidoid library
provided insights into the structures that are best-able to transport siRNAs across the cell
membrane of mammalian cells. Concentrating on these structures - specifically 98N1 2 we synthesized a second-generation library (Fig. 4a) that is composed of structures with
slight chemical modifications that were intended to yield novel lipidoids with even
greater efficacy (enabling lower doses to be administered) and even less toxicity
(enabling higher doses to be administered).
Lessons from the results obtained for the first-generation library were taken into
account in the design of the second-generation library. Because the top-performing in
vivo lipidoids were derived from acrylamides 43 and the objective was to identify
improved vectors for in vivo delivery, the second-generation library focused on nondegradable lipidoids. In particular, shorter acrylamide units were incorporated into the
second-generation library, as the best results from acrylamide-derived first-generation
lipidoids were observed for the shortest member of the library, dodecylacrylamide (N1 2)The backbones of the second-generation library included some of the top-performing
members of the first-generation library (amines 95-109) as well as additional ones that
closely resemble triethylenetetramine (amine 98). The results validated these decisions in
a compelling manner.
25 of the 48 (52%) second-generation lipidoids achieved greater transfection
efficiency than LipofectamineTM 2000 (Fig. 4b). This clearly confirms the great potential
for identifying improved structures through rational library design. Understanding the
lipidoids' mechanism of action will further enable scientists to devise future-generation
lipofection agents deliberately.
The top-performing lipidoids in the second-generation library had several
common characteristics. Firstly, these lipidoids were able to transfect efficiently at lower
lipidoid:siRNA w/w ratios. Specifically, 14 out of the top-18 lipidoids from the secondgeneration library transfected optimally at w/w ratios of 2.5 and 5, whereas only 5 out of
the top-18 lipidoids from the first-generation library transfected optimally below a w/w of
10. An increase in the proportion of lower optimal w/w ratios is important because it
indicates greater efficiency of siRNA binding, demanding less lipidoid for complexation
- translating to reduced cost of goods and potential side effects upon in vivo
administration. Four out of the top-five lipidoids from the first-generation library also
possessed this hallmark, suggesting that it is favorable for good transfection ability.
Secondly, the reduction of alkyl chain length improved transfection ability. Six of
the top-seven lipidoids from the second-generation library were derived from N9, the
shortest acrylamide used. Thirdly, of the top-15 lipidoids, three contained each of the
following backbone amines: 110, 113, and 115. Unsurprisingly, all three are N-alkyl
derivatives of diethylenetriamine, an abridged version of amine 98, triethylenetetramine.
This finding is highly supportive of a discovered convergence of structure.
Specifically, these backbones are all oligoethylene imines. Their larger
counterpart, polyethylene imine (PEI), is a well-known nucleic acid delivery vehicle. In
addition to having an extremely-high charge density, PEI greatly enhances endosomal
escape through its pH-responsive "proton sponge" capacity 48, 50. PEI has been used to
deliver siRNA to the lungs to reduce influenza infection 51. Unfortunately, PEI is
extremely toxic at high doses, as it is not readily biodegradable 52, 53. Cytotoxicity has
been shown to correlate with molecular weight and degree of branching 54. Efforts are
being made to improve the safety profile of PEI by optimizing its structure 55, 56 and by
imparting biodegradable functionalities - reducible disulfide crosslinks 57, 58 or
hydrolysable diacrylates 59, 60 - to liberate smaller PEIs. Excitingly, the lipidoids
described, which are essentially shorter analogs with lipophilic tails, are extremely
efficacious and appear to confer an improved safety profile. In particular, 110N 9-5 (Fig.
5a) affords >99% target gene knockdown of the reporter protein Factor VII following a
single tail-vein injection in mice (Fig.5b).
CONCLUSION
RNAi has the potential to become a new major class of drugs, as it represents a
fundamentally new way of treating disease. Specifically, disease is generally caused by
the inappropriate behavior of proteins, and RNAi allows protein production to be
selectively inhibited. RNAi-based therapeutics can be used to address a broad range of
unmet medical needs. Thus, the development of a novel, effective, and safe vector for
siRNA delivery offers vast opportunities. Because RNAi is a platform technology, one
vector may be applied to sundry diseases, as the lipidoid is presumably indiscriminate to
siRNA sequence. Potential therapeutic applications include Alzheimer's disease,
amyotrophic lateral sclerosis, cirrhosis, coronary heart disease, diabetes, influenza,
cancer, and pain.
This work offers several important findings. The synthetic scheme afforded the
discovery of many siRNA delivery vectors that are efficient in vitro with no apparent
cytotoxicity. While vectors for DNA delivery have been investigated for some time and
many promising vectors already exist, this one study on siRNA delivery yielded a
number of effective compounds that is comparable by number to all of those known in
the literature for this field. Furthermore, in vivo transfection and mRNA and protein
knockdown were achieved. Hopefully, 98N 12-5(1) and 110N 9-5 will enable scientists and
physicians to address some of the aforementioned diseases.
In summary, the combinatorial library approach imparts the ability to determine
structure-activity relationships, which may be used for further improvement of lipidoid
structures. In this work, the synthesis of a second-generation library demonstrated the
principal possibility of the rational design of functional siRNA delivery agents and
enabled the identification of a convergence of structure among such top-performing
materials to be confirmed.
MATERIALS AND METHODS
Materials. Amines were purchased from Sigma-Aldrich (St. Louis, MO) and TCI
America (Portland, OR). Acrylates were purchased from Sigma-Aldrich (St. Louis, MO),
Dajac Monomer-Polymer (Feasterville, PA), Hampford Research (Stratford, CT),
Scientific Polymer (Ontario, NY), and TCI America (Portland, OR). Alkylamines were
purchased from Sigma-Aldrich (St. Louis, MO) and TCI America (Portland, OR).
All library reactions were carried out in 5 mL Teflon-lined screw-top vials with
fresh stirbars. All solvents were ACS grade. Chromatographic purification was performed
as flash chromatography using Merck Silica 60. Solvent removal was performed by
evaporation on a Biichi rotavapor with heating to 40 'C. TLC was performed on Merck
Silica Gel 60 F254 TLC glass plates and visualized with UV 254 and ninhydrin stain.
Acrylamide Synthesis. Acrylamides were synthesized by the drop-wise addition of 3
molar equivalents of acryloyl chloride to the appropriate solution of alkylamine and 4
molar equivalents of triethylamine in dry tetrahydrofuran (0.5 M) at -15 'C with stirring
for several hours. The product was extracted in ethyl acetate, the organic phase was dried
over Na2 SO 4, and the solvent was evaporated under reduced pressure. The crude material
was purified using silica gel column chromatography, running a solvent gradient (petrol
ether:ethyl acetate = 1:0; 3:1; 1:1; 0:1).
Library Synthesis. The ester portion of the lipidoid library was synthesized at a molar
ratio of 2:1 acrylate:amine, with no solvent, unless otherwise specified. The amide
portion of the lipidoid library was synthesized at the maximal ratio of acylamide:amine
for each amine (e.g. 6 acrylamide:amine for amine monomer 98). 200 mg of amine was
added to the corresponding amount of acrylate or acrylamide. The mixture was stirred at
90 oC overnight (for acrylate monomers) or for 7 days (for acrylamide monomers). After
cooling, the lipid mixtures were used further without any purification unless otherwise
specified.
Library Purification and Characterization. The top-performing lipidoids were
synthesized at larger scales and purified into their constituent components: maximal
substitution (e.g. 6 tails for amine monomer 98) and sub-maximal substitution (e.g. 5 tails
for amine monomer 98) for in vivo administration. Chromatographic purification was
performed by flash chromatography using Merck Silica Gel 60. A mixture of
dichloromethane (75%), methanol (22%), and ammonium hydroxide (3%) was combined
with a gradient of increasing additional dichloromethane to yield pure products.
siRNA Transfection Assay. The protocol was adapted from Anderson, D.G., et al. 61
Dual HeLa cells, stably expressing Firefly luciferase and Renilla luciferase were seeded
(14,000 cells/well) into each well of an opaque white 96-well plate (Corning-Costar,
Kennebunk, ME, USA) and allowed to attach overnight in growth medium. Growth
medium was composed of 90% phenol red-free DMEM, 10% fetal bovine serum, 100
units/mL penicillin, and 100 jtg/mL streptomycin (Invitrogen, Carlsbad, CA). Cells were
transfected
with
50
ng
of
Firefly-specific
siRNA
[Sense:
CUUACGCUGAGUACUUCGATT, Antisense: UCGAAGUACUCAGCGUAAGTT)]
complexed with lipidoid at lipidoid:RNA ratios of 2.5:1, 5:1, 10:1, 15:1 (wt:wt) to
determine the optimal ratio for transfection efficiency. Transfections were performed in
quadruplicate.
Working dilutions of each lipid were prepared (at concentrations necessary to
yield the different lipidoid:RNA w/w ratios) in 25 mM sodium acetate buffer (pH 5.2).
25 gll
of the diluted lipid was added to 25 tlof 60 gpg/mL siRNA in a well of a 96-well
plate. The mixtures were incubated for 20 min to allow for complex formation, and then
30 gl of each of the lipidoid/RNA solutions was added to 200 il of media in 96-well
polystyrene plates. The growth medium was removed from the cells using a 12-channel
aspirating wand (V&P Scientific, San Diego, CA), after which 150 [l of the
DMEM/lipidoid/RNA solution was immediately added. Cells were allowed to grow for
24 h at 370 C, 5% CO 2 and were then analyzed for luciferase expression. Control
experiments were performed with LipofectamineTM 2000, as described by the vendor
(Invitrogen, Carlsbad, CA).
Firefly and Renilla luciferase expression were analyzed using the Dual-Glo
Luciferase Assay System (Promega, Madison, WI). Luminescence was measured using a
Victor3TM luminometer (Perkin Elmer, Wellesley, MA). A standard curve for luciferase
was generated by titration of luciferase enzyme (Promega, Madison, WI) into growth
medium in an opaque white 96-well plate. A decrease in Firefly luciferase signal alone
signified sequence-specific knockdown. A decrease in both luciferase signals signified
toxicity.
Lipidoid-siRNA Formulation. Lipidoid-based siRNA formulations comprised lipidoid,
cholesterol, poly(ethylene glycol)-lipid (PEG-lipid), and siRNA. Stock solutions of
98N12-5(1) MW 1273, mPEG2000-Ceramide C16 (Avanti Polar Lipids, Alabaster, AL)
MW 2634, and cholesterol MW 387 (Sigma-Aldrich, St. Louis, MO) were prepared in
ethanol and mixed to yield a molar ratio of 42:10:48. Mixed lipids were added to 200
mM sodium acetate buffer (pH 5.2) to yield a solution containing 35% ethanol, resulting
in the spontaneous formation of empty lipidoid nanoparticles. The resultant nanoparticles
were extruded three times through two 0.08 gtm membranes (Sterlitech, Kent, WA) using
a LIPEXTM Extruder (Northern Lipids, Burnaby, BC, Canada). siRNA in 50 mM sodium
acetate (pH 5.2) and 35% ethanol was added to the nanoparticles at 1:7.5 (wt:wt)
siRNA:total lipids and incubated at 37 'C for 30 min. Ethanol removal and buffer
exchange of siRNA-containing lipidoid nanoparticles was achieved by dialysis against
phosphate-buffered saline using a 3,500 MWCO membrane (Pierce Biotechnology,
Rockford, IL). Particle size was determined using a ZetaPALS dynamic light scattering
detector (Brookhaven Instruments, Holtsville, NY). Resulting particles had a mean
particle diameter of approximately 70 nm and siRNA entrapment efficiency of >95%.
Determination of entrapment efficiency. A modified RiboGreen® (Invitrogen,
Carlsbad, CA) assay was used to quantify entrapment of siRNA. Samples were diluted
1:200 in TE buffer and mixed with Quant-iTTM RiboGreen® RNA reagent (1:200 in TE
buffer) at 37 °C for 30 min in the presence or absence of 0.5% final w/v Triton X-100.
Entrapment efficiency of siRNA in lipidoid nanoparticles was determined by comparing
the fluorescent signal of the lipidoid-siRNA sample in the presence (total siRNA) and
absence (free siRNA) of Triton X-100 detergent. Signal in the absence of detergent yields
the "free" or accessible siRNA fraction, while signal in the presence of detergent yields
the total siRNA content.
In vivo murine Factor VII silencing experiments. All procedures used in animal
studies conducted at Alnylam were approved by the Institutional Animal Care and Use
Committee (IACUC) and were consistent with local, state, and federal regulations, as
applicable. C57BL/6 mice (Charles River Labs, Wilmington, MA) received either saline
or siRNA in lipidoid formulations via tail-vein injection at a volume of 0.01 mL/g. Serum
levels of Factor VII protein were determined in samples collected by retroorbital bleed
using an activity-based chromogenic assay (Coaset Factor VII, DiaPharma Group, West
Chester, OH or Biophen FVII, Aniara Corporation, Mason, OH). All experiments were
performed in triplicate.
FIGURES
Figure 1. a) A first-generation combinatorial lipidoid library was synthesized by reacting
amines with alkyl-acrylates (0) or alkyl-acrylamides (N). b) The Michael-type reaction
scheme proceeds via the conjugate addition of amines to an acrylate or acrylamide.
Treatment vyith methyl iodide yields quarternized amino groups (O+).
Figure 2. a) Percent Firefly luciferase knockdown for the top-100 members of the firstgeneration lipidoid library. b) 25 lipids achieved greater siRNA transfection levels than
LipofectamineTM 2000, as determined by their ability to knockdown Firefly luciferase
expression. Data are shown for the optimal lipidoid:siRNA weight-to-weight ratio for
each lipidoid (2.5:1,
,
, 15:1).
Figure 3. a) The structure of 98NI2-5(1), a five-tailed isomer of triethylenetetramine with
one secondary internal amine. b) Serum Factor VII levels 24 h after a single bolus
injection of 98N 12-5(1)-formulated siRNA in mice (n = 3). Data points represent group
mean ± s.d. Data points marked with asterisks are statistically significant relative to the
control-treated group (*** p< 0.0001, ** p= 0.0005, * p< 0.05; unpaired t-test).
Figure 4. a) A second-generation combinatorial library was synthesized using amines
with similar structures to 98, the backbone of the top-performing lipidoid in vivo from the
first-generation library. b) 25 out of 48 tested lipidoids from the second-generation
library achieved greater siRNA transfection levels than LipofectamineTM 2000, as
determined by their ability to knockdown Firefly luciferase expression. Data are shown
for the optimal lipidoid:siRNA weight-to-weight ratio for each lipidoid (2.5:1,
, 10: ,
15:1). The nomenclature for the second-generation library includes a number following
the lipidoid (e.g. 112N 9-5 as opposed to simply 98N 12 for the members of the firstgeneration library). This number, which indicates the molar ratio of acrylamide to amine
(in this case 5:1) at which the compound was synthesized, indicates the maximal number
of tails that can be conjugated to the amine backbone.
Figure 5. a) The structure of 110N 9-5. b) Serum Factor VII levels 48 h after a single
bolus injection of 110N 9-5-formulated siRNA in mice (n = 3). Data points represent
group mean ± s.d. The asterisk indicates statistical significance relative to the controltreated groups (*** p< 0.0001; unpaired t-test).
Figure la.
28
HO2 /
31
31
HO
32
HO'
33
HO
KNH2
34
HO~\H
NHO5
36
HO
HOO>QH
38
HO
20 HO
H
&
21
0
010
22 .j,
.•NH2
N
22
_
___
_
_
HO,
24
'O
O13
0
_
_O__
NH2
NH,
26
,
NH
2
NH,
_
_
O0
N-v
018
NH2
O/•NH,
HH
HO
0
014
NH,
OH
__
012
^NH2
Monoamines with hydroxyl groups
0
N12
H
N14
HýN
H
7,0
8-.N, N_
H
0
H
9 0
H
H
NH2
9(i
ýN
98
HZN""
99
HN5 -NH-
'
100
H2N
86
HO
87
HO,
HO
O0
NHH
NN
91
H
O0
___/N
,NHz
NH2
NH z
-
H
>N^VNH,
-,
NNH7
93
Q
94
HN
NH
103
HO,_- H N H
109
HN
2
NIN
N-/-NH2
HO
N
N15
-N H
N
95
NH2
N-/
H HC
•-
NH2
i
.N
H
-N,,•N
H
H
0
k,
0
OH
H
CNH,
N\
H
Oligoamines
NH2
'N--
Figure lb.
0
R1-0
R
0 ,0,
R,-OA
+
H 2N-R
,
AT
Mel
N-R 2
2
R1-O
O
0
R1-N
H
AT
H2
NH2
R--
N '
H
H
R1-N.
OH
Oligoamines
with hydroxyl
groups
H
N-R1
0
Figure. 2a.
Percent Knockdown (Firefly Luciferase)
100
90
8C
7(
6(
5
4
Figure 2b.
IUU.U
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
N-OO·_2:~21
N
rc~OO~
~
oL2~G. o\OoQ~2C.I2·~ 2 ~~\~~cO,2!C
j~~
Figure 3a.
H
H
0 H
0 N
O
H
N
H
O
Figure 3b.
1.2
1.0
0.8
T
. 0.6
,,
, 0.4
0.2
-
0.0
II
PBS
L---~--
0.75 mg/kg
1.5 mg/kg
3 mg/kg
Figure 4a.
O
Ng
H
O
N11
H
O
N12
H
O
N13
H
N
H
NH2
HN- H
110
H2N '
NH2
NHg2
N
115
NH2
NH2
111
~
HzN
H
NH2
H
NN'N
H
NH NH2
H
Hz N -,,NH
NH2
HH
HO
H2NH2N *,
N-NH2 N
H
H
N"
oH
113
H2N
N
114
H2N
N
,H
H
N H2
NH2
H2
117
Figure 4b.
I~f·r
r\
I UU.U
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
p
2'
C3'2'~3NN
Figure 5a.
s
p
2'
Pa
,
aE\". ,
pb~;Lq
NNNNNn
2
N~I3`
Figure 5b.
1.4
1.2
2
0.8
0. 6
0.6
e 0.4
0.2
a
0ý
0
0
PBS
110N9-5, 2.5 mg/kg
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CHAPTER 5:
CONCLUSION
RNAi therapeutics afford the possibility of treating disease specifically at the
genetic level. Delivery remains the primary obstacle to the realization of this novel class
of therapy. To address the paucity and general uniformity of current delivery materials, a
combinatorial library comprising diverse chemical functionalities was synthesized using
a Michael-type addition. This reaction scheme obviates the need to use standard steps including protection, deprotection, solvent exchanges, and serial purification - rendering
high-throughput synthesis plausible. More than 1,200 lipid-like materials, termed
"lipidoids", were synthesized and screened for the ability to transfect mammalian cells in
vitro. This work represents an increase in the number of lipofection materials studied to
date by nearly two orders of magnitude. The top-performing materials from the initial
screen were formulated for local and systemic delivery in vivo. Lipidoids conferred
potent, specific, and durable silencing of target gene expression in three cell types and
four species.
More recently, the potential to translate these molecules into the clinic was
validated. Of great importance, this delivery platform was used to demonstrate the safety
of RNAi therapeutics, confirming that siRNA-mediated gene knockdown does not
interfere with the endogenous microRNA pathway '. Specifically, lipidoid-formulated
siRNA did not affect the levels of mature miRNAs or their function in the liver, as
indicated by unchanged levels of their target mRNAs and the proteins that these mRNAs
encode. Additionally, repeat administration in hamsters maintained long-term gene
silencing without altering the levels of the liver-specific miRNA miR-122.
Lipidoid-formulated siRNA was also shown to prevent malarial infection in mice
when administered prophylatically 2. A single intravenous injection recapitulated the
phenotype of heme-oxygenase-l (HO-1) knockout mice. HO-1 is required for the
establishment of malaria infection and is significantly upregulated in hepatocytes
following infection by Plasmodium. The reduction of HO-1 leads to increased levels of
inflammatory chemokines and cytokines known to be involved in liver infection control.
Mice treated with HO-i-targeting siRNA did not develop blood-stage infection after
infection with P. bergheisporozoites, while all infected mice treated with control siRNA
developed patent parasitemia.. Clearly, lipidoids have great potential to contribute to
medicine, particularly in the delivery of RNAi therapeutics.
Thank you for taking the time to read my thesis. I hope that you enjoyed reading
about my research as much as I enjoyed conducting it. ©
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MICHAEL GOLDBERG
EDUCATION
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Graduate Education in Medical Sciences, Harvard-MIT Health Sciences and Technology
Ph.D.: BiologicalChemistry
Supervisor:Institute ProfessorRobertLanger
Cambridge,
MA
2004-2008
* Thesis: "Synthesis, Screening, and Applications of "Lipidoids", a Novel Class of Molecules
Developed for the Delivery of RNAi-based Therapeutics"
* Co-inventor of two licensed patents and one provisional patent
* Member of founding GEMS class, sponsored by the Howard Hughes Medical Institute to encourage
translational research via classroom-based and clinical instruction at Harvard Medical School
* Nominated for Faculty Appreciation Day Award by students
* Awarded Morse Travel Grant
* Student Ambassador
UNIVERSITY OF CAMBRIDGE
Cambridge-MIT Institute
M.Phil.: BioScience Enterprise
*
*
Cambridge,
UK
2003-2004
Thesis: "Critical Factors in the Development of a Successful Biotechnology Cluster"
Focused on biotechnology, technology transfer, and entrepreneurship
UNIVERSITY OF TORONTO
Hon. B.Sc. (Summa Cum Laude): BiologicalChemistry Speciabst
Toronto,
Canada
1999-2003
* Named "Leader of Tomorrow" in convocation literature
* University of Toronto Alumni Association Scholar: Highest honor awarded to graduates
* Gordon Cressy Student Leadership Award: Recognized by Faculty of Arts and Sciences for
outstanding extracurricular contributions
* President, Global Knowledge Foundation: Organized premier GreatMinds at UofT: The University
ProfessorsLecture.Series: designed proposal, presented it to the Dean, invited and scheduled
speakers, acquired sponsorship, coordinated detailed plan for implementation, motivated fellow
students, prepared publicity releases, and edited resulting book (ISBN 0-7727-5200-1)
* University College Merit Award
* President, Mulock House
* 7 other scholarships/awards for excellence in coursework and research
ACADEMIC ACHIEVEMENTS
Perfect Score: Analytical Section, GRE (2002)
9 9 th Percentile: Physical Sciences Section, MCAT (2001)
9 5 th Percentile: Biological Sciences Section, MCAT (2001)
RESEARCH EXPERIENCE
Gained experience in varied fields by conducting research in world-renowned labs since high school:
Biochemistry, drug delivery, materials chemistry, microbiology, oncology, and tissue engineering
100