U.S. patent application number 14/528980 was filed with the patent office on 2015-06-18 for single molecule nucleic acid nanoparticles.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Bradley T. Messmer.
Application Number | 20150166997 14/528980 |
Document ID | / |
Family ID | 43900922 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150166997 |
Kind Code |
A1 |
Messmer; Bradley T. |
June 18, 2015 |
SINGLE MOLECULE NUCLEIC ACID NANOPARTICLES
Abstract
The present technology relates to a nanoparticle platform based
on the unique and varied properties of DNA. Circular DNA can be
replicated using a strand displacing polymerase to generate long
linear concatamers of controllable length that spontaneously fold
into a ball conformation due to internal base-pairing. These balls
of DNA are discreet particles that can be made in variable sizes on
a nanometer size scale in a scalable manner. The particles can be
used in a variety of manners, discussed herein, including specific
targeting, drug delivery to cancer cells, and diagnostics.
Nanoparticles may also serve as multifunctional platforms for the
integration of many currently used cancer therapeutic
techniques.
Inventors: |
Messmer; Bradley T.; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
43900922 |
Appl. No.: |
14/528980 |
Filed: |
October 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13502729 |
Jun 28, 2012 |
8895242 |
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PCT/US2010/053270 |
Oct 19, 2010 |
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14528980 |
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61279408 |
Oct 20, 2009 |
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Current U.S.
Class: |
424/450 ;
435/91.5; 506/16; 506/9; 514/44R; 536/23.1 |
Current CPC
Class: |
G01N 2015/0038 20130101;
A61K 31/7088 20130101; C12N 2310/51 20130101; C12N 2330/31
20130101; C12N 2310/16 20130101; C12N 2310/315 20130101; B82Y 5/00
20130101; C12N 2330/50 20130101; A61K 39/385 20130101; G01N
33/54346 20130101; A61P 35/00 20180101; C12N 15/115 20130101; C12N
15/111 20130101; A61K 31/704 20130101; A61K 9/5146 20130101; A61K
2039/64 20130101 |
International
Class: |
C12N 15/115 20060101
C12N015/115; G01N 33/543 20060101 G01N033/543 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED R&D
[0002] This invention was made with government support under NIH
Grant/Contract Numbers U54CA119933501 and U54CA119335 awarded by
the National Institutes of Health of the United States of America.
The government has certain rights in the invention.
Claims
1.-61. (canceled)
62. A method of making a nanoparticle comprising: contacting a
circular single-stranded nucleic acid template with a nucleic acid
polymerase, wherein said template includes a module sequence which
binds to a target molecule, wherein the module sequence in
monovalent form has low affinity for the target molecule and in
multivalent form binds to the target molecule with a high avidity;
and amplifying said template with said polymerase to produce said
nanoparticle, wherein said nanoparticle comprises a continuous
strand of nucleic acid comprising a concatamer of said module
sequence.
63. The method of claim 62, wherein the target molecule is bound to
a surface.
64. The method of claim 62, wherein said nucleic acid template is
DNA.
65. The method of claim 62, wherein said nucleic acid polymerase is
a strand displacing polymerase.
66. The method of claim 62, wherein said amplifying has a duration
of more than 1 minute.
67. The method of claim 62, further comprising circularizing a
linear nucleic acid template to produce said circular nucleic acid
template.
68. A non-naturally occurring nanoparticle made according to the
method of claim 62.
69. The nanoparticle of claim 68, wherein said nucleic acid
comprises DNA, wherein said DNA is more than 100 kb in length.
70. The nanoparticle of claim 68, wherein said DNA comprises a
sequence encoding a sequence selected from a siRNA, reporter gene,
therapeutic protein, and CpG sequence.
71. The nanoparticle of claim 68, further comprising a nucleic acid
intercalating drug.
72. The nanoparticle of claim 68, further comprising an
oligonucleotide-linked entity selected from the group consisting of
an aptamer, drug, peptide, and siRNA.
73. A liposome comprising the nanoparticle of claim 68.
74. A pharmaceutical composition comprising the nanoparticle of
claim 68.
75. A method of treating cancer comprising administering the
pharmaceutical composition of claim 74 to a subject in need
thereof.
76. A method for identifying nanoparticles comprising a module
sequence capable of binding to a target molecule with high avidity
comprising: generating a library of nanoparticles comprising
putative module sequences using the method of claim 62; contacting
said library to a target molecule; and selecting for a nanoparticle
that binds said target molecule, wherein the module sequence in
monovalent form has low affinity for the target molecule and in
multivalent form binds to the target molecule with a high
avidity.
77. A non-naturally occurring nanoparticle comprising a
single-strand nucleic acid comprising a continuous strand of
nucleic acid comprising a concatameric module sequence which binds
to a target molecule, wherein the module sequence in monovalent
form has low affinity for the target molecule and in multivalent
form binds to the target molecule with a high avidity.
78. The nanoparticle of claim 77, wherein the target molecule is
bound on the surface.
79. The nanoparticle of claim 77, wherein said nucleic acid
comprises DNA, wherein said DNA is more than 100 kb in length.
80. The nanoparticle of claim 79, wherein said DNA comprises a
sequence encoding a sequence selected from a siRNA, reporter gene,
therapeutic protein, and CpG sequence.
81. The nanoparticle of claim 77, further comprising a nucleic acid
intercalating drug.
82. The nanoparticle of claim 77, further comprising an
oligonucleotide-linked entity selected from the group consisting of
an aptamer, drug, peptide, and siRNA.
83. A liposome comprising the nanoparticle of claim 77.
84. A pharmaceutical composition comprising the nanoparticle of
claim 77
85. A method of treating cancer comprising administering the
pharmaceutical composition of claim 84 to a subject in need
thereof.
86. A method of identifying a target comprising: contacting said
target with the nanoparticle of claim 77, wherein said module
sequence selectively binds to said target; and identifying binding
of said module sequence to said target.
87. A library of nanoparticles comprising at least two populations
of nanoparticles, wherein said each of said at least two
populations comprise nanoparticles comprising a single-strand
nucleic acid comprising a continuous strand of nucleic acid
comprising a concatamer of at least one different module sequence
which binds to a target molecule bound, wherein the module sequence
in monovalent form has low affinity for the target molecule and in
multivalent form binds to the target molecule with a high avidity.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/502,729 filed Jun. 28, 2012 which is the U.S. National Phase
of International Application No. PCT/US 2010/053270 entitled
"SINGLE MOLECULE NUCLEIC ACID NANOPARTICLES", filed Oct. 19, 2010
and published in English on Apr. 28, 2011 as WO 2011/050000, which
claims priority to U.S. Provisional Application No. 61/279,408
filed on Oct. 20, 2009, the contents of which are incorporated by
reference in their entireties.
REFERENCE TO SEQUENCE LISTING
[0003] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled UCSD025.TXT, created Oct. 19, 2010, which is
approximately 3.2 KB in size. The information in the electronic
format of the Sequence Listing is incorporated herein by reference
in its entirety
FIELD OF THE INVENTION
[0004] The present technology relates to the fields of molecular
biology, biochemistry, medicine, and cancer therapeutics. In
particular, methods and compositions for making and utilizing
nanoparticles comprising nucleic acids are provided.
BACKGROUND
[0005] DNA is one of the most thoroughly characterized molecules
with regard to structure, chemistry, and modification, and has the
capability to serve a wide variety of functions. It can be, and has
been, used as a scaffold for the integration of varying entities
due to its well defined ability to base-pair hybridize. With the
discovery of aptamers and the realization that DNA has the
capability to structurally and chemically recognize other molecules
with near anti-body specificity at a fraction of the difficultly of
synthesis, a new field of DNA based targeting molecules was born.
Today there are FDA approved therapeutics based solely on DNA, such
as the VEGF aptamer.
[0006] Current nanoparticle-based approaches to treating cancer
include constructs composed of polymer, silica, or gold
nanoparticles, liposomes, and less frequently such platforms as
carbon nanotubes and viral capsids. These structures are coated
with a variety of functionalizing entities such as polyethylene
glycol (PEG) for biocompatibility, various targeting peptides,
antibodies, small molecules, or aptamers and some form of
therapeutic.
SUMMARY
[0007] The present technology relates to a nanoparticle platform
based on the unique and varied properties of DNA. Circular DNA can
be replicated using a strand displacing polymerase to generate long
linear concatamers of controllable length that spontaneously fold
into a ball conformation due to internal base-pairing. These balls
of DNA are discreet particles that can be made in variable sizes on
a nanometer size scale in a scalable manner. The particles can be
used in a variety of manners, discussed herein, including specific
targeting, drug delivery to cancer cells, and diagnostics.
Nanoparticles may also serve as multifunctional platforms for the
integration of many currently used cancer therapeutic
techniques.
[0008] Some embodiments described herein include methods of making
a nanoparticle including contacting a circular single-stranded
nucleic acid template with a nucleic acid polymerase, wherein the
nucleic acid template encodes an aptamer; and amplifying said
template with said polymerase to produce said nanoparticle, wherein
said nanoparticles comprises a concatemer of the sequence of said
template. In such embodiments, the nucleic acid template can be DNA
or RNA. In more embodiments, the nucleic acid polymerase is a
strand displacing polymerase, such as a DNA polymerase, and can be
selected from the group consisting of phi29 polymerase, Klenow
fragment, VENT.RTM. (Exo) DNA polymerase, 9.degree. N.sub.m DNA
polymerase, Bst DNA polymerase, M-MuLV reverse transcriptase, and
AMV reverse transcriptase. In some embodiments, the amplifying step
has a duration of more than about 1, 5, 10, 25, 30, 50, and 120
minutes. Some methods of making a nanoparticle can also include
circularizing a linear nucleic acid template to produce the
circular nucleic acid template. The linear nucleic acid template
can be more than 10, 50, 100, or 1000 bases in length.
[0009] Some embodiments described herein include a nanoparticle
made according to the methods described herein. In such
embodiments, the nanoparticle can include DNA. The DNA can be more
than 1 kb, 10 kb, 100 kb, 1 Mb, 10 Mb, 100 Mb, and 500 Mb in
length. The DNA can encode a sequence selected from a siRNA,
reporter gene, therapeutic protein, and CpG sequence. Some
embodiments include nanoparticles containing a nucleic acid
intercalating drug. Such drugs can include Doxorubicin,
Daunorubicin, and Dactinomycin. More embodiments include
nanoparticles containing an oligonucleotide-linked entity including
an aptamer, drug, peptide, and siRNA.
[0010] Some embodiments include liposomes containing nanoparticles.
More embodiments include pharmaceutical compositions containing
nanoparticles. Particular embodiments include methods of treating
cancer including administering the pharmaceutical compositions
described herein to a subject in need thereof. Even more
embodiments include kits containing the pharmaceutical compositions
described herein and instructions for use of the kit.
[0011] Some embodiments include methods for identifying
nanoparticles containing aptamers including generating a library of
nanoparticles comprising putative aptamers; and screening the
library. The screening can include contacting the library to a
capture probe; and selecting for a nanoparticle that binds the
capture probe. The capture probe can include a tumor cell.
[0012] Some embodiments include nanoparticles containing a
single-strand nucleic acid including a concatemeric sequence
encoding an aptamer. In some embodiments, the nanoparticle can
include DNA. For example, the DNA can be more than 1 kb, 10 kb, 100
kb, 1 Mb, 10 Mb, 100 Mb, and 500 Mb in length. The DNA can encode a
sequence selected from a siRNA, reporter gene, therapeutic protein,
and CpG sequence. Some embodiments include nanoparticles containing
a nucleic acid intercalating a drug. Such drugs can include
Doxorubicin, Daunorubicin, and Dactinomycin. More embodiments
include nanoparticles containing an oligonucleotide-linked entity
including an aptamer, drug, peptide, and siRNA.
[0013] Some embodiments include methods to identify tumor cells.
Such methods can include contacting a tumor cell with a
nanoparticle in which the aptamer selectively binds to the cell;
and identifying binding of the aptamer to the cell. More
embodiments include the identifying binding of the aptamer to the
cell to include identifying a reporter moiety associated with the
nanoparticle. The reporter moiety can include a radioactive probe,
a reporter protein, a reporter gene, and a fluorescent
molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A shows a schematic diagram for uses of DNA
nanoparticles.
[0015] FIG. 1B shows a schematic overview of DNA nanoparticle
synthesis and modular DNA particle library creation. Single module
libraries can be biopanned, whereas the modules within the
multimodule libraries can be re-assorted from round to round,
creating particles with novel combinations of modules.
[0016] FIG. 2 relates to a visualization of DNA nanoparticles.
Panel A shows a photomicrograph of DNA nanoparticles produced by a
30 minute RCA reaction. The particles were labeled after synthesis
with Sybr Green dye and imaged at 100.times. in a fluorescent
microscope. Panel B shows a graph of sorted DNA-NP. DNA
nanoparticles were made by RCA reactions of varying times, labeled
with Oligreen dye, and run on a flow cytometer. The fluorescence
intensity correlates with the size of the particles consequent to
the reaction time. (Graph a: unlabeled beads; graph b: 7.5 min
DNA-NP; graph c: 30 min DNA-NP; graph d: 60 min DNA-NP). Panel C
shows a graph relating to DNA nanoparticles produced with Alexa488
labeled nucleotides and run on a flow cytometer. Panel D shows a
graph relating to unlabelled DNA nanoparticles hybridized with a
complementary oligonucleotide probe labeled with Alexa-647.
[0017] FIG. 3 relates to DNA nanoparticles sized by Dynamic Light
Scattering (DLS). Panel A provides DLS data showing increasing size
with RCA reaction time. The particles rapidly (10 minutes) obtain a
mean size of .about.200 nm. After one hour they are .about.300 nm.
Panel B demonstrates monoexponential autocorrelation decays
indicating significant monodispersity. All data was collected on a
Zetasizer Nano-ZS.
[0018] FIG. 4 shows a schematic of the flow of selection scheme for
DNA nanoparticles that bind human dendritic cells. A 100 base
library with a 60 base random region flanked by 2 20 base primer
sites is ligated and amplified with Rolling Circle Amplification to
produce nanoparticles. The nanoparticles are incubated with the
target cells and washed. Remaining nanoparticles are amplified by
PCR and then asymmetrically PCR (using only one primer) to generate
an excess of the desired strand. The desired strand is re-ligated
and the cycle is repeated.
[0019] FIG. 5 shows a graph of fluorescence and indicates the
selection of DNA nanoparticles that bind to human dendritic cells
(DCs). The pool of DNA nanoparticles after 7 rounds of selection
was regenerated with fluorescent nucleotides and compared to a
similarly labeled non-specific control DNA nanoparticle. The red
curve shows DCs with free labeled nucleotides. There is a clear
shift in the labeling intensity with the selected particles.
[0020] FIG. 6 provides flow cytometry data for 5 positive clones
sequenced from round 7 of selection protocol. Controls included a
DNA nanoparticle that did not bind (N05), dendritic cells alone and
dendritic cells with free Alexa488-dCTP used to label the DNA.
[0021] FIG. 7 relates to a DNA nanoparticle selection process.
[0022] FIG. 8 relates to an embodiment that includes library
generation, ligation, and RCA.
[0023] FIG. 9 relates to an embodiment that includes PCR
amplification and asymmetric PCR.
[0024] FIG. 10 relates to an embodiment that includes multimodule
particle creation from single module libraries.
[0025] FIG. 11 relates to an embodiment that includes non-modular
PCR amplification of multimodule particles to preserve module
combinations.
[0026] FIG. 12 relates to an embodiment that includes subtractive
screening. An irrelevant cell line is used to absorb non-specific
cell binding particles.
[0027] FIG. 13 relates to two methods of introducing paramagnetic
properties to the DNA nanoparticles via iron oxide-oligonucleotide
conjugates.
[0028] FIG. 14 relates to an embodiment of DNA-NP synthesis by
rolling circle amplification.
[0029] FIG. 15 provides data relating to RCA reaction kinetics.
Upper panel: 1 nM ligated oligonucleotides were used as a template
for phi29 polymerase to perform RCA and produce single stranded
particles. Lower panel: incorporation of phosphorothioate
nucleotide. The rate of an RCA reaction was monitored by oligreen
fluorescence. Reactions contained dATP, dGTP, dTTP, and either
dCTP, a 50:50 mixture of dCTP and 2'-deoxycytidine
a-thiotriphosphate, or only 2'-deoxycytidine a-thiotriphosphate.
The reaction proceeds 50% slower when only modified nucleotides are
used for that one base.
[0030] FIG. 16 provides data relating to a visualization of DNA
nanoparticles. Panel A shows a photomicrograph of DNA nanoparticles
produced by a 30 minute RCA reaction. The particles were labeled
after synthesis with Sybr Green dye and imaged at 100.times. in a
fluorescent microscope. Panel B relates to DNA nanoparticles that
were made by RCA reactions of varying times, labeled with Oligreen
dye, and run on a flow cytometer. The fluorescence intensity
correlates with the size of the particles consequent to the
reaction time. Panel C shows a WETSEM image of DNA-NP produced from
a 30 min reaction. The particles are imaged while bound to a
poly-lysine coated membrane in aqueous solution.
[0031] FIG. 17 relates to DNA nanoparticles sized by Dynamic Light
Scattering (DLS). Panel A provides DLS data showing increasing size
with RCA reaction time. The particles rapidly (10 minutes) obtain a
mean size of .about.200 nm. After one hour they are .about.300 nm.
Panel B provides data showing monoexponential autocorrelation
decays indicating significant monodispersity. All data was
collected on a Zetasizer Nano-ZS.
[0032] FIG. 18 relates to the specificity of DC binding clone 3.
Particles were labeled and tested as described herein.
[0033] FIG. 19 provides data relating to specific binding and
uptake of Clone 3 by DC. Panel A relates to Clone 3 DNA-NP
synthesized with alexa488 nucleotides and incubated with DC and
cell lines P815 (mouse mastocytome) and THP1 (human acute monocytic
leukemia). Flow cytometry was performed with clone 3, it's reverse
complement particle, and DCs incubated with just the nucleotide
mix. The same samples were assessed by fluorescent microscopy. The
incubations were done on ice. Panel B relates to DC incubated with
clone 3 particles at 37.degree. C. No fluorescence was seen in P815
or THP1 incubated with clone 3, nor was any observed in DC
incubated with the control reverse complement particle (not
shown).
[0034] FIG. 20 provides data relating to clone 3 DC binding DNA-NP
elicits cytokine secretion and Ca.sup.2+ flux. Panel A relates to
DCs exposed to DC-binding DNA-NPs (DNA(+)) or control DNA-NPs that
do not bind to DCs (DNA(-)), LPS, or media control for 48 h and the
amount of II-6 secreted into the cell culture supernatants was
determined by ELISA. DC binding DNA-NP elicited the most IL-6
production. Panel B relates to Ca.sup.2+ flux followed in real time
after exposure of the cells to either clone 3 (top panel) or the
control (bottom panel) DNA-NP. The FLIPR Calcium 5 assay was used
in conjunction with the FlexStation 3 scanning plate reader, both
from Molecular Devices. DC binding nanoparticles that have been
purified by dialysis into cell culture grade PBS are added to
cell/dye suspensions in a 96 well plate and fluorescent readings
are monitored continuously for approximately 150 seconds following
administration of nanoparticles. Fluorescent signals indicate
increased concentrations of intracellular calcium that is released
upon stimulation by DNA nanoparticles.
[0035] FIG. 21 relates to hybrid DNA nanoparticle formation.
Templates for two particles can be fused by ligation. One ligation
primer is dideoxy terminated so that it can not prime the RCA. The
resulting DNA-NP contains many copies of each sequence, at a
precise 1:1 ratio. If desired the ratio could be tuned by altering
the number of copies of one or the other in the template
construction.
[0036] FIG. 22 provides data relating to 10.sup.5 immature human
DCs generated from HLA.A2. Positive donors were cultured in the
presence of Hp-91 or a control peptide Hp-46 (200 .mu.g/mL), or
left untreated (medium) for 48 h. DCs were washed and cultured with
the melanoma peptide gp100 (500 ng/ml) and gp100-specific responder
cells at a DC to responder ratio of 1:2 overnight. The number of
IFN-.gamma..quadrature. secreting cells was determined 24 h later.
The plate was scanned and the spots were counted automatically
using the image analysis system ELISPOT reader. The data shown is
the number of IFN-.gamma.-spot-forming cells/well, are means
(+/-SEM) of two independent experiments using DCs from different
donors.
[0037] FIG. 23 provides data relating to Hp-91(270 .mu.g), pI:C
(125 .mu.g), or Pam3Cys (125 .mu.g) injected into B16 melanoma and
the mice were sacrificed 24 h after the injection. The tumor was
embedded in OCT and frozen sections were stained for Mac 1
(macrophages), CD11c (DCs), and CD3 (T cells) by
immuno-histochemistry. Pictures were taken in brightfield at
20.times.. Higher doses of pI:C and Pam3Cys (500 .mu.g) still
showed no T cell and DC recruitment (data not shown).
[0038] FIG. 24 relates to embodiments that include a strategy for
selection and combinatorial breeding of multimeric polyvalent DNA
nanoparticles. Step 1. Several libraries with unique ligation and
PCR primers are generated. Step 2. Each library is independently
screened for a few rounds against the target to create an initial
enriched pool. Step 3. The products of the initial screenings are
combinatorially assembled into multimeric templates and polyvalent
DNA particles are generated. Step 4. The particles are subjected to
subtractive screening to enrich the desired binding activity and
eliminate unwanted crossreactivities as described herein. In each
selection step the individual library components are re-assorted,
creating additional combinatorial diversity from which optimal
particles can be selected.
[0039] FIG. 25 shows a photograph of DNA nanoparticles made with
reaction times of 5 and 30 minutes, respectively.
[0040] FIG. 26 shows flow cytometry data illustrating increasing
fluorescence with increasing reaction time indicating larger
particles.
[0041] FIG. 27 shows a spectrograph of Doxorubicin and Doxorubicin
with DNA nanoparticles. Doxorubicin is titrated into .about.500 ng
DNA nanoparticles and the fluorescence is quenched as binds. At low
Doxorubicin concentrations nearly all fluorescence is quenched.
With increasing concentration of Doxorubicin, the DNA nanoparticles
become saturated and free Doxorubicin in solution can
fluoresce.
[0042] FIG. 28 shows a schematic diagram of an example library
template oligonucleotide.
[0043] FIG. 29 relates to library generation and screening. Panel A
shows a schematic diagram of some embodiments that include of a
library generation strategy. Panel B shows a schematic of a
screening strategy for use on touch preparations of primary tumor
samples. Panel C shows a schematic of an amplification and
regeneration method for repeated screenings and enrichment of
target binders.
[0044] FIG. 30 shows a schematic for an example strategy for
selection and combinatorial breeding of multimeric polyvalent
aptamer particles. Step 1 shows several aptamer libraries with
unique ligation and PCR primers are generated. Step 2 shows each
library is independently screened for several rounds against the
target. Step 3 shows the products of the initial screenings are
combinatorially assembled into multimeric templates and polyvalent
DNA particles are generated. Step 4 shows the particles are
subjected to subtractive screening to enrich the desired binding
activity and eliminate unwanted crossreactivities.
[0045] FIG. 31 shows a graph of fluorescence over time. RCA
reactions were run using either dNTPs, a mixture of dNTP and
phosphorothioate backbone cytosine nucleotides (C.alpha.STP) at a
1:1 ratio with dCTP, or with a nucleotide cocktail where all dCTP
was replaced with C.alpha.STP.
[0046] FIG. 32 shows graphs of spectrometer readings vs. drop
count.
[0047] FIG. 33 shows graphs of DNA recovery from drop analysis, and
dialysis of nucleotides from DNA on Millipore MF membranes.
[0048] FIG. 35 shows a graph of cleaning of RCA with centricon
YM-30 at different speeds.
[0049] FIG. 36 shows a graph of digestion of RCA products and
single stranded padlock probes with Exonuclease I and RecJf.
[0050] FIG. 37 shows a graph of fluorescence intensity for various
DNA particles.
[0051] FIG. 38 shows DNA nanoparticles visualized with Sybr Green
dye. Lower panel: 100.times. of 30 min RCA. Upper panel:
100.times.90 min RCA. Particle density is dependent on the spot and
the time it has been under the light (photo-bleaching occurs).
[0052] FIG. 39 shows a graph of incorporated Alexa 488
fluorescence.
[0053] FIG. 40 shows a graph of hybridized probe fluorescence.
[0054] FIG. 41 shows a graph of size distribution by intensity.
[0055] FIG. 42 shows a photograph of an agarose gel.
[0056] FIG. 43A shows graphs of fluorescence intensity, and
includes an example of a cloned particle (shaded) with high
affinity for DC as compared to a control particle (red) or
unstained cells (blue and grey).
[0057] FIG. 43B shows a graph of fluorescence intensity, and
includes an example of clonal particle was assayed against the
MDA-MB-231 breast cancer cell line.
[0058] FIG. 44 shows a graph of fluorescence of particles loaded
with doxorubicin.
[0059] FIG. 45 shows a graph of OD490 over time.
[0060] FIG. 46 shows a graph of IgM secreted from PBMC in the
presence of nanoparticles.
[0061] FIG. 47 relates to production and basic characterization of
DNA nanoparticles. Panel A relates to DNA nanoparticles are
produced by circularizing a 100 nM concentration of a 94 base ssDNA
template with T4 Ligase and a 300 nM concentration of a 31 base
templating primer. Polymerization was done with phi29 DNA
polymerase at 30.degree. C. for 30 minutes and terminated with
EDTA. Discrete particles are stained with SYBR Green and viewed
under a 100.times. oil objective. Panel B: Nanoparticles created
for various reaction times are measured with Dynamic Light
Scattering to validate size and demonstrate positive correlation of
hydrodynamic radius with reaction time.
[0062] FIG. 48 and FIG. 49 relate to flow cytometry assays of DNA
nanoparticles incorporating Alexa488 dCTP. Nine rounds of selection
were performed as summarized in FIG. 9 after which the selected
population was used to generate fluorescent DNA nanoparticles which
were incubated against dendritic cells. From the 9.sup.th round of
selection, individual population members were cloned using a
Promega pGEM-T cloning kit and served as templates for fluorescent
nanoparticle generation which were individually incubated with
dendritic cells. Of these clones, several were observed to bind DCs
with varying degrees of efficacy. The variation can be seen in FIG.
49 which compares several "positive" clones to a "negative" clone
with less binding capability. FIG. 48 provides data relating to a
comparison of the 9.sup.th round selection population with the
whole and negative control. In this case, it was observed that the
population exhibited a net shift over an individual member
indicating that, while not complete, the selection had enriched for
nanoparticles with enhanced binding capabilities. It is important
to note that the incorporation efficiency of Alexa488 OBEA-dCTPs by
phi29 polymerase was calculated to be only .about.1.5%. While it
did not appear to significantly slow the reaction, the poor
incorporation is likely the cause of the smaller shifts in the flow
peaks.
[0063] FIG. 50 provides data relating to flow cytometry data and
bright field/fluorescence microscopy for cell binding assays of
dendritic cell binding DNA nanoparticles. Microscopy images show
bright field, fluorescent and overlays from left to right. For flow
cytometry data, DNA nanoparticles of both the selected sequence
(Clone 3, green) and its reverse complement incorporating
Alexa.sub.488-dCTP were assayed for binding to DCs, P815 and THP1
cell lines. DCs were cultured as described herein. 0THP1 and P815
cells were maintained in RPMI supplemented with 10% FBS and 1% PSG
at 37.degree. C. Fluorescent DNA nanoparticles incubated with
5.times.10.sup.4 cells of each respective cell line were aliquoted
and resuspended in 50 .mu.L, of media (DCs resuspended in their
original media). 50 .mu.L, of the RCA reaction mix containing
synthesized DNA nanoparticles was added directly to each cell line,
mixed gently, and incubated on ice for 20 min in the dark. Cells
were washed 3 times and formaldehyde fixed to be analyzed by flow
and microscopy. Both analyses clearly demonstrate DC specificity as
well as specificity for the selected sequence over its reverse
complement.
[0064] FIG. 51 relates to the generation of ssDNA nano-particles:
.diamond-solid.(upper line): cells+qPCR+RCA; .box-solid.:
cells+qPCR reagent; .tangle-solidup.: cells+qPCR+RCA(-);
.diamond-solid.(lower line): cells. A graph of fluorescence (R) vs.
number of cycles. Eight bio-panning cycles have been made starting
from a random DNA-library. In each bio-panning cycle, MDA-MB-231
cells (epithelial breast cancer cells) were incubated with ssDNA
nanoparticles and the binding particles were amplified by qPCR. The
goal of each cycle was to enrich and amplified the binding
motifs.
[0065] FIG. 52 relates to DNA gel of amplified sequences: After
each bio-panning cycle, the amplified samples were run in a DNA gel
where the amplified products can be visualized. In all the
bio-panning cycles, amplified products were observed only from the
samples corresponding to cells+RCA particles (lane 5). No
amplification of cell DNA was observed (lane 2, 3 and 4). Line 1
represents the molecular weight ladder.
[0066] FIGS. 53A-53C show graphs including FACS analysis to test
the binding and specificity of ss-DNA nanoparticles. ss-DNA
nanoparticles were generated by RCA and incubated with the
epithelial breast cancer cells, MDA-MB-231.
[0067] FIGS. 54A-54C shows graphs including FACS analysis to test
the binding and specificity of ss-DNA nanoparticles. ss-DNA
nanoparticles were generated by RCA and incubated with the
epithelial breast cancer cells, MCF-7.
[0068] FIGS. 55A-55C show graphs including FACS analysis to test
the binding and specificity of ss-DNA nanoparticles. ss-DNA
nanoparticles were generated by RCA and incubated with the
monocytic cell line, THP-1.
[0069] FIG. 56 shows a general example scheme for the production
bi-specific DNA-nanoparticles (NP) that bind to both tumor cells
and T cells.
[0070] FIGS. 57A and 57B summarize the results of the selection of
pancreatic cancer cell line panc02 targeting particles. The mouse
pancreatic lines panc02 was panned with a single module DNA
nanoparticle library. After the 3.sup.rd and 4.sup.th round, the
selected pool was fluorescently labeled and tested on the target
cells by flow cytometry (FIG. 57A). The clones that contain the
AATGGGGCG (SEQ ID NO:12) motif bind specifically to panc02, whereas
the clones lacking the motif (C21 and C50) do not (FIG. 57B). In
the experiment shown, the four clones that show a fluorescent shift
in the left panel all contain the motif whereas the clones without
the motif are no better than controls. No difference is seen
against other epithelial cell lines.
[0071] FIG. 58 provides data relating to an ELISA experiment. Mice
immunized with DC binding DNA-NP, or CpG oligonucleotides
(ODN).
DETAILED DESCRIPTION
[0072] Some embodiments of the present invention include nucleic
acid based nanoparticles comprising multi-kilobase long concatamer
copies of a defined aptameric sequence. These can display a
sequence several hundred times throughout the particle, as well as
on the surface of the nanoparticles. These nanoparticles have the
advantage of a significant increase in the strength of binding over
current aptamer approaches.
[0073] Recent advancements in the understanding of immunity has
brought to light the immunological properties of certain sequences
of DNA, such as CpG sequences, can greatly stimulate the immune
system. CpG sequences can be used as an adjuvant for the delivery
of DNA based vaccines. A DNA based nanoparticle can easily
incorporate these sequences to be displayed hundreds of times on a
single particle potentially increasing the potency several orders
of magnitude.
[0074] More embodiments include nanoparticles comprising small
molecules, for example, drugs such as chemotherapeutics. Many of
the most common cancer chemotherapeutics are natural DNA binding
molecules. Delivery of chemotherapeutics bound to DNA is a method
to sequester such chemotherapeutics for transport to a delivery
site. DNA is also easily modified through altered base composition
to control such parameters as degradation, salt, and pH
responsiveness.
[0075] More embodiments include nanoparticles comprising nucleic
acid encoding sequence information. For example, DNA is an
information carrier that could be utilized for applications such as
gene therapy and siRNA delivery. More embodiments can include DNA
encoding therapeutic proteins and reporter genes.
[0076] One major advantage of this approach includes the ability to
avoid complex conjugation chemistries for targeting specificity,
biocompatibility, and drug incorporation of nanoparticles. Also,
clinical testing may be simpler for nucleic acid based
nanoparticles. For example, whereas the addition of any addition to
other types of nanoparticles may create a new entity for clinical
testing, here, a single particle of DNA can incorporate many
therapeutic functions and remains one type of molecule, namely,
DNA, a molecule that has been already approved for in vivo human
use.
[0077] DNA nanoparticles can be made cheaply in a volumetric
scalable way with simple techniques. Once validated for function
and developed they would most likely meet regulatory guidelines
quickly and be translated into clinical use. In addition to
clinical use, DNA nanoparticles can be used in diagnostics, for
example, as sensing agents utilizing DNA's ability to react to its
microenvironment.
[0078] The basic creation of DNA nanoparticles begins with a
padlock probe of single stranded DNA. The sequence of this probe
can be engineered for a variety of purposes, non-limiting examples
can include, immunogenic stimulation, enzymatic degradation, and
specific hybridization. This probe can vary in length from at least
about 2 bases, at least about 5 bases, at least about 10 bases, at
least about 50 bases, at least about 100 bases, at least about 500
bases, at least about 750 bases, at least about 1000 bases, at
least about 1 kb, at least about 5 kb, at least about 10 kb, at
least about 50 kb, and longer.
[0079] First, the probe is ligated endwise to form a closed loop
circle via a templating primer complementary to the ends of the
probe. The templating primer then serves as a primer for
polymerization with a strand displacing polymerase with the circle
acting as an endless template in Rolling Circle Amplification (RCA)
(FIG. 1A).
[0080] The polymerization can proceed for a period of time, after
which a certain length of single stranded DNA is created comprising
concatamer repeats of the original padlock probe. The concatamer
can spontaneously form a globular shape based on internal base
pairing. The size of the nanoparticles can be controlled by, for
example, the polymerization time of the reaction, the type of
polymerase used, and reaction conditions such as salt concentration
and pH. In some embodiments, the polymerization can proceed for a
length of time according to the length of product desired, where a
longer time can produce a longer product.
[0081] Non-limiting examples for functions of the nanoparticles are
shown in FIG. 1A. The DNA nanoparticle has multiple copies of the
original sequence both internally base-paired as well as displayed
on the surface which can serve as hybridization sites for DNA
conjugated entities or as a multivalent display of an aptameric
sequence that can be used for targeting. They might also be coded
to contain immunostimulatory sequences such as certain CpG
sequences. Furthermore, it may be possible to encode genetic
information in the RCA product and/or encase the DNA nanoparticles
in other nanostructures such as liposomes.
[0082] Some embodiments include nanoparticles with aptamers for use
in therapy. These particles have several advantages. For example,
DNA is essentially non-toxic, biocompatible, and can used as a
scaffold for the attachment of other agents. Also, DNA particles
can be easily loaded with DNA binding chemotherapy agents, such as
Doxorubicin and, if targeted by an aptamer sequence, may represent
an ideal targeting mechanism for such drugs.
[0083] Chemically diverse libraries are a rich source of potential
ligands for biomolecules and cellular targets of interest. When
modular biopolymers such as nucleic acids or polypeptides are used,
the combinatorial diversity of these libraries can become
astronomical and well beyond the capabilities of systemic high
throughput screening methods. Combing these libraries then requires
iterative schemes that couple a selection step with an
amplification step. For peptides, display of a given peptide on a
bacteriophage, virus, or bacteria allows amplification by growth of
the host organism (Scott J K, Smith G P. Searching for peptide
ligands with an epitope library. Science. 1990; 249: 386-390).
Nucleic acids are typically amplified by some variation of PCR
(Tuerk C, Gold L. Systematic evolution of ligands by exponential
enrichment: RNA ligands to bacteriophage T4 DNA polymerase.
Science. 1990; 249: 505-510). These tools have been used to select
peptides and short nucleic acid sequences (aptamers) that can bind
to a wide variety of proteins and cellular targets.
[0084] However, peptide and aptamer libraries have some distinct
limitations. Many peptide display formats, such as phage, present
many copies of each peptide per particle. This can allow the
recovery of relatively low affinity interactions that benefit from
the high avidity of the presentation format. However, it may be
difficult to maintain the desired binding avidity and specificity
when the selected peptides are moved to another particle or
molecule. Aptamer libraries can suffer from the reverse
complication since they are usually presented in a monovalent
format. Aptamers have been most clinically useful when a high
affinity interaction can function in an antagonist manner, though
they have been used as targeting moieties attached to nanoparticle
drug delivery vehicles (Farokhzad O C, et al. Targeted
nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo.
Proc Natl Acad Sci USA. 2006; 103:6315-6320; Bagalkot V, et al. An
aptamer-doxorubicin physical conjugate as a novel targeted
drug-delivery platform. Angew Chem Int Ed Engl. 2006;
45:8149-8152). However, when aptamers that were selected in a
monovalent format are attached to particles in a multivalent way,
specificity can be lost as low affinity interactions gain avidity.
In addition, there is always a concern that the transfer or
attachment of a peptide or aptamer to a new molecule or particle
may alter its conformation and binding affinity for the target of
interest. Thus for targeting moieties on particles, it would be
ideal to select the optimal ligand in the very context in which it
will be used.
[0085] A methodology has been developed for the construction of
large libraries of DNA nanoparticles and a process for the
iterative selection of particles with the desired properties. When
coupled with the other structural, functional, chemical, and
informatic properties of DNA these selectable particles allow the
creation of multifunctional particles for biomedical and
therapeutic use.
Molecular Evolution
[0086] Specific molecular recognition is the basis for most of
life's biological processes, but in the clinic and laboratory the
tools for such are limited to monoclonal antibodies, peptides,
aptamers, small molecules or natural biomolecule ligands. These
tools have not matched well with the problem of specific
recognition of neoplastic cells from their normal counterparts for
several reasons. The primary reason is that unique molecular
structures common to neoplastic cells of a given type but distinct
from normal cells are rare and all of the methods above, in their
simple forms, target only a single molecular shape. Bi-specific or
multi-specific versions have been described, for example diabodies
and bi-specific antibodies, but these are difficult to produce and
not widely used (Kortt A A, et al. Dimeric and trimeric antibodies:
high avidity scFvs for cancer targeting. Biomol Eng. 2001;
18:95-108). High affinity binding moieties can also suffer a loss
of specificity when combined because already high affinity
interactions do no benefit much from coupled or multi-valent
interactions, but previously low affinity interactions can gain
avidity and compromise selectivity.
[0087] Chemically diverse libraries are a rich source of potential
ligands for biomolecules and cellular targets of interest. When
modular biopolymers such as nucleic acids or polypeptides are used,
the combinatorial diversity of these libraries can become
astronomical and well beyond the capabilities of systematic high
throughput screening methods. Combing these libraries requires
iterative schemes that couple a selection step with an
amplification step. For peptides, display of a given peptide on a
bacteriophage, virus, or bacteria allows amplification by growth of
the host organism (Scott J K, Smith G P. Searching for peptide
ligands with an epitope library. Science. 1990; 249:386-390).
Nucleic acids are typically amplified by some variation of PCR.
Subtractive and in vivo selection schemes have been developed for
aptamer and phage displayed peptide libraries that can enhance the
cell specificity of recovered targeting ligands. Cellular targeting
has been demonstrated by libraries of peptides and oligonucleotide
aptamers (Siegel D L, et al. Isolation of cell surface-specific
human monoclonal antibodies using phage display and
magnetically-activated cell sorting: applications in
immunohematology. J Immunol Methods. 1997; 206:73-85; Rasmussen U
B, et al. Tumor cell-targeting by phage-displayed peptides. Cancer
Gene Ther. 2002; 9:606-612; Hicke B J, et al. Tenascin-C aptamers
are generated using tumor cells and purified protein. J Biol Chem.
2001; 276:48644-48654).
[0088] However, peptide and aptamer libraries have some distinct
limitations. Most peptide display formats, such as phage, present
many copies of each peptide per particle. This can allow the
recovery of relatively low affinity interactions that benefit from
the high avidity of the presentation format. However, it may be
difficult to maintain the desired binding avidity and specificity
when the selected peptides are moved to another particle or
molecule. Aptamer libraries can suffer from the reverse
complication since they are usually presented in a monovalent
format. Aptamers have been most clinically useful when a high
affinity interaction can function in an antagonist manner, though
they have been used as targeting moieties attached to nanoparticle
drug delivery vehicles (Farokhzad O C, et al. Targeted
nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo.
Proc Natl Acad Sci USA. 2006; 103:6315-6320; Bagalkot V, et al. An
aptamer-doxorubicin physical conjugate as a novel targeted
drug-delivery platform. Angew Chem Int Ed Engl. 2006;
45:8149-8152). However, when aptamers that were selected in a
monovalent format are attached to particles in a multivalent way,
specificity can be lost as low affinity interactions gain avidity.
In addition, there is always a concern that the transfer or
attachment of a peptide or aptamer to a new molecule or particle
may alter its conformation and binding affinity for the target of
interest. Thus for targeting complex targets like cells where
specificity is a greater concern than raw affinity, it would be
ideal to select the optimal ligand in the very context in which it
will be used.
[0089] Furthermore, while aptamer and phage display technologies
are occasionally referred to as evolutionary processes (one of the
earliest aptamer papers described the process as SELEX--Systematic
Evolution of Ligands by Exponential Enrichment), they are usually a
sequential combing process (thus the term "biopanning") and lack a
key component of Darwinian evolution--the generation of variants.
Even if point mutation is introduced in each round through error
prone PCR or growth in mutator bacteria, the evolutionary potential
is limited (Tuerk C, Gold L. Systematic evolution of ligands by
exponential enrichment: RNA ligands to bacteriophage T4 DNA
polymerase. Science. 1990; 249:505-510; Scott J K, Smith G P.
Searching for peptide ligands with an epitope library. Science.
1990; 249:386-390; Gram H, et al. In vitro selection and affinity
maturation of antibodies from a naive combinatorial immunoglobulin
library. Proc Natl Acad Sci USA. 1992; 89:3576-3580; Irving R A, et
al. Affinity maturation of recombinant antibodies using E. coli
mutator cells. Immunotechnology. 1996; 2:127-143). Rapid evolution
in natural systems occurs when large units of information can
re-assort and recombine, as is the case during meiosis.
[0090] Cell affinity agents are needed in many areas of cancer
research and clinical study. Monoclonal antibodies are the
workhorse of cell labeling, but are mostly useful when a given cell
surface molecule is know. For cancer cell detection in blood or
tissue or for cancer cell capture, single monoclonal antibodies are
rarely sufficient to distinguish cancer cells from neighboring
tissue or other normal cells. Clinically, this can be a problem for
evaluating surgical resection margins and for pathological analysis
of small samples (Blair S L, et al. Enhanced touch preps improve
the ease of interpretation of intraoperative breast cancer margins.
Am Surg. 2007; 73:973-976; Cortes-Mateos M J, et al. Automated
microscopy to evaluate surgical specimens via touch prep in breast
cancer. Ann Surg Oncol. 2009; 16:709-720). There as been
considerable interest of late in circulating tumors cells, but
these are present in very low numbers and require highly processive
and efficient capture methods to be obtainable in sufficient
numbers for down stream analyses. In vivo imaging with cancer
targeted contrast agents would be of obvious utility, and there is
considerable interested and development of tumor targeted
nanoparticles for drug delivery (Nie S, et al. Nanotechnology
applications in cancer. Annu Rev Biomed Eng. 2007; 9:257-288). In
vivo applications require biocompatible materials, and DNA is
obviously one such polymer.
[0091] Accordingly, a DNA nanoparticle library technology has been
developed that uses rolling circle amplification (RCA) of circular
oligonucleotide templates to produce libraries of single stranded
DNA nanoparticles that can be selected for cell binding properties.
By including random nucleotide sequences in the template
oligonucleotide, libraries can be produced and desired functions
selected. In some embodiments multimodal DNA nanoparticles can be
created that specifically bind to cancer cells. In more
embodiments, methods to optimize the creation of multimodal DNA
nanoparticles are described. The desired particles are "bred" by a
novel iterative selection and re-assortment method to create
modular DNA nanoparticles that contain multiple distinct
recognition elements (FIG. 1B).
[0092] A challenge in many areas of cancer research and treatment
is the production of cancer cell specific binding agents. In a few
cases truly cancer cell specific antigens can be targeted by
antibodies or other affinity ligands, but more often cancer cells
distinguish themselves by altered levels of multiple surface
molecules. The multimodal particles proposed here are capable of
multiple interactions with the target cell of interest and can
potentially overcome the limitation of single target agents such as
antibodies, peptides, and aptamers.
[0093] Some embodiments include methods to validate and optimize
combinatorial selection for multi-module particles. Data
demonstrates the selection of cell binding particles from a library
where each DNA nanoparticle contains a single recognition sequence
module. Using a leukemia (K-562) and a non small cell lung cancer
line (NCI-H23) as model systems, the optimal combinatorial strategy
are identified.
[0094] Some embodiments include methods to create particles that
bind to lung cancer and leukemia cell lines. Several different
cells lines derived from each of the two tumor types are used to
select particles with broad specificity to that tumor type. Cell
lines from non-neoplastic origins are used in subtractive screening
strategies if non-specific binders are recovered.
[0095] Some embodiments include methods to demonstrate cancer
specific cell binding of selected particles. Fluorescently labeled
particles are used on tissue arrays for fluorescent microscopy and
on suspension cells for flow cytometry. Particles tagged with
biotin or iron oxide are used for magnetic cell separation.
[0096] While this technology is similar to aptamer technology in
that it uses nucleic acid libraries as the basis for molecular
recognition, it differs in several important ways. Each particle
contains many copies of the sequence elements so there is intrinsic
multivalent display of the modules, allowing avidity to compensate
for low monovalent affinity. The modular nature of the particle
template construction allows multiple distinct recognition elements
to be assembled into a single molecular entity. Furthermore, the
combinatorial selection method allows the optimal particle with
multiple recognition elements to be evolved in the same molecular
context in which it will be used, rather than grafting them on to
some other framework or particle for application. The combinatorial
method also adds an element of true molecular evolution in which
novel combinations of modules can be created by re-assortment, akin
to recombination in meiosis.
[0097] It should be noted that there are many potential clinical
applications of these nanoparticles since DNA can have several
functions in addition to the formation of specific ligands. DNA can
be immunogenic if it contains unmethylated CpG motifs, it can act
as a scaffold for hybridizing other oligonucleotide conjugates, it
can have enzymatic activity, it is easily chemically modified to
allow small molecule or metal ion attachment and metals can be
directly deposited onto DNA, it can carry DNA binding drugs, and it
can carry genetic information (Klinman D M. Adjuvant activity of
CpG oligodeoxynucleotides. Int Rev Immunol. 2006; 25:135-154;
Breaker R R, Joyce G F. A DNA enzyme that cleaves RNA. Chem Biol.
1994; 1:223-229; Bern L, et al. DNA-Templated Photoinduced Silver
Deposition J. Am. Chem. Soc. 2005; 127:11216-11217; Richter J, et
al. Construction of highly conductive nanowires on a DNA template.
Applied Physics Letters. 2001; 78:536; Lund J, et al. DNA Networks
as Templates for Bottom-Up Assembly of Metal Nanowires. 5th IEEE
Conference on Nanotechnology. Nagoya, Japan; 2005:836-840; Zanchet
D, et al. Electrophoretic Isolation of Discrete Au Nanocrystal/DNA
Conjugates. Nano Letters. 2001; 1:32-35). Thus the particles
selected using methods provided herein can serve as the basis for
multifunctional nanoparticles for imaging, drug delivery, or
immunotherapy. DNA has a long clinical history and a favorable
toxicity and biodegradability profile (Fichou Y, Ferec C. The
potential of oligonucleotides for therapeutic applications. Trends
Biotechnol. 2006; 24:563-570).
[0098] Methods and compositions provided here are potentially
transformative in the area of cancer cell study and detection
because they couple the power of random libraries and biopanning
selections with molecular breeding concepts to create
multifunctional molecules for cell binding. In addition to the
conceptual advantages of this approach, discussed herein, once a
particle has been selected and sequenced, other laboratories can
easily create that particle from bacteria containing the cloned
sequence or a synthetic oligonucleotide and a few simple molecular
biology steps. The RCA reaction is scale-able and far less
complicated than hybridoma technology. The pioneering approach of
creating a single molecule nanoparticle with modular functionality
is groundbreaking in its flexibility and potential for development
as a platform for applications beyond just those discussed in
detail here. Some embodiments provided herein are unique and
innovative in at least three major ways. First, the module designs
are unlike any other library format in flexibility and ease of
implementation. Second, selection formats are the same as the
application format meaning the selected particles can be used
immediately without the need to chemically alter or conjugate them
to another molecule or particle. Finally, compared to other
nanoparticle materials, DNA is non-toxic and antisense
oligonucleotides, aptamers, gene therapy, and CpG oligonucleotides
have all been used in human trials.
Production and Characterization of DNA Nanoparticles by RCA
[0099] DNA nanoparticles are produced by enzymatic DNA synthesis
using a strand displacing DNA polymerase, phi29, and a circular
oligonucleotide template. The oligonucleotide circle is typically
produced by ligation of a 100-200 base pair linear oligonucleotide
with a short (30 bp) oligonucleotide complementary to the ends. The
ligation oligonucleotide also serves as the initiating primer for
the RCA reaction. Phi29 polymerase is highly processive (.about.70
kb) and produces a linear increase in single stranded DNA for over
an hour in a typical reaction.
[0100] The resulting RCA products are concatemers complementary to
the template circular oligonucleotide. These long single stranded
products collapse into randomly coiled nanoparticles, a property
that has been exploited for counting individual RCA events (Jarvius
J, et al. Digital quantification using amplified single-molecule
detection. Nat Methods. 2006; 3:725-727). The size of the particles
is a function of the time and efficiency of the RCA reaction. The
reaction can be stopped by the addition of EDTA or heat
inactivation of the phi29 polymerase, though the latter may lead to
aggregation of the DNA particles. The particles can be visualized
with either single stranded or double stranded fluorescent DNA
binding dyes due to the double stranded character that results from
internal base pairing. For analytical purposes the particles can be
made fluorescent by the inclusion of fluorescently labeled
nucleotides during the synthesis. Alternately a fluorescently
labeled oligonucleotide probe can by hybridized to the particles
(FIG. 2, panels A -D).
[0101] It is difficult to size the particles by conventional or
denaturing gel electrophoresis due to their large size and single
stranded character. Dynamic Light Scattering (DLS) is a common
technique for measuring the properties of nanoparticles such as
size and zeta potential. DLS uses the time autocorrelation of a
signal of scattered light to determine the polydispersity and
average diffusion coefficient, which through the Stokes-Einstein
equation is related to the average dynamic radius. RCA reactions
were carried out for four time points (10, 30, 45, 60 minutes) and
were stopped by the heat inactivation of the polymerase at
65.degree. C. for 10 minutes. The samples are then immediately
measured by DLS. For a monodisperse sample the autocorrelation
plots should show a single exponential decay, the exponent
coefficient of which is known as the first moment and is used to
calculate a Z-average size. The second moment is used to calculate
the deviation from monodisperse and is known as the polydispersity
index (PdI), which is a measure of relative peak width of the
Gaussian size distribution. In general if the PdI is greater than
0.25 it is recommended to use a secondary algorithm called
Non-Negative Least Squares (NNLS) which models the autocorrelation
curve as a contribution of several size samples and extracts
individual peak data (FIG. 3, panels A-3B).
Single Component DNA Nanoparticle Library Construction and
Testing
[0102] A method to generate high diversity libraries of DNA
nanoparticles and select for those with desired features through an
iterative screening and re-amplification method is summarized in
FIG. 4.
[0103] The library is generated from a template oligonucleotide
that has a random stretch of bases in the middle, flanked by PCR
primer sites. These ends also bind to the ligation oligonucleotide
to circularize the template for RCA amplification. The random
sequence is 60 nucleotides long and is flanked by defined sequences
that can be used to hybridize fluorescent or otherwise labeled
probes for visualization or purification. Using this design
.about.10 billion unique DNA nanoparticles can be produced in a
small volume (e.g., 50 .mu.l) RCA reaction. The particles are
screened for the desired binding activity and the binders amplified
by PCR using the Stoffel fragment of Taq polymerase that lacks 5'
to 3' exonuclease activity. The use of Stoffel fragment greatly
increased the amplification efficiency, presumably due to the
concatemeric nature of the DNA nanoparticles. Following
amplification several rounds of asymmetric PCR are performed to
increase the copy number of the template strand that is then
circularized by ligation and subjected to RCA to regenerate the DNA
particles. The cycle of screening, amplification, and particle
regeneration is repeated for several (e.g., 5-10) rounds.
[0104] The scheme described herein has been used to select DNA
nanoparticles that bind to human dendritic cells (DCs). DCs were
generated from peripheral blood monocytes. After 5-7 days of
culture, DC were incubated for 1 hour with the DNA nanoparticle
library, and washed several times with cold PBS. After 7 cycles of
selection and re-amplification aggregation of the DCs during the
washing steps was observed, suggesting that binding particles had
been enriched for that were causing agglutination. Flow cytometry
analysis indicated that the population of particles contained in
the 7.sup.th round pool was enriched for particles that bound to
DCs (FIG. 5).
[0105] Individual particles were obtained by cloning the PCR
amplification products from the 7.sup.th round of selection. 15
clones were sequences and all but one were unique. Each clone was
used to generate fluorescently tagged DNA nanoparticles and these
were tested for DC binding by flow cytometry. Several of the clones
showed good binding to the DCs (FIG. 6).
[0106] Further characterization of these DC binding particles is
underway, but the data to date demonstrates the feasibility of the
DNA nanoparticle library selection strategy. In addition,
non-specific uptake or immune activation of non-targeted particles
has not been observed, suggesting that selection of particles with
binding affinity for the target cell type is essential and that
non-specific cell binding is minimal.
[0107] The DNA nanoparticles are stable over the time of the
experiments. However, the particles are fairly resistant to
exonuclease and endonuclease degradation. Since the particles are
formed from a continuous single stranded DNA molecule, each
particle has only one 3' and one 5' end which may account for their
resistance to exonuclease digestion. Most endonucleases, including
DNase, prefer double stranded DNA as a substrate. The nuclease
resistance could increase by polymerizing nucleotides with altered
backbone chemistries. The phi29 polymerase can incorporate
phosphorothioate backbone nucleotides, although the rate of
polymerization is marginally slower.
Research Design and Methods
[0108] Some embodiments provided herein include methods and
compositions to develop multimodal particles that bind to a target
cell type through combinatorial "breeding" of DNA nanoparticles.
The overall strategy is to first optimize the selection
methodology, then apply the method to a panel of cell lines from
each of two cancer types and confirm their usefulness in several
cell binding applications. The rationale for using multiple cell
lines from a couple of cancer types is to allow the selection of
particles that are tumor specific but not cell line specific and to
then be able to cross compare. If necessary, subtractive screening
methodologies are employed to prevent the recovery of non-specific
cell binding particles. Cell lines or normal lines (e.g. NIH-3T3)
are used for subtraction.
Methods to Validate and Optimize Combinatorial Selection
[0109] Two cell lines, K-562 and NCI-H23, are used to test and
optimize the multimodal DNA nanoparticle selection methods. Each
round of selection consists of 4 essential steps and the molecular
biology is the same regardless of selection scheme (FIG. 7).
Library Ligation
[0110] The template oligonucleotide is mixed with the ligation
primer in T4 ligase buffer to final concentrations of 100 nM and
300 nM respectively. The mixture is heated to 95.degree. C. and
allowed to cool slowly to room temperature. T4 ligase is added and
the reaction incubated for 1 hour at 37.degree. C. For multimodal
libraries, there are multiple ligation primers, and all but one
primer are dideoxy terminated so that each multimodal template
circle is primed at only one location.
RCA
[0111] The ligation mixture is added to an RCA reaction mix
containing phi29 polymerase. The final concentration of the ligated
template oligonucleotides is 1 nM, meaning that in a 100 .mu.l
reaction .about.6.times.10.sup.10 DNA nanoparticles are created.
When the initial library is made each of these particles will
contain a unique sequence. The reaction proceeds for 30 minutes at
30.degree. C. and is terminated with EDTA. This produces particles
.about.250 nm in size. The RCA reactions are monitored in real time
with Oligreen, a single and double stranded DNA binding fluorescent
dye, to confirm linear amplification. Since the amount of template
DNA ligated and amplified is the same round to round, this rate
should be constant and thus serves as a quality control checkpoint
(FIG. 8).
Selection
[0112] Step A. The selection step can be performed in several ways,
depending on the application. In one embodiment, the particles are
incubated with the cell target of choice. If non-specific cell
binding is recovered, subtractive approaches can be used in which
the library is first or concurrently counter selected against an
irrelevant cell target to remove non-specific cell binding. This
can be done by preincubating the library with the irrelevant cell
to absorb non-specific binding or, if the target and irrelevant
cells can be easily separated, the library can be added to a
mixture of both with the target cells then later removed. Typical
incubation times are 30 minutes to an hour at either room
temperature or 37.degree. C. 10.sup.5 cells are mixed with the
entire RCA reaction from step 2. Step B. Washing is performed by
centrifuging the cells and aspirating the liquid, then resuspending
the cells and transferring to a new tube for the next wash. Three
to five wash steps will be performed. Transferring to new tubes at
each steps minimizes the recovery of plastic binding particles.
Step C. Particle recovery. Since each particle is a concatemer of
several hundred copies of the basic unit sequence, PCR
amplification of single particles or even particle fragments is
possible. Therefore, the particles can be recovered by lysing the
washed cells followed by 1 hour treatment with proteinase K. The
cell lysate is added to the PCR reaction in the next step, ensuring
recovery of both external bound particles as well as particles that
may have been internalized by the cells.
[0113] Amplification
[0114] One aim of the amplification step is to regenerate the
population of template oligonucleotides that can be ligated and
used to generate a pool of particles reflective of the particles
recovered in the selection step. The main amplification is by PCR
and the desired template DNA strand is secondarily enriched over
the complement by asymmetric PCR using only the primer for the
desired strand. a) Stoffel PCR. PCR amplification of these DNA
nanoparticles is much more efficient when the Stoffel fragment of
Taq polymerase, which lacks 5'-3' exonuclease activity, is used
instead of conventional Taq polymerases, which has 5'-3'
exonuclease activity. This may be because of the concatameric
nature of the DNA particle strand, which could bind many primers in
the initial rounds of PCR. The extending polymerase would run into
the primed strand downstream of it and begin digesting, leading to
very inefficient polymerization from the particle strand. The PCR
reactions are monitored in real time with Sybr green and stopped
once the production of PCR product plateaus. The real time plots
also allow quantitative estimates of the relative amount of DNA,
and by inference the number of particles, recovered in each round.
b) asymmetric PCR. The PCR product is diluted into a new reaction
mixture that contains only the primer that will produce the
ligatable template strand. This primer has a 5' phosphate. The
reaction is run for 10 cycles (FIG. 9).
[0115] After a successful selection, candidate particles are
further analyzed from the final pool. To obtain individual
particles, the final pool are amplified by Stoffel PCR and cloned
into a plasmid sequencing vector. Once cloned, 10-20 candidates are
sequenced to determine the extent of sequence diversity in the
final pool. Each candidate can be regenerated by PCR/asymmetric PCR
amplification from the plasmid.
Library Design and Selection Considerations
[0116] The design of each random module can be subject to some
constraints. The minimum length of an oligo that can effectively
circularize is reported to be around 80 bp. Since the flanking
PCR/ligation primers sites are 30-40 bp, the randomized region can
be at least about 40 bp. The potential diversity of any such
library is much greater than the sample size. For example, a 60 bp
library has 4.sup.60 different possible sequences,
.about.1.times.10.sup.36. Since typically 10.sup.10-10.sup.11
particles can be created in a reasonable volume, only a tiny
fraction is sampled of the possible number, and any particular
batch of the library is a unique subset of the possible with each
individual sequence represented by a single molecule. However,
10.sup.10 particles will likely contain any given 19 bp motif at
least once and smaller motifs will be well represented within the
sampled population.
[0117] When multimodal particles are created there is an additional
combinatorial element. If 3 libraries of the size above are
randomly combined into multimodal particles, then there would be
10.sup.30 different combinations, treating each module as a
discrete entity. Since there may be limits to 10.sup.10 particles
due to the physical constraints of particle synthesis, it may not
be ideal to combine libraries in the first few rounds of selection.
In fact, it is unlikely that any particular combination recovered
in the first round would reform in the second while the remaining
diversity is still high. On the other hand, if particular
combinations of modules would be optimal together but are not
particularly good on their own, then delaying combination of the
library may result in the desirable modules disappearing from the
population before they have a chance to team with the others for
selective advantage. However, in the absence of a rigorous model of
selection kinetics and fitness landscapes, an empirical approach
can be used.
[0118] The overall concept of the modular library screening method
can be divided into a "panning" phase, where binders within the
population are selectively enriched, and a "breeding" phase in
which the multimodal particles are re-assorted in each cycle so
that novel combinations can be generated and the optimal
combinations enriched (FIG. 10).
[0119] Three selection schemes can be tried. In the first, the
libraries are assembled into the multimodal format prior to the
first round and in all subsequent rounds. However, for the first
three rounds the particles are amplified as a single unit and the
modules are not be re-assorted (FIG. 11).
[0120] Non-modular PCR ensures that any particular combination of
modules that comes through the first round will be amplified to
higher copy number before the re-assortment process begins. For
subsequent rounds, the recovered particles are split and some
amplified as a single unit by the non-modular method and the rest
amplified as modules and recombined, ensuring representation of the
selected combinations as well as generating new combinations of
modules.
[0121] The second selection scheme attempts to pre-enrich the
modular pool for desired activity by screening the single component
libraries for several rounds prior to combination in multimodal
format. The individual libraries are screened for 5-10 rounds until
there is evidence in each of enrichment for binding clones
(indicated by an increased in the number of particles recovered in
each round as determine quantification from the real time PCR
amplification step). The combinatorial strategy is then be pursued
for several rounds. The final strategy is a hybrid of the first
two. Three to five rounds of selection are performed with each of
the component libraries, and then the following rounds are done
using only the combinatorial approach.
[0122] Protocols can be optimized against K-562 and NIH--H23 cell
lines. Single module library selections can be run in parallel. To
determine the optimal method, the particles can be evaluated for
cell binding affinity and specificity. The affinity can be measured
by adding equal numbers of particles to the cells and quantitating
the bound number by quantitative PCR after stringent washing.
Specificity can be assessed by performing the same experiment
against the reciprocal cell line and taking the ratio of the two.
If specificity is not achieved, then the selections can be repeated
with a subtractive step using the other line included. Finally, the
individual modules of any multimodule particles can be
independently amplified, ligated, and tested in the single module
particle format to determine if each module is contributing to the
overall binding of the multimodule particle.
[0123] While the library selection schemes do not require a priori
knowledge of the particular molecules on the target cells surface,
particles that are specific for the cell target of interest can be
used to identify the cell surface molecules. The particles can be
used in pull down experiments with target cell extracts and the
bound proteins identified by mass spectrometry (Mallikaratchy P, et
al. Aptamer directly evolved from live cells recognizes membrane
bound immunoglobin heavy mu chain in Burkitt's lymphoma cells. Mol
Cell Proteomics. 2007; 6:2230-2238).
Methods to Create Particles that Bind to Lung Cancer and Leukemia
Derived Cell Lines.
[0124] The optimal selection strategy described herein can be
applied to four additional lung cancer cell lines such as NIH-226,
NCI-H322M, NCI-H460, and NCI522, and four additional leukemia
derived lines, such as, CCRF-CEM, HL-60, Molt-4, and RPMI-8226L.
These cell lines are members of the NCI-60 panel of well defined
cancer cell lines. Since a desirable particle for many applications
can be one that binds broadly to tumors of a particular type, these
panels can be used in two ways. First, selections against each with
subtraction against a normal line or one of the other tumor lines
can be performed (FIG. 12).
[0125] Candidate particles from each of those can be tested against
the other cell lines of the same cancer type to identify cases
where broad selectivity may have been achieved by serendipity.
Conversely, broad specificity can be selected for in the selection
protocol by pooling cell lines and/or alternating the target cell
line or target cell pools in each round, thus providing a selective
advantage to particles that bind to all of the cell lines used.
This approach is applicable to lung cancer lines.
[0126] The leukemia derived lines come from several different types
of leukemia, including acute and chronic myeloid, acute lymphoid,
and plasma cell myeloma. In addition to devising selections that
will favor broad specificity to all of the lines, particles that
recognize the lymphoid derived but not the myeloid derived lines
may be recovered (and vice versa).
Methods to Demonstrate Cancer Specific Cell Binding of Selected
Particles
[0127] Particles selected for using the methods described herein
can be evaluated in at least three formats: flow cytometry,
histology by fluorescent microscopy, and cell capture. These three
techniques are generally reflective of most ex vivo applications of
cell affinity reagents.
Flow Cytometry
[0128] DNA nanoparticles can be coupled to fluorophores in at least
three ways. 1) Fluorescent nucleotides can be used during the
synthesis step, 2) DNA binding fluorescent dyes can be added after
synthesis (though these must be cell impermeant for the
applications here), or 3) fluorescently labeled oligonucleotides
can be hybridized to the particles after synthesis. The first of
these, labeled nucleotides, is has been done successfully with
Alexa488 labeled nucleotides (see data herein).
[0129] Labeled DNA particles can be used in the same way as labeled
antibodies. The particles are incubated with the fixed cells, the
cells washed, and the sample run on a flow cytometer (see data
herein). The particles do not affect the forward or side scatter of
the cells and unbound particles, while detectable by their
fluorescence, do not scatter sufficiently on their own and can be
gated out. Multiple particles can be used if they labeled with
compatible fluorophores.
Histology/Fluorescent Microscopy
[0130] The fluorescently labeled particles can be assessed in
fluorescent microscopy. Initially, the particles can be tested
against the cognate cell lines that have been cytospun onto slides.
Specificity can be assessed by creating slides with mixtures of the
cognate and other cells at defined ratios. Standard cell staining
protocols can be used and the various parameters (incubation times,
DNA nanoparticle concentration, etc) optimized.
[0131] Commercial tissue arrays (MaxArray, Invitrogen) can be used
to determine the specificity of the particles that bind to lung
cancer derived lines. Multi-tumor and normal arrays can be screened
first to determine the specificity, and then lung cancers arrays
can be used to determine the sensitivity against many different
samples. It is possible, if not likely, that different particles
can have different profiles when so compared. For histological
applications it may be useful to use a pool of particles to ensure
the broadest binding to a given cancer type
Cell Capture
[0132] The DNA nanoparticles can be used to capture and isolate
their target cells in several ways. The particles can be labeled
with biotin by either incorporation of biotinylated nucleotides
during the synthesis or by hybridization of a biotinylated
oligonucleotide after the synthesis, as for the fluorophores
described herein. Biotinylated particles can often be captured
along with the cells they are stuck to using streptavidin coated
magnetic beads. Alternately, the particles themselves can be made
magnetic through the coupling of iron oxide nanoparticles. An iron
oxide nanoparticle can be conjugated to the seeding primer in a 1:1
ratio such that when the reaction proceeds the iron oxide will be
embedded inside the DNA nanoparticle. The 1:1 conjugated oligo-iron
oxide structures can be used as hybridizing oligonucleotides to a
DNA nanoparticle scaffold (FIG. 13).
[0133] To assess capture efficiency, the target cell lines can be
mixed with irrelevant cell line and the capture efficiency
determine. The cells can be incubated with the DNA nanoparticles
then, in the case of the biotinylated particles, mixed with
magnetic streptavidin coated beads.
[0134] Some embodiments include methods to validate and optimize
combinatorial selection of libraries. Some embodiments can
demonstrate that 10 DNA nanoparticles each containing 3 unique
recognition elements can be amplified and reassorted to create more
than 50% increase in library diversity i.e. the sequences of more
than 10 out of 20 resorted clones will be different from the
original 10. Some embodiments can identify at least 1 multimodal
particle that binds to the target cell line 10-fold better than a
random control particle. Binding can be defined by quantitation of
recovered DNA by quantative PCR (QPCR) after incubation with the
target cells and washing as in the selection protocol.
[0135] Some embodiments include methods to select particles against
a panel cancer cell lines. Some embodiments can utilize at least 6
of the 10 cell lines, and select at least one binding particle.
Some embodiments can select at least 1 particle that has a 3 fold
increased binding to a targeted cancer cell line relative to the
normal cell line. Some embodiments can select at least 1 particle
that exhibits 3-fold increased binding to 3 or more cell lines from
the same cancer relative to the normal line
[0136] Some embodiments include methods to demonstrate cancer
specific cell binding of selected particles. Some embodiments
include utilizing particles that results in a mean fluorescent
intensity 5 fold greater when used against the target cell line
compared to an irrelevant cell line. Some embodiments include
demonstrating at least one particle or pool of particles that
results in a fluorescent intensity at least 2 fold greater on the
targeted cell line vs a control cell line. Some embodiments include
demonstrating at least one particle or pool of particles that
fluorescently stains lung cancer tissue with a 2 fold greater
intensity than normal tissue. Some embodiments include
demonstrating capture of 1 cell in 100 using a leukemia specific
particle.
Immunotherapy and Targeting Cancer
[0137] Immune therapy of cancer seeks to activate the body's own
immune system to destroy both primary and disseminated tumors. This
attractive idea has not met with much success in the clinic in part
because of the lack of good immune adjuvants. A novel selectable
DNA nanoparticle library technology has been developed. DNA
nanoparticles (DNA-NP) are generated by rolling circle replication
(RCA) of a circular template oligonucleotide. The resulting
complementary single stranded concatemer collapses into a random
coiled nanoparticle. Using this approach, DNA-NP have been
identified that, by virtue of their sequence, bind specifically to
DC, are taken up, and induce Ca.sup.2+ flux and IL-6 secretion. DC
binding DNA-NP can be further refined and tested for their ability
to activate immune responses in a mouse melanoma model. DNA and
other nucleic acids have a long clinical history and offer a
streamlined path to the clinic.
[0138] Some embodiments include methods to develop a DNA-NP capable
of inducing an immune response to an existing tumor. Without
wishing to be bound to any one theory it is believed that targeted
DNA nanoparticles that bind to and stimulate dendritic cells (DC)
can cause immune activation and lead to anti-tumor immune
responses.
[0139] Some embodiments include methods to screen and rank DNA-NP
that activate DC. Data shows the selection of DNA-NP that
selectively bind to and stimulate DCs. The hypothesis that DCs
stimulated with DNA-NP mature into antigen presenting cells capable
of T cell stimulation in vitro can be tested. One of the strengths
of the methods described herein is that multiple distinct sequences
can be easily combined to make combinatorial hybrid particles.
Combinations of DC binding particles can likely cause greater DC
activation than each given individually or in combination as
distinct particles. The most active particle can proceed to in vivo
tests.
[0140] Some embodiments include methods to relating to direct
injection of DNA-NP into tumors that can activate tumor
infiltrating DC. The mouse B16-OVA melanoma model can be used.
DNA-NP can be injected into established tumors and the activity on
tumor infiltrating lymphocytes determined by histology (CD11c &
T) and cytokine analysis. If robust responses are observed the
particles can be optimized for size and backbone composition and
the effect on tumor growth determined.
[0141] Some embodiments include methods that relate to immunization
with DNA-NP and model tumor antigens elicit antigen specific
responses and tumor rejection. The mouse B16-OVA melanoma model can
be used. The hypothesis that immunization with ovalbumin or
peptides derived from it together with the DNA-NP produces an
antigen specific immune response in normal healthy mice can be
tested. If robust responses are observed immunized tumor bearing
mice can be tested. If systemic immune responses are weak, the
hypothesis that intra-tumoral immunization will produce anti-tumor
responses can be tested. The dependence of those responses on T
cells will be analyzed in mice depleted of CD4.sup.+ or CD8.sup.+
cells.
[0142] Methods and compositions provided herein can provide
pre-clinical data important to begin planning human clinical
trials. Possible toxicity of the DNA nanoparticles can be carefully
monitored, particularly excessive inflammatory or autoimmune
responses and, where possible, pharmacodynamics can be
evaluated.
Background and Significance
[0143] Immune therapy in cancer has a long history marked by
striking anecdotal successes but few generally successful
strategies. Anton Chekhov and William Coley noted the relationship
between infection and cancer regression in the late 1800s, with the
latter developing "Coley's Toxin", a mixture of killed
Streptococcus pyogenes and Serratia mercescens as a treatment for
cancer (Gresser I (1987) A. Chekhov, M. D., and Coley's toxins. N
Engl J Med 317: 457). Modern approaches seek to activate the immune
system using defined stimulators of immune cells such as cytokines
or toll-like receptor (TLR) agonists, in place of bacterial
preparations (though Bacillus Calmette-Guerin (BCG) is still used
in the clinical treatment of bladder cancer) (Herr H W, Morales A
(2008) History of bacillus Calmette-Guerin and bladder cancer: an
immunotherapy success story. J Urol 179: 53-6).
[0144] Immunotherapies have shown promising results in several
cancers (Figdor C G, et al., (2004) Dendritic cell immunotherapy:
mapping the way. Nat Med 10: 475-80). Cytotoxic T lymphocytes
(CTLs) play a major role in eliminating malignant cells by
specifically recognizing antigenic peptides presented on MHC class
I molecules by dendritic cells (DC) (Nguyen T, et al., (1999)
Recognition of breast cancer-associated peptides by tumor-reactive,
HLA-class I restricted allogeneic cytotoxic T lymphocytes. Int J
Cancer 81: 607-15). Peptides derived from tumor-associated antigens
(TAA) have been identified for some forms of human cancers (Minev B
R, et al., (2000) Synthetic insertion signal sequences enhance MHC
class I presentation of a peptide from the melanoma antigen MART-1.
Eur. J. Immunol. 30: 2115-2124). Thus far, however, effective
peptide vaccination of cancer patients has been limited to very few
trials and for most cancers the antigens are not identified. DCs
are the most potent antigen presenting cells. In addition to taking
up the antigen, DCs also need to receive a maturation signal in
order to activate CTLs to perform their cytotoxic functions. If DCs
take up antigen without receiving a stimulus, they can induce
tolerance. Two central hurdles in immunotherapy are: (1) In many
instances tumor-specific CTLs though present in patients, are in a
tolerant/nonresponsive state. One of the major challenges for tumor
immunotherapeutic approaches is to break this tolerance to achieve
CTL-mediated killing of tumor cells. (2) For most tumors the
antigens are unknown and possibly patient and tumor specific.
Consequently, methods to overcome these obstacles should lead to a
marked improvement in antigen presentation and induction of potent
anti-tumor CTL.
[0145] Certain immunostimulatory molecules, from TLR agonists such
as CpG DNA and poly(I:C), to cytokines, have been tried in one form
or another, but general principles have been difficult to elucidate
and the need for more potent immune adjuvants remains acute.
Interferon alpha and interleukin 2 are approved for the treatment
of melanoma. Systemic administration of CpG oligonucleodies has
been moderately successful in mouse models and clinical trials,
suggesting that specific tumor antigens need not be included in a
successful therapy.
[0146] The immune system may be far more sensitive to stimulation
with multivalent and multifunctional particle delivery mechanisms,
perhaps mimicking virus and bacteria. The primary cell responsible
for initiating and directing an anti-tumor cytotoxic T cell
response is the dendritic cell, though B cells can also serve in
that role to some extent. Dendritic cells uptake antigen by
endocytosis and pinocytosis, but they must also encounter an immune
stimulus to mature into robust antigen presenting cells. Therefore,
targeting adjuvant activity to DCs is an obvious route to enhance
the potency of an adjuvant. This has been attempted in several
ways, including antibodies that bind to DC restricted membrane
antigens such as DEC-205. Targeting antigen to DCs in lymphoid
tissue using antibodies against DEC-205 in conjunction with an
adjuvant led to increased induction of immune responses. Thus using
molecules that target DC is a very promising approach to enhance
immune response to cancer. However, antibodies, need to be
humanized and are expensive to produce.
[0147] Chemically diverse libraries are a rich source of potential
ligands for biomolecules and cellular targets of interest. When
modular biopolymers such as nucleic acids or polypeptides are used,
the combinatorial diversity of these libraries can become
astronomical and well beyond the capabilities of systematic high
throughput screening methods. Combing these libraries requires
iterative schemes that couple a selection step with an
amplification step. For peptides, display of a given peptide on a
bacteriophage, virus, or bacteria allows amplification by growth of
the host organism. Nucleic acids are typically amplified by some
variation of PCR (Tuerk C, Gold L (1990) Systematic evolution of
ligands by exponential enrichment: RNA ligands to bacteriophage T4
DNA polymerase. Science 249: 505-10). Subtractive and in vivo
selection schemes have been developed for aptamer and phage
displayed peptide libraries that can enhance the cell specificity
of recovered targeting ligands (Siegel D L, et al., (1997)
Isolation of cell surface-specific human monoclonal antibodies
using phage display and magnetically-activated cell sorting:
applications in immunohematology. J Immunol Methods 206: 73-85).
Cellular targeting has been demonstrated by libraries of peptides
and oligonucleotide aptamers (Rasmussen U B, et al., (2002) Tumor
cell-targeting by phage-displayed peptides. Cancer Gene Ther 9:
606-12; Hicke B J, et al., (2001) Tenascin-C aptamers are generated
using tumor cells and purified protein. J Biol Chem 276:
48644-54).
[0148] However, peptide and aptamer libraries have some distinct
limitations. Many peptide display formats, such as phage, present
many copies of each peptide per particle. This can allow the
recovery of relatively low affinity interactions that benefit from
the high avidity of the presentation format. However, it may be
difficult to maintain the desired binding avidity and specificity
when the selected peptides are moved to another particle or
molecule. Aptamer libraries can suffer from the reverse
complication since they are usually presented in a monovalent
format. Aptamers have been most clinically useful when a high
affinity interaction can function in an antagonist manner, though
they have been used as targeting moieties attached to nanoparticle
drug delivery vehicles. However, when aptamers that were selected
in a monovalent format are attached to particles in a multivalent
way, specificity can be lost as low affinity interactions gain
avidity. In addition, there is always a concern that the transfer
or attachment of a peptide or aptamer to a new molecule or particle
may alter its conformation and binding affinity for the target of
interest. Thus for targeting moieties on particles, it would be
advantageous to select the optimal ligand in the context in which
it will be used.
[0149] The paradigm of nanotechnology for applications in the
medical field has been oriented around the framework of bottom-up
construction. Generally, a scaffold of polymer or metal serves as a
basis for the addition of functional moieties to lend the
nanomaterial the desired capabilities such as selective targeting,
transport of therapeutic and imaging agents, and immune evasion.
When biopolymers such as DNA are used, they are often rationally
designed to form a predetermined structure (Zhang C, et al., (2008)
Conformational flexibility facilitates self-assembly of complex DNA
nanostructures. Proc Natl Acad Sci USA 105: 10665-9). However, this
approach has overlooked a powerful tool of molecular biology: the
simple creation and efficient combing of libraries with diversity
of 10.sup.9 or more. Small nucleic acid aptamer sequences have been
identified with binding and enzymatic properties, but their use in
nanoparticle based applications has mostly involved grafting them
onto other materials. An overview of methods of diverse library
selection with nanoparticles to create libraries of DNA
nanoparticles by rolling circle replication of randomized circular
templates and selecting for particles that bind to a target cell
type is shown in FIG. 14.
Production, Characterization, and Purification of DNA Nanoparticles
by Rolling Circle Amplification
[0150] DNA nanoparticles are produced by enzymatic DNA synthesis
using a strand displacing DNA polymerase, phi29, and a circular
oligonucleotide template.
[0151] The oligonucleotide circle is typically produced by ligation
of a 100-200 base pair linear oligonucleotide with a short (30 bp)
oligonucleotide complementary to the ends. The ligation
oligonucleotide also serves as the initiating primer for the RCA
reaction. Phi29 polymerase is highly processive (.about.70 kb) and
produces a linear increase in single stranded DNA for over an hour
in a typical reaction. Phi29 can also incorporate phosphorothioate
backbone nucleotides, although the rate of polymerization is slower
(FIG. 15).
[0152] The resulting RCA produces are concatemers complementary to
the template circular oligonucleotide. These long single stranded
products collapse into randomly coiled nanoparticles, a property
that has been exploited for counting individual RCA events (Jarvius
J, et al., (2006) Digital quantification using amplified
single-molecule detection. Nat Methods 3: 725-7). The size of the
particles is a function of the time and efficiency of the RCA
reaction. The reaction can be stopped by the addition of EDTA or
heat inactivation of the phi29 polymerase, though the latter may
lead to aggregation of the DNA particles. The particles can be
visualized with either single stranded or double stranded
fluorescent DNA binding dyes due to the double stranded character
that results from internal base pairing. For analytical purposes
the particles can be made fluorescent by the inclusion of
fluorescently labeled nucleotides during the synthesis. Alternately
a fluorescently labeled oligonucleotide probe can by hybridized to
the particles (FIG. 16, panels A-C).
[0153] It is difficult to size the particles by conventional or
denaturing gel electrophoresis due to their large size and single
stranded character. Dynamic Light Scattering (DLS) is a common
technique for measuring the properties of nanoparticles such as
size and zeta potential. DLS uses the time autocorrelation of a
signal of scattered light to determine the polydispersity and
average diffusion coefficient, which through the Stokes-Einstein
equation is related to the average dynamic radius. RCA reactions
were carried out for four time points (10, 30, 45, 60 minutes) and
were stopped by the heat inactivation of the polymerase at
65.degree. C. for 10 minutes. The samples are then immediately
measured by DLS. For a monodispersed sample the autocorrelation
plots should show a single exponential decay, the exponent
coefficient of which is known as the first moment and is used to
calculate a Z-average size. The second moment is used to calculate
the deviation from monodisperse and is known as the polydispersity
index (PdI), which is a measure of relative peak width of the
Gaussian size distribution.
[0154] As seen with flow cytometry, the average particle size
increases as the reactions are allowed to proceed for longer. We
have noticed in other batches that the size seems to peak around
300 nm, even with longer reaction times. These measurements are in
good agreement with a freely joined chain model of polymer
condensation which estimates a 60 kb ssDNA strand to have a
hydrodynamic radius of 379 nm. It is suspected that the size of the
particles may be limited by the processivity of the phi29 enzyme
and steric hindrance as the particle grows. However, once reaction
conditions are fixed, the size of the particles is reproducible
batch to batch and can be tuned from roughly 100-300 nm. The
optimal size for a given application must be determined
experimentally, and the notion of "size" may be somewhat of an
anachronism for a flexible polymer condensate. Electron microscopy
is underway to obtain a higher resolution image of the particles at
various stages of growth (FIG. 17, panels A-B).
[0155] DNA-NP are purified by size exclusion chromatography and
dialysis, and concentrated by centrifugal membrane concentration.
After the RCA reaction there are significant excess free
nucleotides that should be removed before the particles are used
for other experiments. Special care is taken to avoid any possible
LPS contamination, including the use of dedicated glassware and
columns Negative control particles are always purified in the same
way on the same apparatus with the same buffers.
DNA Nanoparticle Library Selection Method
[0156] A method to generate high diversity libraries of DNA
nanoparticles and select for those with desired features through an
iterative screening and re-amplification method has been developed.
See Examples.
[0157] Individual particles were obtained by cloning the PCR
amplification products from the 7.sup.th round of selection. 15
clones were sequences and all but one were unique. Each clone was
used to generate fluorescently tagged DNA nanoparticles and these
were screened against DCs by flow cytometry. Several of the clones
showed good binding to the DCs. Clone 3 was chosen for further
evaluation. Particles that bind to breast cancer, ovarian cancer,
and pancreatic cancer derived cell lines as well as particles that
bind to adenovirus have been selected.
Specificity DC Binding DNA-NP
[0158] Further characterization DC binding clone 3 was undertaken.
The specific of the cell binding was investigated with several
other cell type and cell lines. DNA-NP were made fluorescent by the
incorporation of alexa488 tagged nucleotides during the synthesis.
Controls include a particle with the reverse complement sequence to
clone 3 and the reaction mix with the labeled nucleotides. In
addition to the cell types shown in the figure, we have seen no
binding to primary Chronic Lymphocytic Leukemia cells or the RAMOS
cell line. We have observed binding of clone 3 to human monocytes
and macrophages, albeit it at a lower level for the former.
Significantly, the clone 3 DNA-NP binds to mouse as well as human
DC (FIG. 18).
[0159] The results above were confirmed by fluorescent microscopy
(FIG. 19, panels A-B). Since all selections and binding experiments
to this point were performed on ice, it was confirmed that the
clone3 DNA-NP bound DC at 37.degree. C. by flow cytometry (data not
shown). Furthermore, when DC were incubated with clone 3 DNA-NP at
37.degree. C., the pattern of staining suggested that the DNA-NP
had been taken up. Preliminary confocal microscopy has supported
this interpretation but has not been confirmatory to date.
[0160] It was confirmed that clone 3 reproducibly binds by using
separate batches of particles and synthesizing particles from both
the original clone (PCR from bacteria colonies harboring the clone,
followed by assymetric PCR with a 5' phosphate on only the desired
primer for subsequent strand ligation and RCA) or from a synthetic
oligonucleotide template with the same sequence. In addition, it
was tested that the stability of particles kept at -20.degree. C.
and 4.degree. C. for several weeks; no loss of activity has been
observed.
DC Binding DNA-NP Activate DC
[0161] DC binding DNA-NP may cause DC activation through changes in
cytokine secretion, signaling, and surface marker expression. IL-6
secretion is a commonly used indicator that DC have matured into
immune activating cells, though a full cytokine secretion profile
is ultimately desirable to confirm this point. It has been shown
that DC incubated with the clone 3 DC binding DNA-NP secrete IL-6
(representative experiment shown in FIG. 20, panels A-B). In
addition, it has been measured Ca.sup.2+ flux 20 seconds after DC
are exposed to clone 3, but not after exposure to control
DNA-NP.
[0162] In general, non-specific uptake or immune activation of
non-targeted particles was not observed, suggesting that selection
of particles with binding affinity for the target cell type is
essential and that non-specific cell binding is minimal.
Hybrid Particle Formation
[0163] A powerful feature of the DNA-NP methods includes the
template sequence from which the particles are generated can be
easily manipulated. One or more synthetic oligonucleotides can be
used to build the template and beyond a minimum size of 60-80
bases, the RCA reaction proceeds equally well on templates
regardless of size. Therefore, once discrete particle sequences are
identified it is quite straightforward to prepare a hybrid template
by coupling the templates at the ligation step (FIG. 21).
[0164] One concern was that hybrid DNA nanoparticle may lose the
properties of the individual components. To test this, the clone 3
sequence was ligated step-wise into a continuous circle with an
equal length random sequence. The progress of these step-wise
reactions was verified by gel and the final products were observed
to undergo RCA as previously observed with single sequence
ligations. The hybrid nanoparticles still bounding DCs and a hybrid
control did not. This data supports the feasibility of developing
multifunctional DNA nanoparticles from either defined sequences or
during the selection.
Immunostimulatory Peptide Hp91
[0165] A short immunostimulatory peptide, Hp-91, was identified
that causes activation of human and mouse DCs. These peptides will
be used if the DNA-NP do not have sufficient immune activating
properties on their own, either in vitro or in vivo.
[0166] Hp-91 treated DCs induce antigen-specific T cells responses
as measured by IFN-.gamma. secretion in an ELISPOT assay. Hp-91
treated human DCs induced strong melanoma antigen-specific
CD8.sup.+ T cell responses, demonstrating the immunostimulatory
capacity of the peptide Hp-91 (FIG. 22). Similar results were
obtained in the mouse system, where BM-DCs pulsed with OVA-peptide
and exposed to Hp-91, induced strong proliferation of OVA-specific
CD8.sup.+ OT-I cells.
Intra-Tumoral Injection of Hp-91 Peptide Causes Recruitment of T
Cells and Dendritic Cells to the Tumor.
[0167] The number of lymphocytes and DCs within tumor has been
shown to correlate with good prognosis. In certain subsets of
breast tumors the presence of tumor-associated macrophages is
associated with better prognosis. Thus, in order to maximize the
anti-tumor immune response we tested two TLR agonists; pI:C (TLR3
agonist) and Pam3Cys (TLR2 agonist), and our immunostimulatory
peptides for their ability to recruit immune cells to the tumor.
Hp-91, pI:C, or Pam3Cys were injected into B16 melanoma, the mice
were sacrificed 24 h after the injection and the tumor was frozen
and sections stained for the indicated markers. Although all three
adjuvants caused recruitment of macrophages (MacI+) into the tumor,
only Hp-91 also caused the recruitment of DCs (CD11c+) and T cells
(CD3+) (FIG. 23). Further characterization using cell type specific
antibodies, suggests that these are CD8+ cells. No CD4+ T cells or
FoxP3+ cells (Treg) were detected (data not shown). This is a very
promising result as Hp-91 will not only contribute to the
recruitment of DCs, but also mature the arriving DCs. Thus in the
context of antigen release by concurrent local cytotoxic therapy,
we expect to create a very favorable environment for the uptake of
tumor antigen by DCs and their subsequent activation. In addition,
CD8 T cells after being primed in the draining lymph nodes are
expected to be recruited in higher numbers to the tumor site via
the peptide leading to a strong immune response and tumor
killing.
Coupling of Hp91 to DNA-NP
[0168] A method to attach peptides to DNA-NP has been developed.
The peptide is synthesized as a c-terminal conjugate to a 15 base
oligonucleotide that is complementary to the sequence of the DNA-NP
ligation or priming site. A Cy5 labeled Hp91-oligo conjugate was
synthesized with a complementary sequence to clone 3. An estimation
of the number of potential hybridization sites on the DNA
nanoparticles was made from the total DNA quantitation. The
nanoparticles were then hybridized at 37.degree. C. for 30 minutes
with increasing concentrations of the conjugate. After
hybridization, the mixture was purified by low pressure size
exclusion chromatography for which the elution profiles of the DNA
nanoparticles and the free Cy5 labeled peptide-oligo conjugate had
been previously established. At high ratios of Cy5 peptide-oligo
conjugate to DNA nanoparticles, a significant fraction of the
conjugate remained unhybridized as indicated by a strong peak at
the free conjugate retention time. As the ratio of the labeled
conjugate to the estimated hybridization sites on the DNA
nanoparticles dropped, the free conjugate elution peak began to
drop, eventually disappearing at a ratio of 1:2 (labeled
conjugate:DNA nanoparticle sites) indicating that the DNA
nanoparticles can be loaded to saturation by hybridization when 50%
of their available sites are occupied.
Research Design and Methods
[0169] Some embodiments will utilize existing DC binding DNA-NP to
find those that best stimulate DCs into antigen presenting cells as
measured by T cell activation. If none of the existing particles
are sufficient new ones can be selected by several strategies or,
if that fails, combine DC binding DNA-NP with the potent peptide
adjuvant Hp91. A unique feature of the technology can be exploited
to make hybrid particles and determine if they offer improved
performance over single sequence particles. Once the most promising
candidate particle is identified it can be tested in the B16-OVA
mouse melanoma model by both intra-tumoral and systemic
administration, with and without the model tumor antigen. Initial
studies of immune responses can direct pilot studies of anti-tumor
responses, with success leading to larger, statistically powered
studies.
Methods to Screen DNA-NP that Activate DC.
[0170] Five DNA-NP that bind to DC have been identified. These can
be compared for their ability to activate DC. The DC stimulatory
capacity of DNA-NPs can be assessed on myeloid (CD11c+) bone
marrow-derived DCs (BM-DCs) in vitro, as these are known to
function similarly to human monocyte-derived DCs.
[0171] DC activation and maturation is characterized by altered
surface expression of characteristic molecules, production of large
amounts of cytokines and enhanced T cell stimulatory capacity.
Therefore, the dendritic cell stimulatory capacity of the
DC-binding DNA-NPs can be evaluated in three ways: 1) their ability
to alter the expression of surface molecules on immature dendritic
cells that are classically up or down regulated upon maturation; 2)
their ability to induce secretion of inflammatory cytokines, and
finally 3) their ability to mature DCs into effective antigen
presenting cells that activate naive antigen-specific T cells. The
activity of the DNA-NPs can be compared to PBS (negative control)
and LPS as positive control. Bone marrow DCs can be generated from
primary mouse bone marrow cells depletion of other cell types and
culture with GM-CSF (Inaba K, et al., (1993) Granulocytes,
macrophages, and dendritic cells arise from a common major
histocompatibility complex class II-negative progenitor in mouse
bone marrow. Proc Natl Acad Sci USA 90: 3038-42). Each DNA-NP can
be tested at final concentrations of 10 and 100 ng/ml, which
corresponds to rough 10.sup.9 and 10.sup.19 particles per ml.
[0172] After 48 h, the CD11c.sup.+ CD11b.sup.+ B220.sup.- myeloid
DCs can be analyzed for surface expression of MHC-II, CD86, CD40,
and CD80 by flow cytometry. Furthermore, the cell culture
supernatants can be assayed for the content of IL-12, IL-6,
TNF-.alpha., IL-8, IL-10, TGF-.beta. and IL-1.alpha. by ELISA. To
demonstrate functional maturation of DCs stimulated by the DNA-NPs,
DCs can be tested for their capacity to activate antigen-specific
syngeneic T cells. Bone marrow-derived DCs generated from C57BL6
mice will be stimulated with DNA-NPs, LPS (positive control) or PBS
(negative control) for 24 h. The next day the DCs can be pulsed
with OVA.sub.257-264 peptides (for CD8 T cells) and OVA.sub.323-339
(for CD4 T cells) and co-cultured with TCR transgenic OVA-specific
OT-I and OT-II transgenic T cells for 38-50 h. C56BL/6 mice are
chosen, because antigen presentation can be readily monitored using
CD4+ and CD8+OVA-specific OT-II and OT-I transgenic T cells. T cell
activation can be assessed for a) proliferation by measuring the
uptake of [.sup.3H]-thymidine during the remaining 16 h of culture,
and b) Th1 and Th2 cytokines by measuring IL-4, IL-2, IL-10 and
IFN-.gamma. levels of the cell culture supernatants by ELISA and
intracellular IL-4, IL-2 and IFN-.gamma. levels by flow cytometry.
For intracellular staining of cytokines, the cells can be incubated
with brefeldin A to prevent leakage of the cytokines before
permeabilization.
Particle Ranking and Hybrids
[0173] The particles can be ranked according to their activity in
the assays herein. Since T cell stimulation is the ultimate goal,
significant weight can be given to those results, followed by
up-regulation of co-stimulatory molecules. However, any particle
that shows activity in any of the assays can be included in the
hybrid particle matrix herein.
[0174] As described herein, methods for producing DNA-NP with two
or more discrete sequence components are provided. Individual DC
binding particles that show activity can be combined to further
enhance their potency. The counter hypothesis would be that the
particles all function via the same mechanism and that there will
be no advantage to combining them. Hybrids pairs can be produced
from all sequences that produced positive results above. If all
five of the particles tested show activity, that would require 10
unique hybrids. These can be assayed and ranked as described above.
These experiments can be the most interesting if different
particles show a different spectrum of activation (ie. cytokine
secretion but not phenotypic maturation, T cell stimulation in the
absence of cytokines). However, if none of the hybrids shown
superior activity to the composite monomers then only the monomers
may be used.
Mechanism
[0175] Mechanistic studies are performed to address whether: 1)
endocytosis is required for the DNA-NP activity, and 2) the DNA-NP
activity occurs with TLR/MyD88. The first question is addressed
using a panel of endocytosis inhibitors concurrent with DNA-NP,
using cytokine secretion and phenotypic maturation as readouts. The
inhibitors are brefeldin A and colchicine, both of which interfere
with vesicle trafficking, filipin, which is known to inhibit
caveolae-mediated endocytosis by binding to cholesterol and
disrupting caveolae structure and function, and sucrose, which
generates hyperosmolarity that blocks membrane internalization and
clathrin recycling via the coated-pit pathway. Confocal microscopy
confirms the intracellular localization of the DNA-NP.
[0176] The potential role of TLR signaling is addressed by using DC
from TLR 3,4,7, and 9 as well as MyD88 knockout mice. An hypothesis
is that the DNA-NP bind to the surface of the DC by some DC
specific protein or glycoprotein and are then internalized by
endocytosis. While in the endosome they signal through TLR9 and,
downstream, MyD88. However, it is possible that DC activation
occurs by other mechanisms and the initial binding event could
trigger MyD88 independent pathways. The rapid calcium flux would be
consistent with this model. These experiments provide a basic
working model for the DNA-NP activity.
In Vitro Selection Against Mouse DC
[0177] A pool of DC binding particles were selected against human
DC. While at least one clone also binds to mouse DC, activation of
mouse DC was not confirmed. Bone marrow-derived DCs are generated
from C57BL/6 mice. These bone marrow-derived DCs are generated in
the presence of GM-CSF and yield only myeloid not plasmacytoid DCs
(PDCs), since GM-CSF prevents the development of PDCs. Further
purification are achieved by positively selecting CD11c.sup.+ cells
from the day 6-8 cultured using magnetic beads. If non-specific
cell binders are recovered, an adherent mouse fibroblast cell line
is used for counter selection. The adherent cells absorb
non-specific cell binders and are removed from the non-adherent
DCs.
In Vivo Selection
[0178] In vivo selection of targeting ligands has been well
established with phage displayed peptide libraries. While seemingly
a complicated and potentially difficult proposition, in vivo
selections have two significant advantages. The first is that the
selection is being performed in the very same environment that the
ultimate product will be used. The second is that the rest of the
animal acts as a subtractive substrate that will remove any
non-specific particles. We will perform in vivo selections by
injecting the DNA particle library subcutaneously and recovering
the draining lymph nodes several hours later. The lymph nodes will
be treated with collagenase to create single cell suspensions, and
the MHC-class II+ antigen presenting cells (including DCs and B
cells) will be isolated by magnetic bead separation. Subsequently
the particle recovery, re-amplification, and ligation is as
described herein.
Combinatorial Library Screening
[0179] FIG. 24 shows a strategy for selection and combinatorial
breeding of multimeric polyvalent DNA nanoparticles. Step 1.
Several libraries with unique ligation and PCR primers are
generated. Step 2. Each library is independently screened for a few
rounds against the target to create an initial enriched pool. Step
3. The products of the initial screenings are combinatorially
assembled into multimeric templates and polyvalent DNA particles
are generated. Step 4. The particles are subjected to subtractive
screening to enrich the desired binding activity and eliminate
unwanted cross-reactivities as described herein. In each selection
step the individual library components are re-assorted, creating
additional combinatorial diversity from which optimal particles can
be selected.
[0180] The hybrid particle concept can be introduced at the library
screening step. The specificity of particles for cancer cells can
be improved if more than one ligand is targeted, creating an "AND"
type function for binding. This can be implemented by performing
several selections against a given target cell population using
libraries with different circularization and PCR primer sequences.
The products of each selection can be combinatorially assembled by
creating particles that consist of one unit from each library. In
this way particles can be "bred" that optimize selectivity and
exploit the potential that each DNA unit might recognize a distinct
component of the target cell. In each round, the three component
pieces are re-assorted so that the optimal combination enrich over
several rounds of selection.
Candidate Particle Cloning and Binding to Target Cells
[0181] After a successful selection, candidate particles are
further analyzed from the final pool. To obtain individual
particles, the final pool are amplified by Stoffel PCR and cloned
into a plasmid sequencing vector. Once cloned, 10-20 candidates are
sequenced to determine the extent of sequence diversity in the
final pool. Each candidate can be re-generated by PCR/asymmetric
PCR amplification from the plasmid. The candidate particles from a
selection are tested individually for target cell binding by making
fluorescently tagged particles. The labeled clones are incubated
with lymph node suspension cells on ice for 1 h and co-stained with
antibodies against B220 (B cells), CD11c (myeloid DCs), and PDCA-1
(plasmacytoid DCs) to identify the bound target cells and
demonstrate specificity. Labeled cells are analyzed cell binding by
flow cytometry and fluorescent microscopy.
Addition of Immunostimulatory Peptide Hp91
[0182] If no particle is obtained with the ability to activate DC,
the DC binding DNA-NP is used as a carrier for peptide Hp91 as
described in the preliminary data. It is determine if the
DNA-NP/Hp91 formulation retains or improves the activity of the DC
stimulatory activity of the peptide. Even if there is not an
improvement with the DNA-NP in the in vitro assays, in vivo
experiments are performed as it is possible that the DNA-NP
targeting could be far more relevant in the in vivo.
Methods to Test the Hypothesis that Direct Injection of DNA-NP into
Tumors Activate Tumor Infiltrating DC
Tumor Model
[0183] All in vivo experiments are conducted in a transplantable
mouse model of melanoma using the mouse melanoma cell line B16-OVA
that expresses chicken ovalbumin, which serves as a tumor marker to
monitor immune responses. When injected s.c. into C57/BL6 mice,
B16-OVA produces a local tumor growth. The experiments are
hierarchically designed to reveal that 1) the DC binding DNA-NP can
perform that function in vivo, 2) that immune activation via DNA-NP
within the tumor can awaken a tumor specific immune response, and
3) that immune response will lead to tumor rejection.
[0184] C57/BL6 mice (n=5 per group) are inoculated with
5.times.10.sup.5 B16 cells s.c. Once the tumors reach 3-5 mm in
size they receive intra-tumoral injection of: 50 .mu.l of PBS, DC
binding DNA-NP, or a control DNA-NP. DNA-NPs is injected at 1 and
10 .mu.g/ml (.about.10.sup.10 and 10.sup.11 particles) suspended in
PBS. 24 hours later the mice are sacrificed and the tumor, draining
lymph nodes, blood, liver, and spleen are collected.
[0185] Histology is performed on the tumor and the number of
infiltrating lymphocytes compared (CD3+ and CD11c+) to controls.
Single cell suspensions are made by treating the tissue with
collagenase and the expression of IL-12, IFN.gamma., TNF alpha, and
RANTES by sorted CD11c+ cells determined by RT-PCR. The particle
characteristics are optimized.
Nanoparticle Optimization
[0186] The size of the DNA nanoparticles can be tuned by the RCA
reaction time (see data herein). The DNA-NP are synthesized under
reaction conditions that produce a mean size of 100, 200, and 300
nm as measured by dynamic light scattering. Aliquots are reserved
for confirmation by WETSEM (see data herein for example image). The
different size batches are equalized for total particle number and
compared as described herein. The optimal size thus determined is
used for all other experiments.
[0187] In addition, the nuclease sensitivity can be manipulated in
several ways. The particles are inherently resistant to
exonucleases since they are a continuous single strand with only
one 5' and one 3' end. Furthermore, these ends can be made
exonuclease resistant. The 5' end is created by the ligation primer
and can therefore be made with an altered base (e.g.
phosphorothioate) at the time of oligonucleotide synthesis. The 3'
end can be made resistant by adding a modified dideoxy nucleotide
triphosphate at the end of the RCA reaction, such that the growing
strands are terminated by an exo resistant base. The particles are
insensitive to endonuclease because of their primarily single
stranded character. However, endonuclease resistance can be
increased by the incorporation of phosphorothioate nucleotides
during the RCA reaction. Furthermore, degradation independently
increases activity of CpG oligonucleotides containing
phosphorothioate backbones. Particles are synthesized that contain
entirely a phosphorothioate backbone and are compared as described
herein.
Tumor Regression Studies
[0188] A long term tumor monitoring study is performed. Groups of
mice (n=5) are inoculated with tumor and injected with DNA-NP or
controls as described herein. 7-14 days after the initial injection
mice receive a second injection identical to the primary one. The
mice are observed bi-weekly and the primary tumor size measured
using a set of calipers: A.times.B.sup.2/2 (A=long axis, B=short
axis) over a period of 20 days. After 20 days or if the tumors
reach 1.5 cm in diameter, whichever occurs first, mice are
sacrificed. At time of euthanasia mice are monitored for potential
pathological effects. The spleen and kidney are weighed and tissue
analysis is performed.
Methods to Test the Hypotheses that Immunization with DNA-NP and
Model Tumor Antigens Elicit Antigen Specific Responses and Tumor
Rejection
[0189] Co-injection of the most potent DNA-NPs identified herein
with antigen can lead to the induction of an immune response. The
potency of DNA-NPs as vaccine adjuvants is compared to alum to
measure the quality as well as quantity of the immune response by
using the same route of immunization. Ovalbumin (OVA) and influenza
haemagglutinin (HA) have been suggested to study the adjuvanticity
of new formulations. Since aluminum compounds do not exhibit an
adjuvant effect when used with HA, whole OVA protein are used as
antigen, to establish the adjuvanticity of the DNA-NP.
[0190] The adjuvanticity of the DNA-NPs is assessed and compared to
aluminum hydroxide. Groups of C57BL/6 mice (n=10) receive s.c.
injections of OVA/PBS (as negative control), OVA/aluminum hydroxide
(Alhydrogel.sup.R, aluminum hydroxide, from Superfos Biosector,
Vedbaek, Denmark) (positive control), or OVA/CpG (positive
control), and 3 different doses of OVA/DNA-NPs (1-100 .mu.g/mouse),
that show DC activation in preliminary studies. Optimal
formulations of antigen adsorbed to aluminum adjuvant are prepared
to correctly evaluate new adjuvants. To minimize variation and to
avoid non-reproducibility due to different preparations of aluminum
components, a specific preparation of Alhydrogel.sup.R, aluminum
hydroxide, from Superfos Biosector, Vedbaek, Denmark, are used. The
complete absorption of the antigen on aluminum adjuvant is verified
by measuring antigen/protein levels before and after adsorption in
the supernatants. The conditions are optimized to reach the WHO
recommended adsorption of 80%. Two to three weeks after
immunization the animals receive a second "booster" immunization
performed exactly as the first injections. Blood is obtained from
mice at three time points: before immunization for base antibody
levels, before the booster immunization and 1-2 weeks after the
second "booster" immunization. Plasma IgG and IgM levels specific
for the injected antigen are measured by direct ELISA using plates
coated with antigen. The antibody results are determined in
arbitrary units against an ELISA reference serum in order to
reliable compare results obtained on different days. The type of
immune response is further characterized by measuring the subclass
antibody concentrations. In mouse, the production of IgG2a is
recognized as characteristic of a Th1 response, whereas the
production of IgG1 is characteristic of a Th2 response. Therefore,
the assessment of the type of immune response is performed by
measuring IgG1, IgG2a and IgG2b levels by ELISA. The ratio of
IgG2a/IgG1 antibody titers is used as indicator of Th bias. A Th2
response is also characterized by the secretion of Th2 type
cytokines, such as IL-4, IL-5, whereas a Th1 type response is
characterized by the secretion of IL-2 and IFN-.gamma..
[0191] The in vivo induced T cell responses is detected in vitro
using a variety of assays. 1) Proliferation assays are performed by
adding OVA.sub.257-264 peptides (to stimulate CD8 T cells) and
OVA.sub.323-339 (to stimulate CD4 T cells), PBS/no peptide
(negative control) or ConA (positive control) to un-separated
lymph-node cells from the draining lymph node, which contain T
cells and antigen presenting cells, and measuring the uptake of
[.sup.3H]-thymidine after 4 d. To measure T cell responses,
un-separated lymph-node cell cultures are set up as described
herein, but the positive control will be phorbal myristate acetate
(PMA) and soluble anti-CD3, since ConA is not a potent stimulus for
Th2 cytokines. After 24 h the culture supernatants are assessed for
IL-4, IL-2, IL-5, IL-10, TGF-.beta. and IFN-.gamma. levels by
ELISA. IFN-.gamma. ELISPOT and intracellular flow cytometry assays
for IL-4, IL-2 and IFN-.gamma. are used to measure the number of
cytokine secreting T cells and double labeling for CD4 and CD8 to
detect the type of T cells responding. For intracellular staining
of cytokines, the cells are incubated with brefeldin A to prevent
leakage of the cytokines.
Tumor Rejection Studies
[0192] C57/BL6 mice are inoculated with 5.times.10.sup.5 B16 cells
s.c. Once the tumors reach 3 mm in size, groups of mice (n=20)
receive s.c. immunizations of Ovalbumin mixed with: PBS, or 10
.mu.g DNA-NPs. 7-14 days after the initial injection mice receive a
second injection identical to the primary one. Half the mice from
each group (n=10) are sacrificed to analyze immune responses and
the other half are monitored for tumor progression.
[0193] Ten days after the final immunization, half the mice from
each group are sacrificed and tumor draining lymph nodes and
spleens are harvested. Unfractionated lymph node cells and
splenocytes are cultured in medium only and re-stimulated with
mitomycin C-treated (50 .mu.g/ml) B16-OVA cells. B16-OVA cells are
exposed to mitomycin C for 20 min, washed and co-cultured with
lymph node cells. As a positive control lymph node cells and
splenocytes are stimulated with Concanavalin A (5 .mu.g/ml). After
16-40 h the cell culture supernatants are assessed for IL-2, IL-4,
IL-5, IL-10, TGF-.beta. and IFN-.gamma. levels by multiplex-luminex
assay or ELISA. IFN-.gamma. ELISPOT is used to measure the number
of cytokine secreting T cells. To further investigate the
contribution of CD8+ and CD4+ T cells to the cytokine secretion,
each cell type is depleted from the splenocytes separately using
specific antibodies prior to in vitro culture. To measure the
generation of functional CTL responses, unfractionated lymph node
cells and splenocytes are re-stimulated in vitro with the mitomycin
C-treated (50 .mu.g/ml) B16-OVA cells. The cells are expanded in
24-well plates for 6 days at a concentration of 3.times.10.sup.6
cells in 1.0 mL of medium with the addition of recombinant mouse
IL-2 (50 U/ml) after 24 h of culture. Cytolytic activity is
assessed at day 6 by culturing expanded lymph node cells and
splenocytes with B16-OVA target cells and B16 (=negative control
target cells), using a standard 4 h LDH assay. The absorbance
values from supernatants are recorded at OD 490 nm. The percent of
specific lysis is calculated as follows:
(D.sub.EXp.-OD.sub.spon.E-OD.sub.Spon.T/OD.sub.Max.T.-OD.sub.Spon.T).time-
s.100, where OD.sub.Exp is the OD related to the experimental LDH
release, OD.sub.Spon.E the OD related to the spontaneous release of
LDH from the effector cells only, OD.sub.Spon.T is the OD related
to the spontaneous LDH release from target cells only, and
OD.sub.maxT. is the OD related to the maximum LDH release from
target cells using lysis buffer.
[0194] The other half of mice from each group (n=5) are observed
bi-weekly and the primary tumor size is measured using a set of
calipers: A.times.B.sup.2/2 (A=long axis, B=short axis) over a
period of 20 days. After 20 days or if the tumors reach 1.5 cm in
diameter, whichever occurs first, mice are sacrificed. At time of
euthanasia mice will also be monitored for potential pathological
effects see below. The spleen and kidney will be weighed and tissue
analysis will be performed by our molecular pathology core.
[0195] If there is a low frequency of melanoma-specific T cells a
second round of in vitro activation of the lymph node cells might
be necessary. Cells are cultured for 12-16 days with periodic
re-stimulation of the cultures with tumor-lysate pulsed irradiated
syngeneic BM-DCs. After 2-3 rounds of re-stimulation, the lymph
node cells are mixed with target cells and assayed for their
killing capacity as described herein. If no specific lysis of
target cells is observed in the LDH assay, target cells are
pre-treated with 20 ng/ml IFN-.gamma.24 hours prior to use in
cytotoxicity assays, which is known to increase the HLA expression.
HLA molecules are monitored by FACS.
Pharmacodynamics and Toxicity of DNA-NPs In Vivo.
[0196] The immunization experiments provide an opportunity to gain
insight into the in vivo distribution and half life of the DNA NPs,
as well as any associated toxicity. Organs and blood are recovered
from the sacrificed animals and tissue extracts prepared. An
attractive feature of the DNA-NP is that they can be very
sensitively and precisely quantified by real time PCR. rtPCR is
used to surmise the biodistribution and circulating levels of the
particles. DNA-NP were injected into mice intravenously and no ill
effects were observed.
[0197] Groups of mice receive DNA-NPs, that show activity in the
tumor model described herein, in 3 different doses s.c. (1-100
.mu.g/mouse). All mice are monitored daily for necrotic areas at
the site of injection and for signs of distress and death over the
course of all experiment. Any mice that die or are euthanized due
to distress or discomfort is evaluated. Furthermore, serum is
assayed for anti-nuclear antibodies by standard immunofluorescence
approaches, for anti-DNA antibodies by L. crithidia assays, and for
rheumatoid factor and anti-HMGB1 antibodies by ELISA once every two
weeks until 2-3 months after the injection of adjuvant. At time of
euthanasia mice are also be monitored for potential pathological
effects. The spleen and kidney will be weighed and tissue analysis
will be performed.
[0198] It is possible that high levels of DNA-NPs could potentially
cause side effects, such as local tissue necrosis or lead to the
induction of anti-DNA antibodies. If tissue necrosis occurs it will
likely be mild and transient, since the DNA-NPs are not
administered chronically. If side effects occur, lower doses are
tested.
Concurrent Cytotoxic Therapy
[0199] An hypothesis for antigen agnostic approach is that DNA-NP
will stimulate the tumor infiltrating DC which will, in turn,
awaken tumor specific CTLs. However, other immunotherapy studies in
this system s have shown improved results when combined with
cytotoxic therapy. If the DNA-NP alone do not induce tumor
regression, experiments are repeated with concurrent treatment
using 5-azacytidine(5-aza). 5-aza is given i.p. at a dose of 0.2
mg/kg for three cycles, each cycle consisting of a daily i.p.
injection for 5 consecutive days followed by 2 days rest.
EXAMPLES
Example 1
Generation of Nanoparticles
[0200] This example demonstrates the characterization of the DNA
nanoparticles. Nanoparticles were created through RCA with a
variety of different encoding sequences. Nanoparticles were made
into discreet particles as imaged by microscopy (FIG. 25) and were
made in varying sizes as determined by DNA binding dyes in a flow
cytometry setting (FIG. 26). Discreet DNA nanoparticles were made
with modified fluorescent nucleotides as an internal labeling
method with little to no background. In addition, the DNA binding
drug Doxorubicin was loaded on the DNA nanoparticles (FIG. 27).
Example 2
Generation of Aptamer Particle Libraries
[0201] Multivalent aptamer containing particles will be generated
from circular oligonucleotide templates by rolling circle
amplification. FIG. 28 shows the library template oligonucleotide
design. The ends contain PCR primer binding sites for the
amplification of the library during rounds of selection. These ends
also bind to the ligation template oligonucleotide to circularize
the template for RCA amplification. The random sequence is 60
nucleotides long and is flanked by defined sequences that can be
used to hybridize fluorescent or otherwise labeled probes for
visualization or purification.
[0202] The generative library oligonucleotide templates will be
circularized by annealing to a complementary ligation target. That
target oligonucleotide will also serve as the primer to initiate
rolling circle amplification (RCA) by phi29 polymerase. RCA
produces a single stranded concatemer that is the complement of the
library template oligonucleotide (FIG. 29, panel A). The number of
repeats contained in a given strand is a function of the size of
the template and the amount of time the RCA reaction is allowed to
proceed. Initially, 30 minute to one hour reactions will be used to
produce strands containing several hundred repeats. These strands
collapse into random coiled balls that are 100-1000 nm in diameter.
Each particle should have many copies of the aptamer on or near the
surface of the particle, enabling multivalent interactions with
targets.
Example 3
Screening Aptamer Particle Libraries
[0203] The particle library will be panned against touch
preparations of breast tissue containing both normal and malignant
cells. Slides used will be selected as those with small numbers of
clearly identifiable tumor cells in a background of many normal
cells. The normal cells will serve to sponge away aptamer particles
that bind to targets found on both normal and tumor cells. Tumor
specific aptamer particles will be recovered by either positive or
negative selection using a laser capture microscopy (FIG. 29, panel
B).
Example 4
Amplification and Regeneration of Aptamers
[0204] Aptamers bound to the target cells will be amplified by real
time PCR. The amplified aptamers will then subjected to several
rounds of asymmetric PCR to enrich for the template strand. The
templates will be re-circularized using the ligation template from
above and RCA used to generate particles as originally done (FIG.
29, panel C). Multiple rounds of panning will be performed. The
real time PCR amplification will give a rough quantitative estimate
of the number of bound particles in each round. Successful
enrichments will be indicated by a substantial increase in the
number of bound particles (for example, 100-1000 fold).
[0205] Once a pool of aptamers has been enriched against the breast
tumor cells, individual aptamer sequences will be determined by
cloning the final amplified products into a plasmid vector and
sequencing. Clonal populations of aptamer particles derived from
individual aptamers can be recreated from these clones by the same
PCR/asymmetric PCR approach used in the screening rounds.
[0206] The aptamer particles can be readily labeled either with DNA
binding fluorescent dyes (for example, Sybr gold, or oligreen) or
by hybridization of a fluorescently labeled oligonucleotide probe.
The clonal populations will be tested for specific binding to
breast tumor cells by fluorescent in situ histochemistry.
Individual aptamer particles stained with a DNA dye such as
Oligreen, are sufficiently bright to be detected by a flow
cytometer.
Example 5
Development of Multimeric Polyvalent Aptamer Particles
[0207] The modular nature of the DNA particles allows for multiple
discrete aptamer sequences to be displayed on a given particle. The
specificity of particles for cancer cells should be improved if
more than one ligand is targeted, creating an "AND" type function
for binding. This will be implemented by performing several
selections against a given target cell population using libraries
with different circularization and PCR primer sequences (FIG. 30).
The products of each selection can then be combinatorially
assembled by creating particles that consist of one unit from each
library. In this way we can "breed" particles that optimize
selectivity and exploit the potential that each aptamer unit might
recognize a distinct library. In each round, the three component
pieces will be resorted so that the optimal combination will enrich
over several rounds of selection.
Example 6
DNA Nanoparticle Libraries for Imaging and Therapeutic
Applications
[0208] Rolling Circle Amplification (RCA) of a circular DNA
template produces a continuous single stranded complementary
concatemeric nanoparticle. These particles may have applications in
biological sensing, detection, and therapeutics. RCA reactions were
monitored in realtime and the relationship between reaction times
and conditions and particle size determined by dynamic light
scattering and gel electrophoresis. DNA binding fluorescent dyes
were used to visual the particles in flow cytometry and fluorescent
microscopy. A purification strategy based on size exclusion
chromatography was developed. The nuclease sensitivity profiles of
the particles were determined for both endo and exo nucleases:
these could be attenuated by the incorporation of phosphorothioate
nucleotides during the RCA reaction or by modifying the 5' or 3'
bases of the strand. The stability of the particles in human serum
and plasma was evaluated and the interactions with serum proteins
was profiled. These experiments lay the foundation for further
development of DNA nanoparticles for biomedical applications.
Example 7
Production & Characterization of DNA Particles
Incorporation of Phosphorothioate Nucleotides
[0209] RCA reactions were run using either dNTPs, a mixture of dNTP
and phosphorothioate backbone cytosine nucleotides (C.alpha.STP) at
a 1:1 ratio with dCTP, or with a nucleotide cocktail where all dCTP
was replaced with C.alpha.STP. The results indicate that the phi29
DNA polymerase can incorporate C.alpha.STP in place of dCTP during
the RCA process, albeit with some loss of efficiency (FIG. 31).
Method to Purify the Particles by Size Exclusion Chromatography
[0210] Protocol for size exclusion resins: 1. Template circles were
RCAed at 1 nM in 50 .mu.l volumes for 30 minutes and stopped with 5
.mu.l 500 mM EDTA. Oli Green 1.times. was included in the reaction.
2. EconoColumns (BioRad) 0.5 cm internal diameter.times.5 cm length
were filled with Bio-Gel P-100 Medium Resin (90-180 .mu.m hydrated
radius, fraction range 5-100 kDa). The resin was hydrated in PBS
and was packed to a column height of 3.7 cm, total column volume
726 .mu.l. PBS elution buffer was drained to the top of the column
and 50 .mu.l of sample was applied and 1 drop was released from the
bottom of the column to let sample enter resin--column was then
closed. 1 mL of PBS was added to provide hydrostatic pressure (this
was kept constant as column flowed) and drops were collected on
full flow rate. Drops were then measured for A260 and Ex 480 Em 520
showing clear fractionation between nucleotides and DNA created
(FIG. 32).
Method to Purify Small Volumes of Particles by Drop Dialysis &
Centrifugal Concentration
[0211] Protocol for drop dialysis: 1. RCA products were made at 1
nM in 100 .mu.l with ligations from the streptavidin padlock probe
for 5, 10, 30 and 60 minutes respectively and stopped with 10 .mu.l
500 mM EDTA. 2. Three Millipore MF membranes 0.05 .mu.m VMWP (2.5
cm) were floated on 100 mL TBS in a glass dish until hydrated. 30
.mu.l from each time point was applied for each time point. 20
.mu.l of each sample was recovered at the appropriate time point.
Time points were 30, 60, and 120 minutes. A zero time point was not
applied but kept aside. 3.10 .mu.l of each recovered sample from
each time point was mixed with 40 .mu.l TBS and 0.125 (1.times.)
stock Oli Green and measured at ex 480 em 520.
[0212] FIG. 33 shows a drop dialysis recording of 30 minute
unlabelled RCA reaction (normal dNTPs) along side a different drop
containing Alexa-488 nucleotides. Every hour 1 .mu.l of each drop
was removed and diluted in 50 .mu.l PBS and measured. The Alexa-488
nucleotides were measured by simple fluorescence and the DNA was
measured by Oli Green.
[0213] DNA particles were cleaned with centricon YM-30 column at
different speed. Speed 14,000 rpm and 5,000 rpm gave similar amount
of flow through, but 1,000 rpm did not seem sufficient to drive the
liquid through the membrane in a reasonable time (FIG. 35).
[0214] Method for DNAse and Exonuclease Resistance Profiles
[0215] Step 1. Template circles were RCAed at 1 nM using
phosphodiester dNTPs for 10 min at 30.degree. C. and heat
inactivated at 65.degree. C. for 10 min. Step 2. RCA products were
mixed 1:1 with 1.times.NEB buffer 2 containing 2.times. OliGreen
stock dye. Step 3. Similarly, ss probe (unligated linear templates)
was diluted to 100 nM in phi29 buffer and mixed 1:1 with
1.times.NEB buffer 2 containing 2.times. OliGreen. Step 4. Each
sample was divided into 3 equal aliquots of 100 .mu.l and each
aliquot received either 20 units Exonuclease 1 or nothing. Samples
were monitored by Ex 480 Em 520 at 37.degree. C. over 1 hour with
20 second interval reads. Step 5. Percent digestion is calculated
as the 1-(the ratio of the digested signal to blank signal). Step
6. RecJ may not be optimal. Also note that the ExoI digestion was
already very progressed by the time readings started (FIG. 36).
[0216] More experiments to characterize DNA particles include
experiments where serum stability and interactions are tested. The
incorporation of nucleotides with free carboxy, thiol, amine for
conjugating other small molecules is tested. Incorporation of
biotinylated nucleotides is tested. Attenuation of nuclease
sensitivity by incorporation of modified DNA or RNA backbones is
tested.
Labeled with DNA Binding Dye (Oligreen) by Flow Cytometry &
Microscopy
[0217] DNA particles were made by RCA reactions of varying times,
labeled with Oligreen dye, and run on a flow cytometer. The
fluorescence intensity correlates with the length of the reaction
and presumably the size of the particles (FIG. 37).
[0218] DNA nanoparticles visualized with Sybr Green dye (FIG. 38).
LEFT: 100.times. of 30 min RCA, RIGHT: 100.times.90 min RCA.
Particle density is dependent on the spot and the time it has been
under the light (photo-bleaching occurs).
Labeled with Incorporated Fluorescent Nucleotides or Hybridized
Probe by Flow Cytometry.
[0219] This is an example of Alexa-488 labelled DNA balls. The
experiment was to hybridize on Alexa-647 oligos and see dual
fluorescence. Sample 1 was the non labelled DNA with no oligo.
Sample 3 was Alexa-488 labelled DNA hybridized to Alexa-647 tagged
oligo. The incorporated Alexa 488 fluorescence is shown in FIG. 39,
while the hybridized probe fluorescence is shown in FIG. 40.
Size Distribution by Dynamic Light Scattering
[0220] A Zetasizer Nano instrument was used to measure the size
distribution of DNA nanoparticles produced by RCA reactions of
varying time, using dynamic light scattering (FIG. 41). We have
noticed in other batches that the size seems to peak around 250 nm,
even with longer reaction times. It is suspected that the size of
the particles may be limited by the processitivity of the phi29
enzyme. Dynamic light scattering may also underestimate size due to
the low index of refraction of the particles.
Agarose Gel Electrophoresis
[0221] RCA reactions were run for time points 30 sec, 1 min, 2 min,
5 min, 10 min across left to right from the 1 kb ladder (FIG. 42).
The top band on the ladder is 10 kb. The RCA reactions were stopped
on time by EDTA and then mixed with alkaline denaturing buffer.
They were then heated to 95.degree. C. for 5 min. The gel was a
0.7% agarose alkaline gel. Samples were run for 3 hours at 40 V and
stained with GelRed. The products (including the ladder) should be
rendered single stranded by the alkaline conditions.
[0222] In more experiments to visualize DNA nanoparticles,
nanoparticles are visualized using electron microscopy.
Example 8
Generating a DNA Particle Library
Methods to Produce, Select, Amplify, and Reproduce DNA
Particles
[0223] An overview of the strategy is shown in FIG. 6. (A)
LIGATION: (1) Prepare the buffer and add the library and the gluing
primer (gp). (2) Boil a water bath, mix solution well and place in
water bath. Allow water to cool to RT slowly. (3) Once about
33.degree. C., add 1 .mu.l T4 ligase and ligate at RT for 1 hour.
TABLE 1 summarizes components of the reaction.
TABLE-US-00001 TABLE 1 Component Volume buffer Water 41.5 .mu.l T4
ligase 10 X buffer 5 .mu.l 500 mM DTT 0.5 .mu.l Total: 47 .mu.l 10
uM library 0.5 .mu.l 10 uM gp 1.5 .mu.l T4 ligase 1 .mu.l
[0224] (B) VERIFICATION OF THE RINGS (RCA): (1) Add 49 .mu.l of the
master mix with oh green and add 0.5 .mu.l of ligated sample. (2)
Ensure that the TECAN is at 30.degree. C. and add 0.5 .mu.l phi 29
polymerase. (3) Mix together and run in TECAN with program 3 Hour
RCA FAM to visualize linear amplification--note slope of curve. (4)
Run a reference sample with: 0.5 .mu.l sal inv pp (Standard for
RCA). TABLE 2 summarizes components of the reaction.
TABLE-US-00002 TABLE 2 Component Volume Water 40.5 .mu.l 101 .mu.l
413.1 .mu.l 10 X phi buffer 5 .mu.l 12.5 .mu.l 51 .mu.l dNTPs 10 mM
3 .mu.l 7.5 .mu.l 30.6 .mu.l 500 mM DTT 0.5 .mu.l 1.25 .mu.l 5.1
.mu.l Oli Green from stock 0.125 .mu.l 0.313 .mu.l 10.2 Ligated
sample (1 nM) 0.5 .mu.l Phi29 polimerase 0.5 .mu.l
[0225] (C) CREATION OF DNA BALLS: (1) Add to the 49 .mu.l of the
Master Mix 0.5 .mu.l of ligated sample. (2) Right before starting
the program add 0.5 .mu.l phi 29 polymerase. (3) Run all tubes at
30.degree. C. for 30 minutes. (4) Stop the reactions with 5 .mu.l
500 mM EDTA--Mix well and store at 4.degree. C. TABLE 3 summarizes
components of the reaction.
TABLE-US-00003 TABLE 3 Component Volume PCR water 40.5 .mu.l 101.25
.mu.l 141.75 .mu.l 413.1 .mu.l 10 X phi buffer 5 .mu.l 12.5 .mu.l
17.5 .mu.l 51 .mu.l dNTPs 10 mM 3 .mu.l 7.5 .mu.l 10.5 .mu.l 30.6
.mu.l 500 mM DTT 0.5 .mu.l 1.25 .mu.l 1.75 .mu.l 5.1 .mu.l Ligated
sample 0.5 .mu.l Phi29 polimerase 0.5 .mu.l
[0226] (D) ELISA WELLS: (1) Rinse the Elisa well 2 times with
TBS-Tween and 2 times with TBS. (2) Add the solution (.about.55
.mu.l) in the well and incubate for 20 min in a shaker. (3) Rinse 3
times with TBS-Tween and 3 times with TBS. (4) Elute with 26.6
.mu.l of 0.04% biotin in DI-Water. (5) Let it rest in a shaker for
20 min. The 55 .mu.l can be divided into two parts and select with
those. This decreases the risk of loosing some of the positive
samples. In addition the two parts can be taken along and at the
end, at the PCR cleaning, be re-combined again.
[0227] (E) PCR: (1) Take the 26.5 .mu.l of the selected RCA and add
22 .mu.l of PCR mmx and 1 .mu.l of VB and VF; (2) Add 0.5 .mu.l of
Stoffel enzyme. (3) Program: Open SYBR Green with Dissociation
curve protocol. Select the correct wells that your samples. Thermal
profile: START: 120 sec at 95.degree. C.; CYCLES: 35 cycles, 30 sec
at 95.degree. C., 60 sec at 61.degree. C., 20 sec at 72.degree. C.
END: 60 sec at 95.degree. C., 30 sec at 55.degree. C., 30 sec at
95.degree. C. (4) Remove 25 .mu.l from each tube and store in new
tubes to be run in a gel for analysis--use a 1.5% precast Gel Red
agarose gel run at 115 V for 1 hr. (5) Remove 15 .mu.l from each of
the two remaining and transfer to a new tube and store at
-20.degree. C. for future reference. (6) Put aside 10 .mu.l of the
solution for the asymmetric PCR. TABLE 4 summarizes components of
the reaction.
TABLE-US-00004 TABLE 4 Component Volume PCR Water 26.5 .mu.l 66.25
.mu.l 92.75 .mu.l 278.25 .mu.l Stoffel 10 X Buffer 5 .mu.l 12.5
.mu.l 17.5 .mu.l 52.5 .mu.l 10 mM dNTPs 5 .mu.l 12.5 .mu.l 17.5
.mu.l 52.5 .mu.l 25 mM MgCl.sub.2 10 .mu.l 25 .mu.l 35 .mu.l 105
.mu.l 100 X SYBR (final conc 2X) 1 .mu.l 2.5 .mu.l 3.5 .mu.l 10.5
.mu.l 10 .mu.M vB (200 nM final conc) 1 .mu.l 2.5 .mu.l 3.5 .mu.l
10.5 .mu.l 10 .mu.M vF protected Phos 1 .mu.l 2.5 .mu.l 3.5 .mu.l
10.5 .mu.l Stoffel Fragment 0.5 .mu.l
[0228] (F) GEL: Create gel: 100 mL 0.5.times.TBE buffer, 2.5 g
Agarose. Boil: 60 sec MIX, 30 sec MIX, 10-30 sec and mix until
liquid. Add 10 .mu.l of 10000.times. GelRed dye. Pour gel
(.about.50 mL) on the tray and add the comb. Let it cool
(.about.20-30 min) Only once cooled remove the comb. Solutions for
the gel: 15 .mu.l sample. 6 .mu.l diluted dye. (160 .mu.l water+40
.mu.l of Bluejuice 10.times. and 40 .mu.l Bluejuice 10.times.). Run
1.5 .mu.l only for the ladder (100 bp) vs the 20 .mu.l total of
sample w/dye. Load a bit of 0.5 TBE buffer on the platform, add the
gel in the tray and cover it with buffer. Run at 70V for 80 min
[0229] (G) ASSYMMETRIC PCR: Mix together 10 .mu.l of the PCR
product with 37.5 .mu.l of a PCR mmx and 2 .mu.l of VF protected
phosphate. Add 0.5 .mu.l of Stoffel Fragment, flick and put in the
machine. Program: Start: 120 sec @ 94.degree. C.; Cycles: 10
cycles, 30 sec at 94.degree. C., 60 sec at 61.degree. C., 20 sec at
72.degree. C. Rest: 4.degree. C. After the asymmetric PCR, PCR
clean each sample. TABLE 5 summarizes components of the
reaction
TABLE-US-00005 TABLE 5 Component Volume PCR water 21.5 .mu.l 53.75
.mu.l 225.75 .mu.l 10X Stoffel Buffer 4 .mu.l 10 .mu.l 42 .mu.l
dNTP 10 mM (2.5 mM ea) 4 .mu.l 10 .mu.l 42 .mu.l MgCl.sub.2 (25 mM)
8 .mu.l 20 .mu.l 84 .mu.l vF protected phos (final conc. 400 nM) 2
.mu.l 5 .mu.l PCR product 10 .mu.l Stoffel fragment 0.5 .mu.l
[0230] (H) LIGATION 2 and after: To the cleaned sample add 5 .mu.l
of T4 ligase 10.times. buffer, 0.5 .mu.l of 500 mM DTT and 1.5
.mu.l of gluing primer added T. Boil water and place mix in water
and allow to cool to RT. Add 1 .mu.l T4 Ligase and ligate for 1
hour.
[0231] (I) VERIFICATION OF THE RINGS considerations from round 2 on
(RCA): Ligation2Slope/slope standard=x. Ligation3Slope/slope
standard=y. To get back to the same concentration you need to
dilute: 100 x/y.mu.L of ligated sample over 100 .mu.l of DI
water.
[0232] (J) ELISA WELL consideration from round 2 on: If the sample
on the PCR spikes too early, try to dilute it. After run 2 or 3 run
the RCA in an empty container to see eventual non-specific
binding.
Example 9
Selection Against a Cellular Target (Primary Human Dendritic
Cells)
[0233] Procedure: 1. Count 105 cells to use in 1 mL. 2. Spin 3 min
at 3000 rpm remove supernatant and add 50 .mu.l of RCA. 3. Let
react for 1 hr on ICE. 4. At the same time put 1.2 mL of Cell media
on as many eppendorf as the planned rinses steps and keep it in the
ice box. 5. Spin the cells 3 min at 3000 rpm. 6. Remove the
supernatant and rinse with 500 .mu.l of COLD 5% BSA in PBS. 7.
Remove the cell media from one of the test tube and add the liquid
with the cells. 8. Discard the empty tube. 9. Repeat it as many
times as your rinses steps. 10. Remove the supernatant and add 50
.mu.l of Hypotonic Lysis buffer and 3 .mu.l of Proteinase K (10
mg/mL). 11. Put in heat block at 56.degree. C. for 1 hr. 12. Move
to a second heat block in order to stop the protease at 95.degree.
C. for 15 min 13. The sample is ready to be used.
[0234] Results of a selection against dendritic cells (DC). The
final pool of particles after 7 rounds of selection by the
procedure above was labeled with alexa nucleotides and added to
dendritic cells (green line). An irrelevant particle was used as a
control (blue line). The red line is the cells alone. The shift in
fluorescence, as measured by flow cytometry, indicates enrichment
of DC binding particles. The pool of particles was cloned into a
plasmid vector and individual clones selected, sequenced, and
regenerated by PCR, ligation, and RCA to create individual clonal
particle populations. These were then assayed as above. FIG. 43A
shows an example of a cloned particle (shaded) with high affinity
for DC as compared to a control particle (red) or unstained cells
(blue and grey).
[0235] The same clonal particle was assayed against the MDA-MB-231
breast cancer cell line (FIG. 43B).
[0236] The particle originally selected against DC does not seem to
bind the breast cancer cell line, suggesting that some level of
cell specificity may have been achieved even without a counter
selection strategy., This example shows selection resulting in
unique sequence particles that bind to the cells much better than
control particles
[0237] In more experiments to select against a cellular target
(primary human dendritic cells), resulting in unique sequence
particles that bind to the cells much better than control particles
the following experiments are carried out: Selective enrichment of
particles that bind to one cell type preferentially over another is
performed. In vivo selection for tumor targeting particles in a
mouse model is carried out. Multi-library combinatorial selection
is carried out. Selection for enzymatic activity is carried
out.
Example 10
Functional experiments
Loading of Doxorubicin into DNA Particles
[0238] The fluorescence of doxorubicin is quenched with the
addition of DNA nanoparticles, indicating doxorubicin binding to
the particles (FIG. 44).
Protection of Cells from Dox when Untargeted Particle is Used
[0239] The IC.sub.50 was determined to be 0.1 .mu.g of Doxo for 104
cells. When this amount of Doxo is incubated with the DNA
particles, the survival is almost 100%, indicating that DNA
absorbed the Doxo and protect cells from the toxicity of Doxo. This
preliminary data indicate two things: (1) DNA is not able to get
into the cells by itself. Targeted DNA particles may be selected or
a targeting moiety added. (2) At the same time, DNA nanoparticles
absorbed Doxo, which is good for our purpose of loading DNA with
drugs (FIG. 45).
Lack of Non-Specific Activation of Immune Cells with Untargeted
Particles
[0240] DNA nanoparticles containing an immunogenic CpG sequence
were incubated with PBMCs and IgM secretion measured. The K3
oligonucleotide is a positive control and contains the same
stimulatory sequence as the DNA nanoparticles. "DNA" in the FIG. 46
refers to the nanoparticles. The experiments were conducted in both
primary serum and heat inactivated serum.
[0241] In more experiments to demonstrate function of DNA
nanoparticles the following experiments are carried out Immune
activation by targeted particle containing CpG motifs is performed
Immune activation by targeted particle hybridized to an
immuno-stimulatory peptide is performed Immune activation by
chemical conjugation of a TLR3 agonist is performed Immune
activation by combinations of the above is performed. Cell
targeting by hybridization of a targeted peptide is performed.
Example 11
Applications: Therapeutic
[0242] In experiments to demonstrate therapeutic applications of
DNA nanoparticles the following experiments are carried out. Cancer
therapy by targeted delivery of doxorubicin is performed. Cancer
therapy by immune activation is performed. Cancer therapy by direct
killing of tumor cells is performed. Vaccine adjuvant for
protective vaccines is performed.
Example 12
Applications: Imaging
[0243] In experiments to demonstrate imaging applications of DNA
nanoparticles the following experiments are carried out. Ex vivo
imaging of cancer cells with tumor specific particles fluorescently
labeled or biotinylated for histochemistry is performed. In vivo
imaging with particles that hold contrast agents is performed.
Example 13
DeNAno: Selectable Deoxyribonucleic Acid Nanoparticle Libraries
[0244] DNA nanoparticles of approximately 250 nm were produced by
rolling circle replication of circular oligonucleotide templates
which results in highly condensed DNA particulates presenting
concatemeric sequence repeats. Using templates containing
randomized sequences, high diversity libraries of particles were
produced. A biopanning method that iteratively screens for binding
and uses PCR to recover selected particles was developed. The
initial application of this technique was the selection of
particles that bound to human dendritic cells (DCs). Following 9
rounds of selection the population of particles was enriched for
particles that bound DCs, and individual binding clones were
isolated and confirmed by flow cytometry and microscopy. This
process, which has been termed DeNAno, represents a novel library
technology akin to aptamer and phage display, but unique in that
the selected moiety is a multivalent nanoparticle whose activity is
intrinsic to its sequence. Cell targeted DNA nanoparticles may have
applications in cell imaging, cell sorting, and cancer therapy.
[0245] The paradigm of nanotechnology for applications in the
medical field has been oriented around the framework of bottom-up
construction. Generally, a scaffold of polymer or metal serves as a
basis for the addition of functional moieties to lend the
nanomaterial the desired capabilities such as selective targeting,
transport of therapeutic and imaging agents, and immune evasion
(Ferrari, M. Cancer nanotechnology: opportunities and challenges.
Nat Rev Cancer 5, 161-171 (2005)). When biopolymers such as DNA are
used, they are often rationally designed to form a predetermined
structure (Zhang, C. et al. Conformational flexibility facilitates
self-assembly of complex DNA nanostructures. Proc Natl Acad Sci USA
105, 10665-10669 (2008)). However, this approach has overlooked a
powerful tool of molecular biology: the simple creation and
efficient combing of libraries with diversity of 10.sup.9 or more
(Wilson, D. S. & Szostak, J. W. In vitro selection of
functional nucleic acids. Annu. Rev. Biochem. 68, 611-647 (1999);
Smith, G. P. & Scott, J. K. Libraries of peptides and proteins
displayed on filamentous phage. Methods Enzymol. 217, 228-257
(1993); Clackson, T., Hoogenboom, H. R., Griffiths, A. D. &
Winter, G. Making antibody fragments using phage display libraries.
Nature 352, 624-628 (1991)). Small nucleic acid aptamer sequences
have been identified with binding and enzymatic properties, but
their use in nanoparticle based applications has mostly involved
grafting them onto other materials (Tuerk, C. & Gold, L.
Systematic evolution of ligands by exponential enrichment: RNA
ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510
(1990); Ellington, A. D. & Szostak, J. W. In vitro selection of
RNA molecules that bind specific ligands. Nature 346, 818-822
(1990); Bartel, D. P. & Szostak, J. W. Isolation of new
ribozymes from a large pool of random sequences [see comment].
Science 261, 1411-1418 (1993); Huang, C. C., Huang, Y. F., Cao, Z.,
Tan, W. & Chang, H. T. Aptamer-modified gold nanoparticles for
colorimetric determination of platelet-derived growth factors and
their receptors. Anal. Chem. 77, 5735-5741 (2005)). In this study,
the concepts of diverse library selection methods with
nanoparticles have been have fused by creating libraries of DNA
nanoparticles by rolling circle replication of randomized circular
templates and selecting for particles that bind to a target cell
type.
[0246] Rolling circle replication of a circular oligonucleotide
template using a strand displacing DNA polymerase produces a
continuous single strand of DNA that is the concatemeric complement
of the template. The single strand condenses into a discrete
particle that can be visualized by fluorescent microscopy and flow
cytometry if fluorescently labeled (FIG. 47) (Blab, G. A., Schmidt,
T. & Nilsson, M. Homogeneous detection of single rolling circle
replication products. Anal. Chem. 76, 495-498 (2004); Jarvius, J.
et al. Digital quantification using amplified single-molecule
detection. Nat Methods 3, 725-727 (2006); Larsson, C. et al. In
situ genotyping individual DNA molecules by target-primed
rolling-circle amplification of padlock probes. Nat Methods 1,
227-232 (2004)).
[0247] The processivity of the strand displacing enzyme most
commonly used, phi29 DNA polymerase, is .about.60 kb so that a
particle produced from a 100-200 oligonucleotide template will
consist of several hundred complementary copies. The size of the
particles is a function of the reaction kinetics and can be
controlled by stopping the reaction with saturating amounts of EDTA
and/or heat in activation of the polymerase. Dynamic light
scattering (DLS) estimates that particles produced from reactions
of 10-60 minutes have hydrodynamic radii between 217-338 nm with
polydispersity indices of 0.228-0.333 (FIG. 8). These measurements
are in good agreement with a freely joined chain model of polymer
condensation which estimates a 60 kb ssDNA strand to have a
hydrodynamic radius of 379 nm (Austin, R. Nanopores: The art of
sucking spaghetti. Nat Mater 2, 567-568 (2003)). Because of their
large size and chaotic single stranded structure, the particles
will not migrate in an agarose gel.
[0248] The library screening process consists of three major steps
which are performed iteratively: particle synthesis, selection, and
amplification. A random library template sequence
(5'-Phos-GCGCGGTACATTTGCTGGACTA-N.sub.60-TGGAGGTTGGGGATTTGATGTTG 3;
SEQ ID NO:13) (Integrated DNA Technologies, Coralville, Iowa) was
circularized with a template sequence (TCC AGC AAA TGT ACC GCG CCA
ACA TCA AAT CCC CAA CCT; SEQ ID NO:14) using T4 DNA ligase (New
England BioLabs, Ipswich, Mass.) and polymerized with phi29 DNA
polymerase (NEB) for 30 minutes at 30.degree. C. and terminated by
addition of 50 mM EDTA. The initial library particle synthesis
reaction produced over 10.sup.10 unique nanoparticles and was used
to begin a selection directed against primary human dendritic cells
with an eye towards vaccine or cancer immunotherapy applications
(Fong, L. & Engleman, E. G. Dendritic cells in cancer
immunotherapy. Annu Rev Immunol 18, 245-273 (2000)). Bound
particles were amplified by PCR using primers that bound to the
sequences flanking the random region. Because each particle
contains several hundred copies of the sequence unit, PCR
amplification from a single particle is robust. To regenerate the
library, the desired single strand template was enriched after
symmetric PCR by adding a 20 fold excess of the desired strand's
phosphorylated primer v6F (5'-Phos-GCG CGG TAC ATT TGC TGG ACT A;
SEQ ID NO:15). The regenerated single strands were then
circularized to form a pool of template circles for the next round
of particle synthesis and selection (FIG. 4). Briefly, DNA
nanoparticle iterative selection scheme. ssDNA libraries are
ligated with T4 ligase and polymerized with phi29 DNA polymerase.
3'-5' exonuclease activity of phi29 DNA polymerase ensures
nanoparticle purity from extraneous DNA. Immature DCs were cultured
in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 mM
2-mercaptoethanol, 10 mM HEPES, penicillin (100 U/mL), streptomycin
(100 mg/mL), 5% human AB serum, 1000 U GM-CSF/mL and 200 U IL-4/mL
and harvested in days 5-7. Cell incubation and washing followed by
QPCR (200 nM primers, 95.degree. C. 2 min, cycle 95.degree. C. 30
sec, 61.degree. C. 1 min, 72.degree. C. 20 sec to completion. 5
.mu.L of resultant reaction was added to 45 .mu.L fresh PCR buffer
with 400 nM phosphorlyated template primer v6F. 10 additional
cycles of PCR generate an excess of the desired single strand. DNA
was purified with a QIAquick Nucleotide Removal Kit (Qiagen,
Valencia, Calif.), eluted into T4 DNA Ligase Buffer and
recircularized to begin the next round. Nine rounds were produced
after which sequences were cloned using a pGEM-T cloning kit
(Promega, Madison, Wis.).
[0249] After nine rounds of selection the pool of selected
sequences served as templates for the generation of fluorescent DNA
nanoparticles by replacing 10% of total dCTPs with ChromaTide.RTM.
Alexa Fluor.RTM. 488-7-OBEA-dCTP (Invitrogen, Carlsbad, Calif.) in
the polymerization reaction and incubating for 30 minutes at
30.degree. C. followed by inactivation by EDTA. These fluorescent
nanoparticles were used in all analyses of binding by flow
cytometry and microscopy. An increase in total population
fluorescence was observed compared to a negative DNA nanoparticle
control, suggesting that cell binding particles had become enriched
(FIG. 49).
[0250] Individual population members were cloned, sequenced,
regenerated as fluorescent particles, and similarly tested for
binding by flow cytometry. Several clones were found to bind to DCs
more than an irrelevant particle control with some of them
demonstrating similar binding patterns. The multivalent binding
nature of these nanoparticles may lend them the ability to bind a
pattern of surface markers on a cell surface rather than a single
target. In the four clones tested in FIG. 3, there is definitive
homology in the binding characteristics of Clones 3 and 4 that
differs significantly from Clones 10 and 12. It is possible that
subpopulations of nanoparticles have been selected that bind to
unique but distinctive cell surface patterns. It is also
interesting to note that even among clones that exhibited similar
binding patterns by flow, there was no obvious primary sequence
homology. The shape space of such long concatemers is enormous and
it likely that even divergent primary sequences may accommodate
similar cell surface targets. Consequently, a single clone
TABLE-US-00006 (Clone 3; SEQ ID NO: 01)
(5'-GCGCGGTACATTTGCTGGACTATGCATGTTCGTAGTTATATAGGG
GGATTGTTTGATAGTCGGAACCGCTGTGCTCAAAGTTTGGAGGTTGGGG
ATTTGATGTTG-3')
was pursued for additional validation (primer sites underlined).
Particles with the sequence of Clone 3 were independently generated
from a synthetic oligonucleotide template for all subsequent
experiments. A control particle made from the reverse complement of
the Clone 3 template was also produced. While the selection scheme
used did not include a subtractive or counter-selective step to
exclude generic cell binding, the selected DNA nanoparticles bound
only to DCs and not to human THP1 (acute monocytic leukemia) and
mouse P815 (mastocytoma) cell lines (FIG. 50).
[0251] Both flow cytometry and fluorescent microscopy supported the
conclusion that the selected particles bind to DCs specifically
while the reverse complement control particle did not. Other cell
types tested including K562 (chronic myelogenous leukemia) and
primary CLL cells (chronic lymphocytic leukemia) also showed no
difference between control and selected nanoparticles (data not
shown). Cell binding could be completely abrogated by incubation of
the nanoparticles with oligonucleotides that hybridize to the
selected random regions, though hybridizing a smaller
oligonucleotide to the flanking sequence did not affect the DC
binding (data not shown). This suggests that the binding is a
consequence of the single stranded nature of the particle,
presumably due to specific secondary structure. It is important to
note that the DC specificity that we observed was an inadvertent
result that cannot be assumed in most positive selection
mechanisms. Both the power and weakness of random library
selections against complex targets such as cells is that the
binding target need not be known in advance so there is no reason
to believe that any selected ligand would bind a target unique to a
particular cell type. However, subtractive or counter-selective
screens against non-specific cell types can be used if necessary to
enrich for cell specificity.
[0252] An important component of many biological nanoparticle
applications for in vivo use is the ability to selectively target
the desired cells or tissue. Monoclonal antibodies are the primary
tool for biomolecular recognition both experimentally and in vivo.
However, the general immunogenicity of non-human antibodies and the
immune clearance of nanoparticle aggregated humanized antibodies
raise concerns about this approach with nanoparticles. As a result,
many nanoparticle applications have turned to molecular selection
of aptamers and peptides for targeting ligands in place of
antibodies. However, since each of these methods produces a small
affinity ligand, the transition to a multivalent platform is
commonly performed by the relatively crude method of simply
attaching several monomers to a common surface, assuming the
coupling can be performed without losing the binding activity of
each monomer ligand. A potential problem with this approach is that
weak non-specific binding can gain sufficient avidity to dilute the
desired specificity. In contrast, because DNA nanoparticles
described herein are composed of concatemeric repeats of a sequence
they offer a native multivalent platform in a single particle that
allows us to perform a selection on whole particles in the same
context of ultimate usage.
[0253] DNA has a unique complement of overlapping biochemical,
structural, and functional activities when compared to other
polymers typically used in nanoparticle synthesis. DNA motifs can
act as ligands to specific biomolecules, DNA can be immunogenic if
it contains unmethylated CpG motifs, it can act as a scaffold for
hybridizing other oligonucleotide conjugates, it can have enzymatic
activity, it is easily chemically modified to allow small molecule
or metal ion attachment and metals can be directly deposited onto
DNA for imagining, and it can carry DNA binding drugs (Klinman, D.
M. Adjuvant activity of CpG oligodeoxynucleotides. Int. Rev.
Immunol. 25, 135-154 (2006); Breaker, R. R. & Joyce, G. F. A
DNA enzyme that cleaves RNA. Chem. Biol. 1, 223-229 (1994); Bern,
L., Alessandrini, A. & Facci, P. DNA-Templated Photoinduced
Silver Deposition J. Am. Chem. Soc 127, 11216-11217 (2005)). DNA
has a long clinical history and a favorable toxicity and
biodegradability profile (Fichou, Y. & Ferec, C. The potential
of oligonucleotides for therapeutic applications. Trends Biotechnol
24, 563-570 (2006)). Cell specific DNA nanoparticles are a
potential affinity reagent for research work and are an attractive
platform for targeted imaging or therapeutic applications.
Example 14
ssDNA-Nanoparticles as Molecular Marker for Detection of Cancer
Cells
[0254] Cancer cells have molecular alterations that can be used for
their identification at early stages of the disease.
ssDNA-nanoparticles are designed able to bind to these cell
alterations. These particles can be created by rolling cycle
amplification (RCA) from a template DNA. Different DNA templates
can be combined together to create a multivalent DNA nanoparticle
able to recognize more than one alteration. Starting from a random
DNA-library, MDAMB-231 cells (epithelial breast cancer cell) were
incubated with DNA-sequences for several biopanning cycles to
enrich the binding sequences. After donning and sequencing of the
motifs, these were incubated with 3 different cell lines (2 breast
cancer cell lines, MDA-MB-231 and MCF-7, and one monocytic cell
line, THP-1) to test the specificity of the ssDNA-nanoparticles.
Forty clones have been analyzed and at least 2 of them are
candidates for specific binding to MDA-MB-231. These methods and
ssDNA nanoparticles are useful in breast cancer and other types of
cancers, for example, lung, pancreatic, brain. The ssDNA
nanoparticles contribute to the development of new tumor markers
and help to consolidate the use of a new technology,
Nano-technology, for diagnosis of cancer.
[0255] FIG. 51. Generation of ssDNA nano-particles:
.diamond-solid.(upper line): cells+qPCR+RCA; .box-solid.:
cells+qPCR reagent; .tangle-solidup.: cells+qPCR+RCA(-);
.diamond-solid.(lower line): cells. A graph of fluorescence (R) vs.
number of cycles. Eight bio-panning cycles have been made starting
from a random DNA-library. In each bio-panning cycle, MDA-MB-231
cells (epithelial breast cancer cells) were incubated with ssDNA
nanoparticles and the binding particles were amplified by qPCR. The
goal of each cycle was to enrich and amplified the binding
motifs.
[0256] FIG. 52. DNA gel of amplified sequences: After each
bio-panning cycle, the amplified samples were run in a DNA gel
where the amplified products can be visualized. In all the
bio-panning cycles, amplified products were observed only from the
samples corresponding to cells+RCA particles (lane 5). No
amplification of cell DNA was observed (lane 2, 3 and 4). Line 1
represents the molecular weight ladder.
[0257] FACS analysis of the binding ss-DNA nanoparticles to test
specificity of the particles are shown in FIG. 53-55. ss-DNA
nanoparticles were generated by RCA and incubated with 3 different
cell lines: Two epithelial breast cancer cells, MDA-MB-231 (FIG.
53A-53C) and MCF-7 (FIG. 54A-54C), and a monocytic cell line, THP-1
(FIG. 55A-55C). This test was repeated five times. Of 40 clones
analyzed, 2 of them, L5C22 and L5c37, were specific for MDA-MB-231
cells as is shown in the FACS plots (blue line, background
((-)RCA); red line, specific signal). The last column represents
the average of the results of five experiments.
Example 15
Bi-Specific DNA-Nanoparticles
[0258] This example illustrates the production bi-specific
DNA-nanoparticles (NP) that bind to both tumor cells and T cells.
Binding particles that bind the pancreatic tumor cell line, panc02,
are used as the tumor binding moiety. In this example, T cell
binding DNA-NP are selected using a DNA-NP library technique to
identify DNA-NP that bind to mouse and human T cells. Each moiety
is selected against separately. Construct and validate Hybrid
particle that bind to T cells and pancreatic cancer cells are
constructed and validated. In addition, the effects of bi-specific
DNA-NP on tumor growth and metastasis are evaluated. The scheme is
summarized in FIG. 56.
[0259] DNA-NP differ from other affinity reagents in several ways.
Each particle contains many copies of the sequence elements so
there is intrinsic multivalent display of the modules, allowing
avidity to compensate for low monovalent affinity. Molecular
modeling suggests that the repeating units that make up a particle
can adopt more complex secondary structures than simply repeating
the predicted structure of the monomer. The modular nature of the
particle template construction allows multiple distinct recognition
elements to be assembled into a single molecular entity.
Furthermore, the selection method allows the optimal particle to be
evolved in the same molecular context in which it will be used,
rather than transplanting them to some other framework or particle
for application.
[0260] DNA-NP are produced using methods described herein. Briefly,
DNA-NP are produced by enzymatic DNA synthesis using a strand
displacing DNA polymerase, phi29, and a circular oligonucleotide
template. The resulting RCA products are concatemers complementary
to the template circular oligonucleotide. These long single
stranded products collapse into randomly coiled nanoparticles.
DNA-NP can be readily analyzed by Dynamic Light Scattering (DLS)
and a low polydispersity index. A typical 30 minute reaction
produces particles .about.250 nm in size, with an estimated DNA
length of .about.30 kb.
[0261] DNA nanoparticle libraries are constructed using methods
described here. A general scheme is shown in FIG. 7. Particles that
bind to human breast cancer cell line MDA-MB-231 and mouse
pancreatic cell line panc02 have been selected. In each case,
enrichment of cell binding particles was observed after 4 rounds of
selection and clones taken from the 6.sup.th round were shown to
have cell line specific binding. Strikingly, a clearly sequence
motif was apparent among the clones that bound to panc02. The
particles do not bind to several other epithelial derived cell
lines tested.
[0262] FIGS. 57A and 57B summarizes the results of the selection of
pancreatic cancer cell line panc02 targeting particles. The mouse
pancreatic lines panc02 was panned with a single module DNA
nanoparticle library. After the 3.sup.rd and 4.sup.th round, the
selected pool was fluorescently labeled and tested on the target
cells by flow cytometry (FIG. 57A). A shift indicating enrichment
of binding clones was observed. After the 5.sup.th and 6.sup.th
round, clones were generated from the selected pool and sequenced
(TABLE 6). 10 of the 12 sequenced clones contained a specific
sequence motif: AATGGGGCG (SEQ ID NO:12). In two of the clones the
motif appears in the same frame (C45 and C58). Two independent
clones for each we recovered with the sequence of clone 40 and
46.
TABLE-US-00007 TABLE 6 SEQ ID Clone Sequence NO: C33
TGCTTTTTGGAACTCCTGCTAGATGATGGAATATCA 02
AGGCTGATTAAACGGGGCGTTTCCTGAAATGTATTA CTTGTTGAGGTGACGTTGAGTTGGATCC
C39 TGCTTTTTGGAACTCCTGCTTAGCAGTAAGAAAGTA 03
CAATGGGGCGATAACCCCAATCATGACTAAAAATAT GATTCGGAGGTGACGTTGAGTTGGATCC
C40 TGCTTTTTGGAACTCCTGCTAAACAAAAGAGGATTG 04
TATGGGGCGTATCAGTTCGACTATCTGGTAGAGCAA AGAAAAGTGGTGACGTTGAGTTGGATCC
C43 TGCTTTTTGGAACTCCTGCTAACCGGAAGTTCGTAT 05
GGCCAAAGCTGATTAAAACGGGGCGTTTACACAAGG TGTATGTGGGTGACGTTGAGTTGGATCC
C44 TGCTTTTTGGAACTCCTGCTATAGCTGAAGGATATT 06
GGATCGGGGAGTTTTGGATTTACGATTTAGATTTGT
TATGTTCTCTTGGGTGACGTTGGGATCCGGTGACGT TGAGTTGGATCC C45
TGCTTTTTGGAACTCCTGCTGAATAGAGAACAACTA 07
AATTCTGCAATGATGTTGCGTAGTGACTAANGATCA AATGGGGCGGTGACGTTGAGTTGGATCC
C46 TGCTTTTTGGAACTCCTGCTGATCAGGTTATAAAGC 08
GTTAATAGCTTAATAAAACTTGAAAGGTAATAAATG GGGCGTCTGGTGACGTTGAGTTGGATCC
C58 TGCTTTTTGGAACTCCTGCTAAAGAGTACGAGGTAG 09
AAATATGAGAAACTTTAAATTTGTCCAGCAGATCCT AATGGGGCGGTGACGTTGAGTTGGATCC
C21 TGCTTTTTGGAACTCCTGCTAGACGTTAGATGTATC 10
TGACCTTACGACTTCAACTTCCTTCTAAATCTGCCC ACAACGATGGTGACGTTGAGTTGGATCC
C50 TGCTTTTTGGAACTCCTGCTCAACTTGTGTCCTCTT 11
GAAAGAGTCGGTCATACCTATAAGAATACTTTTATA
CAGCCAAAGGTGACGTTGAGTTGGATCC
[0263] The clones that contain the AATGGGGCG (SEQ ID NO: 12) motif
bind specifically to panc02, whereas the clones lacking the motif
(C21 and C50) do not (FIG. 57B). In the experiment shown, the four
clones that show a fluorescent shift in the left panel all contain
the motif whereas the clones without the motif are no better than
controls. No difference is seen against other epithelial cell
lines.
Selecting for T Cell Binding DNA-NP
[0264] DNA-NP that bind to an implantable mouse tumor cell line
(panc02) are used. Therefore, DNA-NP that bind the T-cell binding
motif also, are selected. Three approaches are used. (1) Primary
mouse T cells obtained from PBMCs by negative selection are
screened. Binding particles are checked for binding to human T
cells. (2) A mixture of human and mouse T cells is screened, with
the notion that a crossreactive particles would have a selective
advantage during the screening. Candidate particles are checked
against both mouse and human T cells. (3) Alternate mouse and human
will be alternated in each round of screening.
[0265] Once a particular screening is performed for 6-8 cycles, the
pool of particles present at that stage is analyzed by
incorporating fluorescent nucleotides and checking the binding by
flow cytometry. If the population looks like it has enriched for
binding, individual candidate particles are recovered by subcloning
the PCR amplified templates into bacteria. Candidates are sequenced
and regenerated by asymmetric PCR/RCA for further testing as
particles. The specificity of binding clones is analyzed by
incubating the particles with PBMCs and anti-CD3 antibodies. The
particle fluorescence should be confined to the CD3+
population.
Construct and Validate Hybrid Particle that Bind to T Cells and
Pancreatic Cancer Cell.
[0266] The templates for the T cell and panc02 binding motifs are
ligated together. Adding an additional module to a cell binding
particle does not significantly reduce the cell binding. At least
five unique combinations of panc02 and T cell binding motifs are
produced and tested for binding to both cell types independently by
flow cytometry. T-cell/panc02 cell crosslinking is evaluated in
several ways. First, varying numbers of particles with an equal
mixture of both cell types are incubated together. High
concentrations of particles will lead to wholesale crosslinking and
essential agglutinate the cells are expected, whereas lower
concentrations will form primarily heterocellular dimers. The
former is evaluated by microscopy and the later by flow cytometry
using differently labeled anti-CD3 and anti-EpCAM antibodies to
distinguish hetero and homocellular aggregates.
[0267] In addition, direct cell lysis during co-culture of the
panc02 with T cells from the same mouse strain (C57BL/6) from which
the line was derived, using either LDH or chromium release assays
is evaluated. The stoicheometry of particle to target cells is
optimized by titrating the particles and the relative number of
cells, and the time course for cell lysis determined. In addition,
the effect, if any, of particle size is evaluated, by producing
particles of .about.50, 100, and 200 nm as measured by dynamic
light scattering. With this panel of assays, which hybrid DNA-NP
combinations are most potent is determined.
Evaluate the Effects of Bi-Specific DNA-NP on Tumor Growth and
Metastasis.
[0268] Hybrid particles are analyzed for circulating half-life in
normal mice, as a function of particle size. In addition, particle
accumulation in liver, spleen, and lymph nodes are measured. In all
cases, the particles can be easily quantitated by real-time PCR,
with the same primers used to amplify the particles during the
selection. Indeed, since each particle consists of many copies of
the repeated sequence, single particle sensitive is achievable.
Some of these methods allow the de-selection of any particle that
has exceptionally rapid clearance, and establish the optimal size
for subsequent tumor studies.
Example 16
Targeting Dendritic Cells with DNA-Nanoparticles
[0269] Dendritic cell (DC)-based immune therapy for cancer has met
with some success using ex vivo approaches of injecting
antigen-pulsed mature DCs into patients. However, in vitro
generation of DCs is costly, cumbersome, and difficult to
standardize. Thus, activation of DCs in situ is an attractive
approach but requires agents that can both specifically target and
activate DC. DNA-NP library technology is described herein that can
select cell specific binding NP made solely of single stranded DNA.
Using such techenology, DNA-NP have been identified that bind
specifically to DCs, are taken up, induce Ca.sup.2+ flux, and IL-6
secretion by DCs, and can act as vaccine adjuvants in mice. These
results show a DC targeting molecule that also carries intrinsic
adjuvant properties. This example illustrates targeted DNA-NPs that
bind to and stimulate DCs, and cause immune activation and prevent
or retard tumor growth.
Production, Characterization, and Purification of DNA
Nanoparticles
[0270] DNA nanoparticles were produced by methods described herein.
High diversity libraries of DNA-NP were generated using methods
described herein, and DNA-NP that bind specifically to DCs were
selected through an iterative screening and re-amplification.
[0271] DC binding DNA-NP was verified using separate batches of
particles and synthesizing particles from both the original clone
(PCR from bacteria colonies harboring the clone, followed by
asymmetric PCR with a 5' phosphate on only the desired primer for
subsequent strand ligation and rolling circle amplification (RCA)
or from a synthetic oligonucleotide template with the same
sequence. The stability of particles kept at -20.degree. C. and
4.degree. C. for several weeks; no loss of activity was
observed.
DC Binding DNA-NP Activate DC
[0272] DC binding DNA-NP cause DC activation as measured by
cytokine secretion, and Ca.sup.2+ signaling, and surface marker
expression. IL-6 secretion is a commonly used indicator that DC
have matured into immune activating cells, though a full cytokine
secretion profile is ultimately desirable to confirm this point.
DCs exposed to several of the DC binding DNA-NPs secrete IL-6,
IL-12, and TNF-alpha. In addition, Ca.sup.2+ flux 20 seconds after
DC were exposed to DC binding DNA NP was detected, but not after
exposure to control DNA-NP.
[0273] An immunization study was carried out in which DNA-NP were
mixed with ovalbumin and injected s.c. into mice, boosted two weeks
later, and analyzed a week after the final boost for antibody
responses by serum IgG titers. Robust antibody responses were seen
in all mice immunized with DC binding DNA-NP even though the dose
is much lower than typical immunization protocols with CpG
oligonucleotides (ODN) (FIG. 57). Since our particles are single
molecules made up of contameric repeats (n=.about.300) of the
complement of the template circle from which they are produced, the
dose can be expressed as either the number of particles (68 fmol),
or as the number of complement repeats (20 pmol).
Screening DNA-NP that Activate DC
[0274] Five 5 DNA-NP that bind to DC were identified. All 5 DC
binding DNA-NPs are compared for their ability to activate myeloid
(CD11c+) bone marrow-derived DCs (BM-DCs) in vitro, as these are
known to function similarly to human monocyte-derived DCs. BM-DCs
will be generated (Telusma G, et al. Dendritic cell activating
peptides induce distinct cytokine profiles. Int Immunol. 2006;
18:1563-1573). DC activation and maturation is characterized by
altered surface expression of characteristic molecules, production
of large amounts of cytokines and enhanced T cell stimulatory
capacity. DC stimulatory capacity of the DC-binding DNA-NPs is
evaluated in three ways: 1) their ability to alter the expression
of surface molecules on immature DCs that are classically up or
down regulated upon maturation; 2) their ability to induce
secretion of inflammatory cytokines, and finally 3) their ability
to mature DCs into effective antigen presenting cells that can
activate antigen-specific T cells. The particles are ranked
according to their activity in these assays.
[0275] In vivo selection of ligands that bind to cells or soluble
proteins has been well established, for example, with phage
displayed peptide libraries. In vivo selections have two
significant advantages. The first is that the selection is being
performed in the very same environment that the ultimate product
will be used. The second is that the rest of the animal acts as a
subtractive substrate that will remove any non-specific
particles.
[0276] In vivo selections are performed with DNA-NP by injecting
the DNA particle library subcutaneously and recovering the draining
lymph nodes several hours later. The lymph nodes are treated with
collagenase to create single cell suspensions, and the CD11c+DCs
are isolated by magnetic bead separation. Subsequently the particle
recovery, re-amplification, and ligation will be as described
herein.
Injection of DNA-NP into Tumors to Activate Tumor Infiltrating
DC
[0277] All in vivo experiments are conducted in a transplantable
mouse model of melanoma using the mouse melanoma cell line B16-OVA
that expresses chicken ovalbumin (OVA), which serves as a tumor
marker to monitor immune responses. When injected s.c. into C57/BL6
mice, B16-OVA produces a local tumor growth.
[0278] C57/BL6 mice (n=5 per group) are inoculated with
5.times.10.sup.5 B16 cells s.c. Once the tumors reach 3-5 mm in
size, they receive intra-tumoral injection of: 50 .mu.l of PBS, DC
binding DNA-NP, or a control DNA-NP. DNA-NPs are injected at 1 and
10 .mu.g/ml (.about.10.sup.10 and 10.sup.11 particles) suspended in
PBS. 24-48 hours later the mice will be sacrificed and the tumor,
draining lymph nodes, blood, liver, and spleen are collected.
Histology is performed on the tumor and the number of infiltrating
lymphocytes compared (CD3+ and CD11c+) to controls. Single cell
suspensions are made by treating the tissue with collagenase and
tumor infiltrating DCs are analyzed for the expression levels of
co-stimulatory and adhesion molecules, e.g. CD80, CD86, MHC class
II, CD40, by flow cytometry. The expression of IL-12, IFN.gamma.,
TNF-.alpha. and RANTES is determined by intracellular staining
combined with surface CD11c and analyzed by flow cytometry.
[0279] Long term tumor monitoring is performed. Groups of mice
(n=5) are inoculated with tumor and injected with DNA-NP or
controls. The mice receive 5-10 daily injections of DC-targeting
DNA-NPs, control non-targeting DNA-NPs or PBS. Mice are monitored
for tumor size a set of calipers: A.times.B.sup.2/2 (A=long axis,
B=short axis) daily until the last injection and then bi-weekly
over a period of 20 days. After 20 days or if the tumors reach 1.5
cm in diameter, whichever occurs first, mice are sacrificed. The
spleen, liver, and kidney will be weighed and tissue analysis will
be performed by our molecular pathology core. If the pilot study
indicates potential tumor retardation or regression in the mice
that received the DNA-NP without overt toxicity then a larger study
will be designed in coordination with the biostatistics core.
Immunization with DNA-NP and Model Tumor Antigens
[0280] Co-injection of the most potent DNA-NPs with tumor antigen
is likely to lead to the induction of anti-tumor immune response.
OVA serves a model tumor antigen for which tool to measure immune
responses have been developed and thus allows one to easily monitor
the potency and type of induced immune response.
[0281] Immunizations: Groups of C57BL/6 mice (n=10) receive s.c.
injections of OVA/PBS (as negative control), OVA/IFA (positive
control), or OVA/CpG (positive control), and 3 different doses of
OVA/DNA-NPs (1-100 .mu.g/mouse), that demonstrate DC activation.
Two to three weeks after the primary immunization, the animals
receive a second "booster" immunization performed exactly as the
first injections. Blood is obtained from mice at three time points:
before immunization for base antibody levels, before the booster
immunization and 1-2 weeks after the second "booster" immunization.
Plasma IgG and IgM levels specific for the injected antigen is
measured by direct ELISA using plates coated with antigen=OVA
protein. The antibody results are determined in arbitrary units
against an ELISA reference serum in order to reliable compare
results obtained on different days. The type of immune response is
evaluated by measuring the subclass antibody concentrations. In
mouse, the production of IgG2a is recognized as characteristic of a
Th1 response, whereas the production of IgG1 is characteristic of a
Th2 response. Therefore, the assessment of the type of immune
response is done by measuring IgG1, IgG2a and IgG2b levels by
ELISA. The ratio of IgG2a/IgG1 antibody titers is used as indicator
of Th bias.
[0282] The in vivo induced T cell responses is detected in vitro
using the following assays: (1) Proliferation assays are performed
by adding OVA.sub.257-264 peptides (to stimulate CD8 T cells) and
OVA.sub.323-339 (to stimulate CD4 T cells), PBS/no peptide
(negative control), or ConA (positive control) to splenocytes from
immunized mice which contain T cells and antigen presenting cells,
and measuring the uptake of [.sup.3H]-thymidine after 4 d. (2) A
Th2 response is also characterized by the secretion of Th2 type
cytokines, such as IL-4, IL-5, whereas a Th1 type response is
characterized by the secretion of IL-2 and IFN-.gamma..sup.46-47.
To measure type of T cell responses, splenocytes are set up as
described herein using phorbol myristate acetate (PMA) and soluble
anti-CD3, since ConA is not a very potent stimulus for Th2
cytokines. After 24 h the culture supernatants are assessed for
IL-4, IL-2, IL-5, IL-10, TGF-.alpha. and IFN-.gamma. levels by
ELISA. (3) IFN-.gamma. ELISPOT and intracellular flow cytometry
assays (using brefeldin A to prevent leakage of the cytokines for
the latter) for IL-4, IL-2 and IFN-.gamma. are used to measure the
number of cytokine secreting T cells and double labeling for CD4
and CD8 to detect the type of T cells responding. (4) To measure
the generation of functional CTL responses splenocytes are cultured
in medium only and re-stimulated for with mitomycin C-treated (50
.mu.g/ml) or irradiated B16-OVA cells and IL-2 for 6 days. As
positive control splenocytes are stimulated with concavalin A (5
.mu.g/ml). Expanded splenocytes are cultured with B16-OVA target
cells and B16 (=negative control target cells), using a standard 4
h LDH assay.
Tumor Rejection Studies
[0283] Conditions that gave the strongest immune responses are used
for tumor rejection studies. Prophylactic setting: 7 days after the
final immunization C57/BL6 mice (10 per group) are challenged with
5.times.10.sup.5 B16 cells s.c. and tumor growth are monitored as
described herein. Therapeutic setting: C57/BL6 mice are inoculated
with 5.times.10.sup.5 B16 cells s.c. Once the tumors reach 3 mm in
size, groups of mice (n=20) receive s.c. immunizations of OVA
protein mixed with: PBS, or the DNA-NPs conditions that induced
strongest immune responses. Half the mice from each group (n=10)
are sacrificed 7 days after the final immunization to analyze
immune responses and the other half are monitored for tumor
progression.
Pharmacodynamics and Toxicity of DNA-NPs In Vivo
[0284] Immunization experiments provide an opportunity to gain
insight into the in vivo distribution and half life of the DNA-NPs,
as well as any associated toxicity. Organs and blood are recovered
from the sacrificed animals and tissue extracts prepared. DNA-NP
are quantified by real time PCR to evaluate the biodistribution and
circulating levels of the particles.
[0285] A powerful feature of the DNA-NP methods described herein is
that the template sequence from which the particles are generated
can be easily manipulated. One or more synthetic oligonucleotides
can be used to build the template and beyond a minimum size of
60-80 bases, the RCA reaction proceeds equally well on templates
regardless of size. Therefore, once discrete particle sequences are
identified a hybrid template can be prepared by coupling the
templates at the ligation step. Certain DC binding particles that
show activity can be combined to further enhance their potency.
[0286] All references cited herein, including but not limited to
published and unpublished applications, patents, and literature
references, are incorporated herein by reference in their entirety
and are hereby made a part of this specification. To the extent
publications and patents or patent applications incorporated by
reference contradict the disclosure contained in the specification,
the specification is intended to supersede and/or take precedence
over any such contradictory material.
[0287] The term "comprising" as used herein is synonymous with
"including," "containing," or "characterized by," and is inclusive
or open-ended and does not exclude additional, unrecited elements
or method steps.
[0288] All numbers expressing quantities of ingredients, reaction
conditions, and so forth used in the specification are to be
understood as being modified in all instances by the term "about."
Accordingly, unless indicated to the contrary, the numerical
parameters set forth herein are approximations that may vary
depending upon the desired properties sought to be obtained. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of any claims in any
application claiming priority to the present application, each
numerical parameter should be construed in light of the number of
significant digits and ordinary rounding approaches.
[0289] The above description discloses several methods and
materials of the present invention. This invention is susceptible
to modifications in the methods and materials, as well as
alterations in the fabrication methods and equipment. Such
modifications will become apparent to those skilled in the art from
a consideration of this disclosure or practice of the invention
disclosed herein. Consequently, it is not intended that this
invention be limited to the specific embodiments disclosed herein,
but that it cover all modifications and alternatives coming within
the true scope and spirit of the invention.
Sequence CWU 1
1
151105DNAArtificial SequencePrimer, vector, or motif 1gcgcggtaca
tttgctggac tatgcatgtt cgtagttata tagggggatt gtttgatagt 60cggaaccgct
gtgctcaaag tttggaggtt ggggatttga tgttg 1052100DNAArtificial
SequencePrimer, vector, or motif 2tgctttttgg aactcctgct agatgatgga
atatcaaggc tgattaaacg gggcgtttcc 60tgaaatgtat tacttgttga ggtgacgttg
agttggatcc 1003100DNAArtificial SequencePrimer, vector, or motif
3tgctttttgg aactcctgct tagcagtaag aaagtacaat ggggcgataa ccccaatcat
60gactaaaaat atgattcgga ggtgacgttg agttggatcc 1004100DNAArtificial
SequencePrimer, vector, or motif 4tgctttttgg aactcctgct aaacaaaaga
ggattgtatg gggcgtatca gttcgactat 60ctggtagagc aaagaaaagt ggtgacgttg
agttggatcc 1005100DNAArtificial SequencePrimer, vector, or motif
5tgctttttgg aactcctgct aaccggaagt tcgtatggcc aaagctgatt aaaacggggc
60gtttacacaa ggtgtatgtg ggtgacgttg agttggatcc 1006120DNAArtificial
SequencePrimer, vector, or motif 6tgctttttgg aactcctgct atagctgaag
gatattggat cggggttgga tttacgattt 60agatttgtta tgttctcttg ggtgacgttg
agttggatcc ggtgacgttg agttggatcc 1207100DNAArtificial
SequencePrimer, vector, or motif 7tgctttttgg aactcctgct gaatagagaa
caactaaatt ctgcaatgat gttgcgtagt 60gactaangat caaatggggc ggtgacgttg
agttggatcc 1008100DNAArtificial SequencePrimer, vector, or motif
8tgctttttgg aactcctgct gatcaggtta taaagcgtta atagcttaat aaaacttgaa
60aggtaataaa tggggcgtct ggtgacgttg agttggatcc 1009100DNAArtificial
SequencePrimer, vector, or motif 9tgctttttgg aactcctgct aaagagtacg
aggtagaaat atgagaaact ttaaatttgt 60ccagcagatc ctaatggggc ggtgacgttg
agttggatcc 10010100DNAArtificial SequencePrimer, vector, or motif
10tgctttttgg aactcctgct agacgttaga tgtatctgac cttacgactt caacttcctt
60ctaaatctgc ccacaacgat ggtgacgttg agttggatcc 10011100DNAArtificial
SequencePrimer, vector, or motif 11tgctttttgg aactcctgct caacttgtgt
cctcttgaaa gagtcggtca tacctataag 60aatactttta tacagccaaa ggtgacgttg
agttggatcc 100129DNAArtificial SequencePrimer, vector, or motif
12aatggggcg 913105DNAArtificial SequencePrimer, vector, or motif
13gcgcggtaca tttgctggac tannnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
60nnnnnnnnnn nnnnnnnnnn nntggaggtt ggggatttga tgttg
1051439DNAArtificial SequencePrimer, vector, or motif 14tccagcaaat
gtaccgcgcc aacatcaaat ccccaacct 391522DNAArtificial SequencePrimer,
vector, or motif 15gcgcggtaca tttgctggac ta 22
* * * * *