Download Whole-Animal Selection of Aptamers Targeting Murine Spinal Cord

Whole-Animal Selection of Aptamers
Targeting Murine Spinal Cord
Jamie S. Harper, G. Thomas Caltagirone, PhD
Aptagen, LLC
RESULTS
METHODS
INTRODUCTION
The ALS Foundation Project
Aptamers are stable, single-stranded DNA or RNA
molecules that are capable of eliciting strong affinities
for a variety of targets.
An aptabodyTM, a functionalized aptamer, is an
aptamer conjugated to a known drug or compound.
AptabodiesTM are the next step in aptamer research.
aptabody™
aptamer
Naked RNA
Conjugated RNA
Failure sequences removed and remaining template purified via
PAGE purification, crush-soak method and cartridge purification
Concentration of template was than determined
LCR (ligase chain reaction) of template and splint to produce the
initial Generation 0 (G0) of circular aptamer library (1nmol scale
LCR produces a ~1014 ssDNA library)
Exonuclease treatment of post-LCR product
10% Denaturing PAGE gel of LCR results
in vivo selection
Weighed balb/c mouse, then injected G0 library into mouse via tail-vein
3
Effective Drug
qPCR Data from Whole-Animal Selection, Generation 3
The tissues were harvested 100 min post tail-vein injection
4
Figure 1b. 34.62 pmol scale LCR of
linear template APT(PO4)55/M13F(40)w/M13Ra/90 and Gx splint oligo (36
Figure 1a. Ligase Chain Reaction (LCR) nt) to generate the Generation-0 (G0)
of the aptamer APT(PO4)55/M13F(circular library. Lane 3- circularization
40)w/M13Ra/90 (90 nt) and the G0
reaction not exposed to exonucleases.
splint oligonucleotide (35 nt). Linkages Lane 4- same reaction treated with
only occur between proximal 5’exonucleases. 10% Denaturing PAGE
phosphate and 3’-hydroxyl ends.
gel, GelStar poststained and destained.
Functionalized RNA
After indicated post-IV harvesting time, mouse was euthanized via
cervical dislocation. A cardiac puncture was then performed
Delivery
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linear aptamer template
C A G C AC T G A C C C T T T T G – PO4 – 5’
5’-g t c g t g a c t g g g a a a a c
forward qPCR primer-17nt
What is an aptamer? An aptabodyTM?
In-house synthesis of template and splints. Primers were
outsourced.
1
reverse qPCR primer-18nt
3’-c g t a t c g a c a a a c g a c a c
G C A T A G C T G T T T G C T G T G – 3’
ALS (Amyotrophic lateral sclerosis or Lou Gehrig’s
Disease) is a neurodegenerative disorder affecting the
nerve cells that control voluntary movement. There is
no cure for ALS but currently treatments are available
for the control of symptoms and the prolonging of life.
Delivery of ALS treatments via aptamers is expected
to improve specificity of the treatment to the desired
target and may also improve the pharmacokinetic
properties of the drug.
Design of APT(PO4)55/M13F(-40)w/M13Ra/90 aptamer template,
corresponding forward and reverse PCR primers, & corresponding
G0 and Gx splint oligonucleotides
Circularization of ssDNA aptamer library for in vivo stability
Figure 3a. Ten replicate samples from the spinal cord were qPCR-amplified,
producing uniform amounts of product to use as template for the production of the
G4 circular library. The earlier a sample amplifies, the more concentrated the
sample. Not unexpectedly, the CT increased from a mean value of 21.62 for G2
(results not shown) to 25.56 in G3. Both negative control curves (NTC’s-blu & grn)
were flat line.
Figure 3b. Ten replicate samples from the spinal cord were qPCR-amplified.
Samples exhibited a tight melting curve, demonstrating reproducibility of data. The
amplification was stopped at the PCR plateau to prevent heteroduplexing (no 620C
peak). Both negative control curves (NTC’s-blu & grn) were flat line.
qPCR Data from Whole-Animal Selection, Initial Round 0
The tissues were harvested 30 min post tail-vein injection
Improve PK/PD
The affinity of aptamers to a target can be compared
to the binding affinity of an antibody to an antigen,
but there are many advantages of aptamers and
aptabodiesTM over antibodies: they are inexpensive,
easy to produce in large quantities, their high
specificity can be extended to functional groups other
than proteins, and they have been observed to have
higher binding affinities than those of monoclonal
antibodies and random peptide libraries.
The ultimate goal of the ALS project is to
develop an aptabodyTM targeting epitopes of the
spinal cord with high specificity, deliver ALS
treatment and be released. Before selection
with aptabodiesTM can begin, we must first
demonstrate that it is possible with an aptamer
alone.
The target tissue, the spinal cord, was then harvested first and then
the six background tissues
The tissues were then weighed and minced to a pulp. The entire
spinal cord and equivalent weights of background tissues were
processed. Again, the spinal cord was processed first.
Tissues were lysed according to in-house SOP
Figure 2a. qPCR amplification curves from
(1:2 dilution),
and G0 background tissues (
,
,
,
,
, and
), and a
. The earlier a sample amplifies, the more concentrated
the sample is. The NTC is flat line.
Aptamers were isolated and purified from the tissue lysates
Remaining tissue samples were normalized to 2mg of tissue per
PCR tube
Analytical qPCR performed, results analyzed
If successful, this will be the first demonstrated case
of whole-animal in vivo selection of aptamers
targeting a tissue type with high specificity.
1. To select for aptamers that bind to the
spinal cord with a higher specificity
than six background tissues (brain,
heart, lung, spleen, liver, kidney).
2. To select for aptamers that bind to the
spinal cord for 30+ minutes
Figure 3c. Duplicate samples of 5 of the 6 background tissues were amplified. The
spleen was unable to be processed in this round. CT values for this tissue panel
range from 27.62 to 29.71, which are higher than the mean CT observed for spinal
cord tissue (25.56). This lead us to believe that the G3 aptamer library targeted the
spinal cord tissue with more specificity than the background tissues.
qPCR- mass amplification of spinal cord aptamers
Combined post-PCR replicates, cleaned-up material, concentration
determined
LCR of selected spinal cord aptamers to generate to library for the
next round of selection
Repeat from indicated step until greater enrichment of the spinal
cord as compared to the background tissues can be seen
Figure 2b. Melting curve profile from qPCR of
(1:2 dilution), and
G0 background tissues (
,
,
,
,
, and
), and a
. Two melting peaks are exhibited (Tm 620C and 830C): The
620C peak is a heteroduplexing artifact resulting from excessive cycling. The 830C
peak is the desired amplicon.
LITERATURE CITED
Caltagirone, G. T. 2007. Perspective: Aptamers as
Drugs. MS. Aptagen, L.L.C., Jacobus PA.
Shamah, S.M., Healy, J. M., & Cload, S. T. 2008.
Complex Target SELEX. Accounts of chemical
research 41:130-138.
ACKNOWLEDGEMENTS
I would like to thank Dr. Robert Farrell and Dr. Rich Bamford, for helping with analyzing results
and endless troubleshooting, and also Dr. Kaltreider for being my York College mentor.
Also, Derek Jendras.
Figure 3d. Duplicate samples of 5 of the 6 background tissues were amplified. The
spleen was unable to be processed in this round. Contamination in the NTC does
not appear to be consistent with background tissue amplicons as seen from the
bimodal nature of the NTC peak. Some variance between background tissues can
be seen.
1. Although a 20-fold greater enrichment of
spinal cord tissue as compared to background
tissues was seen in G3, the separation of
affinities was lost after subsequent rounds.
2. Selection will be restarted from G0 with a new
random aptamer library, and perfusion with
PBS-T will be employed to reduce nonspecific
binding to background tissues.
Administer
Molecular
Library
Normal/healthy animal
Selection Scheme
to Overcome
Delivery Issues
reselect
Propagate
molecules that
survive selection
Positive Selection:
Characterize
‘enriched’
Library
aptabody™
aptamer
Naked RNA
Conjugated RNA
Functionalized RNA
Effective Drug
Delivery
Improve PK/PD
Isolate and harvest spinal
cord tissue containing
absorbed molecules
‘Molecular Bullet’
Candidates