design of apt(po 4 )55/m13f(-40)w/m13ra/90 aptamer template, corresponding forward and reverse pcr...
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Delivery Improve PK/PD
Effective Drug
aptameraptabody™
Naked RNA Conjugated RNA Functionalized RNA
Design of APT(PO4)55/M13F(-40)w/M13Ra/90 aptamer template, corresponding forward and reverse PCR primers, & corresponding
G0 and Gx splint oligonucleotides
In-house synthesis of template and splints. Primers were outsourced.
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
Weighed balb/c mouse, then injected G0 library into mouse via tail-vein
After indicated post-IV harvesting time, mouse was euthanized via cervical dislocation. A cardiac puncture was then performed
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
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
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
Whole-Animal Selection of Aptamers Targeting Murine Spinal Cord
Jamie S. Harper, G. Thomas Caltagirone, PhD Aptagen, LLC
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.
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.
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.
If successful, this will be the first demonstrated case of whole-animal in vivo selection of aptamers targeting a tissue type with high specificity.
INTRODUCTION
OBJECTIVES1. 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
RESULTS
C A G C AC T G
A C C C T T T T G – PO
4 – 5’
G C A T A G
C T G T T T G
C T G T G
– 3’
5’-g t c g t g a c t g g g a a a a c
3’-c g t a t c g a c a a a c g a c a c
-
G0 Splint
forward qPCR prim
er-17nt
reverse qPCR primer-18nt
linear aptamer tem
plateFigure 1a. Ligase Chain Reaction (LCR) of the aptamer APT(PO4)55/M13F(-40)w/M13Ra/90 (90 nt) and the G0 splint oligonucleotide (35 nt). Linkages only occur between proximal 5’-phosphate and 3’-hydroxyl ends.
Figure 1b. 34.62 pmol scale LCR of linear template APT(PO4)55/M13F(-40)w/M13Ra/90 and Gx splint oligo (36 nt) to generate the Generation-0 (G0) circular library. Lane 3- circularization reaction not exposed to exonucleases. Lane 4- same reaction treated with exonucleases. 10% Denaturing PAGE gel, GelStar poststained and destained.
Circularization of ssDNA aptamer library for in vivo stability
qPCR Data from Whole-Animal Selection, Initial Round 0The tissues were harvested 30 min post tail-vein injection
Figure 2a. qPCR amplification curves from G0 Spinal Cord (1:2 dilution), and G0 background tissues (spleen, brain, heart, liver, lung, and kidney), and a negative control (NTC). The earlier a sample amplifies, the more concentrated the sample is. The NTC is flat line.
Figure 2b. Melting curve profile from qPCR of G0 Spinal Cord (1:2 dilution), and G0 background tissues (spleen, brain, heart, liver, lung, and kidney), and a negative control (NTC). 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.
What is an aptamer? An aptabodyTM?
The ALS Foundation Project
METHODS
CONCLUSIONS1. 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.
LITERATURE CITEDCaltagirone, 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.
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, Generation 3The tissues were harvested 100 min post tail-vein injection
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.
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.
ACKNOWLEDGEMENTSI 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.
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in vivo selection
Positive Selection: ‘‘Molecular BulletMolecular Bullet’’CandidatesCandidates
Administer Molecular
Library
Characterize ‘enriched’
Library
Propagate molecules that survive selection
reselect
Isolate and harvest spinal cord tissue containing absorbed molecules
Selection Scheme Selection Scheme to Overcome to Overcome Delivery IssuesDelivery Issues
Normal/healthy animal
Delivery Improve PK/PD
Effective Drug
aptameraptabody™
Naked RNA Conjugated RNA Functionalized RNA
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