Transcript nf aggregato

Nucleic acid Aptamers as Potential
Therapeutic and Diagnostic agents
for Lymphoma
Presented by
Naila Sajjad
PhD Biochemistry
10-arid-1766
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List of Abbreviation used
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ALCL
PEI
BCL2
AML
ALL
Anaplastic large cell lymphoma
Polyethyleneimine
B cell lymphoma 2
Acute myeloid lymphoma
Acute lymphoid lymphoma
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Aptamers
• Oligonucleotide or peptide molecules that bind
to specific target molecules
• Created by selecting them from large random
sequences
• Used for research and clinical purposes as
macromolecular drug
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Conti…..
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Classification of Aptamers
DNA/RNA aptamers
• Consist of short strand of oligonucleotide
Peptide aptamers
• Consist of short variable peptide domain
attached at both ends to a protein scaffold
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Nucleic acid Aptamers
• Nucleic acid species engineered through
repeated rounds of in vitro selection or SELEX
(Systematic evolution of ligand by exponential
enrichment)
• Bind to molecular targets such as small
molecule, protein, nucleic acid
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SELEX
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LYMPHOMA
• Blood cancers develops in the lymphatic system
arise from affecting lymphocyte
• There are at least 35 types of lymphoma which
are grouped based on cell histology, genetic
mutations and expression of certain genes
• Hodgkin’s and non-Hodgekin’s lymphoma are
two major categories
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Aptamers
• As a potential “next generation” of diagnostic
tools
• Therapeutic applications of aptamers in
lymphoma
• Aptamer “small bomb” mediates delivery of
drugs to cancer cells
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Aptamers as a potential “nextgeneration” of diagnostic tools
• Lymphoma is fatal if left untreated or diagnosed at a
late stage
• Burkitt lymphoma (BL)
Methods to diagnose
• Immunophenotyping by flow cytometry (IFC)
• Karyotyping analysis of peripheral blood & bone
marrow
• Detection of malignant cell mutations through PCR
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BL
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Conti…
Limitations
• IFC requires the use of monoclonal antibodies. Complicating
diagnose and increasing cost
• PCR can sensitively examine RNA expression of certain genes to
detect cancer cells have very low signal levels
Advantages of nucleic acid aptamers over antibodies
• tolerant of chemical and temp changes
• Longer shelf lives
• Reduce manufacturing cost
• Better tissue penetration & cellular uptake
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Anti-CD30 Aptamers
CD30 Antigen
(Transmembrane protein
Receptors of TNF)
expressed in activated T & B
cells but over expressed in ALCL
Hodgkin’s lymphoma
Marker for diagnosing
these lymphoma
Anti-CD30 antibodies
used in
Anti-CD30RNA Aptamers
binds to
(identified using extracellular
domain of receptor activator of
nuclear factor KB)
CD30 expression
CD30 Proteins
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ALCL
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Use of anti-CD30 Aptamers as probe
• Explored using flow cytometry and immunostaining
Fluorescently labeled
anti-CD30 aptamers
tested with
cultured ALCL & Hodgkin’s
lymphoma
flow ctometry & fluoroscene
microscopy shows
binding to lymphoma cells
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Immunostaining
Anti-CD30 Aptamers
& antibodies
binds to
CD30 Proteins
serves as
replace
ment of
CD30
antibodies
Biotinylated anti-CD30 Aptamers
Used to study expression of CD30 on lymphoma cells subsequently visualized
by horseradish peroxidase
Specifically stain the lymphoma cells and required a shorter incubation time
and lower temp
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TD05 Aptamers
Burkitt’s lymphoma (BL)
• Acute blood cancer
• Diagnosed through morphological inspection
of blood cells & biopsied tissue sample
• TD05 aptamers have been designed to
diagnosed BL
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Conti……..
TD05 Aptamers
(Evolved by applying cell-SELEX
Against BL cell lines i.e. Ramos cells
Not bind
To normal CD19 B
cells or other bone marrow cells
Subsequent experiments
activated receptor)
initially panel of DNA aptamers
were selected for Ramos cells
only recognize
Ramos cells
shows strong binding
affinity
immunoglobulin heavy mu chain (B-cell
expressed on the surface of BL cells
binding target for TD05 Aptamers
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Aptamer nanoparticle strip
biosensors
• Flow based detection of BL
• Development of simple, sensitive dipstick test
• Litmus test type of assay
Gold nanoparticles aggregate
Responsive to Ramos cells
Was designed
Test zone of strip
immobiized on
immobilized
thiolated TD05
aptamers
TE02 aptamer
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Principle of assay
sample solution or
capillary action
If in the presence of Ramos cells
move along strip
TD05 aptamers immobilized on Au
nanoparticles interact with Ramos cells
Au- nanoparticles
aptamers cell complex
complex was captured by
TE02 immobilized on strip
producing red line
migrate along strip
complex accumulation at test zone
migration control zone
Complex captured by DNA sequence complimentary to TD05 aptamer
2nd red line
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Conti…
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In vivo studies by TD05 Aptamers as
molecular probe
• TD05 aptamers were labeled with fluorescent
dyes such as cy 5 & injected into mice
engrafted with Ramos cells
• Cy 5 labeled TD05 Aptamer accumulate in
tumor tissues
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PEI Aptamer probe
• Used in vivo cancer imaging
• Degradation ratio of aptamer is reduced when TD05
aptamer are immobilized on PEI Polymer
• Fluorescence signal remained elevated for five hours
post injection while signal when naked TD05 only
stayed for 45 minutes
• Feasible method to image tumor in vivo with high
specificity and sensitivity
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Sgc 8 Aptamers
• DNA aptamers generated for specific recognition of
leukemia cells
• Two tumor cell lines were selected as ligands
• An ALL cell line, CCRF-CEM
• Ramos cell line was used as counter selection
• Protein tyrosine kinase 7 (PTK 7) is a molecular target
of sgc 8 aptamers
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Conti..
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In vivo visualization of tumor
• Reporter bearing probes are used
• Cell recognizing ligend are labeled with reporter mol such as fluorescent
dye & then bind to target cancer cells
• Signals are generated
Acivable aptamer reporter probe
• Tumor targeting
• A poly T linker
• Short DNA sequence complementary to part of aptamer that has a
fluorophore & a quencher linked at either terminus
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Conti….
• In absence of target protein probe is in active form
• It keeps fluorophore & quencher in close proximity leading to quenched
fluorescence
• In presence of cancer cells conformation of aptamer probe is recognized on
binding
• Fluorophore get separated from quencher
• Strong signals are generated in response to interaction of probe with target
cells
• Thus sgc 8 aptamers diagnosed cancer cells shorten the diagnostic time
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Therapeutic Applications of
Aptamers in Lymphoma
• In past radiotherapy & chemotherapy were
used
• Aptamers based therapeutic have emerged as
an alternative
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AS1411 Aptamer
• A 26-nt G quadruplex DNA aptamer identified
as a first anticancer nucleic acid agent for
treatment of renal cell carcinoma
AS1411+nucleolin+nuclear
Factor KB essential
modulator
activate
transcription factor
NK-KB
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Conti…..
Nucleolin
(multifunctional protein
Roles in cell growth & death)
Binds Au-rich element
In 3 UTR of
Bcl2 mRNA
Protect
over expressed in cancer cells
modulate interleukin 9
expression in T- cell lymphoma
Anti-apoptic Bcl2 from
degradation & stabilized Bcl2 mRNA
overproduction of Bcl2 in
cancer cells
AS1411 inhibit proliferation of tumor cell lines & suppressed tumor growth
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Conti….
• Phase I clinical trials (involving 30 patients) were
completed in 2006 and indicated that AS1411 was safe
and well-tolerated
• Phase II trials for AML are showing positive interim
results (ClinicalTrials.gov Identifier: NCT00512083
and NCT01034410)
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Recent research has explored the use of AS1411 as a
carrier for the delivery of cancer drugs specifically into
cancer cells by nucleolin mediated internalization
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NOX-A12 Spiegelmers
• 45-nucleotide long (L Steroisomer) RNA aptamer
• High binding affinity, selectivity to the target,
passive immunogenicity
• Evolved from natural D-RNA library by using
mirror image target D-amino acid
• Synthesized as L-RNA to bind natural target (L
amino acid)
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Conti….
NOXA12
Antagonize CXCL12/SDF-1
CXCR5 & CXCR7
metastasis
pathophysiology of chronic
lymphocytic leukemia
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Conti…
• A Phase 1 clinical trial involving 48 healthy subjects was launched
in 2009 and showed that NOX-A12 spiegelmers were safe and welltolerated
• Recently, two Phase IIa clinical studies of NOX-A12 have begun for
hematological oncology indications, including non-Hodgkin’s
lymphoma, chronic lymphocytic leukemia and multiple myeloma
(Clinical- Trials.gov Identifier: NCT01486797 and NCT-01486797)
• This study will involve 33 relapsed CLL patients who will receive
NOX-A12 spiegelmers in combination with a background therapy of
chemotherapeutic agents. In-terim results are expected to be
available in early 2013.
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Aptamer “smart bomb” mediates
delivery of drugs to cancer cells
• Antibody drug conjugates have a cytotoxic potential of
chemotherapy
• Two antibody –drug conjugates have been approved by
FDA for treatment of hematological cancer
• Gemutuzumab ozogamium was 1st FDA approved
designed for patients with replased AML
• It target CD33 antigen expressed on leukemia blast
cells
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Conti….
• Brentuximab vedotin was 2nd FDA approved
for treatment of Hodgkin’s lymphoma
• It target CD30 which is a establish marker for
hodgkin’s lymphoma
• Aptamers have advantages over antibodies
used in target delivery, particularly for cancer
therapy
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TD05 Aptamer-mediated delivery of
photosensitizer
• TD05 aptamer were engineered with photosensitizer for
selective photodynamic therapy of targeted tumor cells
• It exploit the ability of non toxic light sensitive compound
called photosensitizer to produce cytotoxic reactive oxygen
species
• An aptamer photosensitizer conjugate (APC) was
engineered
• Conjugate contain cytotoxic porphyrin-based
photosensitizer reagent chlorin 6
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Conti….
• APC was selective to Ramos cells
• TD05 aptamers were designed to attach a lipid
tail at the end of aptamers to form selfassembled aptamer-micelle nanostructure
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AS1411 Nucleolin Aptamer-mediated
drug delivery
• Nuclear delivery
• It acts as a shuttle to transfer molecules
between cell surface & nucleus
• Scientist have reported a novel strategy of
using AS1411 aptamers as drug carrier to
target cancer cells for photodynamic therapy
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Conti…
AS1411 aptamer+
AS1411apt6photodynamic molecules TMPy4
multivalent
TMPy4 conjugate
nucleolin expressing cells
damage cells upon exposure
to light
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Lipid polymer hybrid nanoparticles
(LPHN)
• These lipid-polymer hybrid nanoparticles were
composed of 6 elements
• 1) a hydrophobic polymeric core made of poly D,
L lactic-co-glycolic acid (PLGA)
• 2) a chemotherapeutic drug, paclitaxel
• 3) a fluorescent dye
• 4) a lipid layer made of lecithin,
• 5) a PEGylated phospholipid, DSPE-PEG2000COOH and
• 6) AS1411 aptamers on the surface
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Theranostic probe (TP)
• Scientist developed a cancer-targeting probe that can
detect & inhibit cancer cells at the same time
• Probe used micro-RNA as a cancer biomarker &
magnetic fluorescence nanoparticle conjugated with
AS1411 aptamers and an miRNA-221 molecular
beacon to simultaneously target cancer cells
• Collectively, resulting nanoparticles displayed superior
selectivity in nucleolin expressing cancer cells
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Conti….
• When incubated with target cells, the
nanoparticles were internalized and released the
miR-221 molecular beacon in the cytoplasm
• In the presence of miR-221, the unloaded miR221 molecular beacon hybridized with
endogenous miR-221, resulting in a strong
fluorescence signal & simultaneously miRNA221 inhibition, thereby reducing expression of
oncogenes
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Conti…
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Anti-CD Aptamer-mediated siRNA
Delivery
• Used to deliver siRNA to Anaplastic lymphoma kinase (ALK)
positive ALCL,which is an aggressive T-cell lymphoma that over
expresses ALK oncogenes & CD30 surface proteins
• Nanoparticle of 140nm were formed
• ALK ,siRNA and CD30 aptamer were incorporated onto cationic
PEI-Citrate carriers to form nanoparticles
• Inside the cell ALK siRNA were released from the nanoparticles to
specifically silence ALK gene expression
• Result in growth arrest and apoptosis in CD30-expressing ALCL
cells
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Conclusions
• Lymphoma are cancers that arise from white blood cells &
usually present as solid tumors
• Aptamers are
oligonucleotides
single
stranded
DNA
or
RNA
• The immense diversity in function and structure of nucleic
acids enable numerous aptamers to be generated through in
vitro selection technique known as SELEX
• Aptamers may inhibit the function of target proteins, and
can be used as potential therapeutic and diagnostic agents
for lymphoma
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References
• Shum., K. T., J. Zhou and J. J. Rossi. 2013. Nucleic acid aptamers as
potential therapeutic and diagnostic agents for lumphoma. J. Cancer
therapy. 4: 872-890.
• Hu, S. Z., Y. Xu-Monette, A. Balasubramanyam, G. C. Manyam, C. Visco,
A. Tzankov. 2013. “CD30 Expression Defines a Novel Subset of Diffuse
Large B-Cell Lym- phoma with Favorable Prognosis and Distinct Gene Expression Signature: A Report from the International DLBCL RituximabCHOP Consortium Program Study,” Blood. 4: 2715-2724.
• Mori., T. A. Oguro, T. Ohtsu and Y. Nakamura.2004. “RNA Aptamers
Selected against the Receptor Activator of NF-kappaB Acquire General
Affinity to Proteins of the Tumor Necrosis Factor Receptor Family,”
Nucleic Acids Research. 32. 6120-6128.
• Shieh., Y. A., S. J. Yang, M. F. Wei and M. J. Shieh.2010. “Ap- tamerBased Tumor-Targeted Drug Delivery for Photo- dynamic Therapy,” ACS
Nano, Vol.3: 1433-1442.
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