Tranosyl - Infinity Medical Engineering

Transcription

Tranosyl™
t-RNA & Ribosomal RNA Inhibitors
Against Acute Myeloid Leukemia
Immunotrex Biologics, Inc
Syed K. Hasan M.D. Ph.D. – CEO/President
Michael McDaniel – CEO/President Infinity Medical Engineering
Immunotrex Biologics, Inc.
Infinity Medical Engineering, LLC
Tranosyl™ t-RNA & rRNA Inhibitor
Cancer Therapeutic Technology
Overview
The Central Dogma: A Brief History
RNA Interference
Tranosyl™: Mechanism of Action
Methodology
Tranosyl™: Selective Targeting
Tranosyl™ Attributes
03/22/2014
© 2014 Immunotrex Biologics
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The “Central Dogma” of Molecular Biology
In 1958, Francis Crick explained the flow of genetic information within a biological
system. Information is stored within an organism’s DNA, transcribed into RNA and
translated into protein. An organism regulates its biological function at the molecular
level in response to cellular needs.
Mutations within the DNA often lead to the synthesis of irregular or dysfunctional protein.
Since proteins are the molecular actuators and regulators of cellular process, errors in
protein production directly affect cell viability.
A great deal of energy and intellectual
effort has been expended in pursuit of
the “holy grail” of direct manipulation of
the DNA “source code” in an attempt to
prevent and/or cure genetic disease.
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DNA is an elusive target
The difficulty of manipulating specific
DNA sequences can be largely
attributed to its location and packaging.
DNA is housed within the nucleus of the
eukaryotic cell, and is complexed with
histones in a densely packed state.
This difficulty in accessing and effecting
changes in DNA has focused efforts on the
second stage of the “central dogma” – RNA.
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Image courtesy of the Center for Cancer Research:
www.ccr.cancer.gov
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Messenger RNA is specific
Messenger RNA (mRNA) is a
rational therapeutic target.
Messenger RNA codes
specifically for each protein
produced by a cell.
If you can influence a
specific messenger RNA,
you can regulate a specific
protein…
Used with permission: [email protected]
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RNA INTERFERENCE
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Small Interference RNA (siRNA): A Regulatory Mechanism
The 2006 Nobel Prize in Physiology or Medicine was awarded to Andrew Fire and
Craig C. Mello for their discovery of RNA interference in 1998. RNA interference
effectively silences specific gene expression by binding messenger RNA (mRNA).
A bound mRNA is biologically inactive; it
RNA Interference Overview
cannot be translated into its intended
protein. mRNA interference inhibits specific
protein production.
Micro RNA (miRNA) was discovered in the
1990s, but was only understood to be an
endogenous regulatory mechanism in the early
2000s. Regulation by miRNAs is another form
of RNA interference.
http://www.uni-konstanz.de/FuF/chemie/jhartig/
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Interference: Endogenous microRNA Interference
Elements of endogenous RNA interference by microRNA (miRNA):
• Endogenous miRNA interference is semi-specific—It is believed that a
relatively small number of miRNAs (~1000) are responsible for
regulating a majority (~60%) of human genes.
• A single miRNA modulates multiple mRNA transcripts. This is
accomplished through variability in the binding complementarity.
• Variable binding complementarity results in “transient” binding
of an mRNA, and transient protein regulation.
The ability to regulate gene expression (protein production) by microRNA
interference has been well documented.
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Interference: Exogenous siRNA Interference
Exogenous small interfering RNA (siRNA)—both double-stranded RNA
(dsRNA) and small hairpin RNA (shRNA)—share the following
characteristics:
• Exogenous small interfering RNA (siRNA) interference is specific—Each
exogenous siRNA binds a single mRNA transcript. This is accomplished
through full complementarity in the binding region.
• Efficacy of exogenously induced mRNA interference is limited due to
its specificity. Specificity results in enhanced binding, but poses
difficulty in targeting within the cellular matrix.
• Targeting diverse populations of mRNA with exogenous siRNA
requires multiple therapeutic siRNAs.
The ability to regulate gene expression (protein production) by exogenous
siRNA interference has been demonstrated.
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TRANOSYL™
MECHANISM OF ACTION
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Ribosomes: The protein factory of the cell
The ribosome is the site of protein
production within the cell. Ribosomal
complexes (40S + 60S subunits) form in
response to the presence of messenger
RNA (mRNA). Messenger RNA is
translated in ribosomal complexes.
Proteins are formed by the addition of
amino acids as directed by the
messenger RNA. The primary structure
of protein is formed by elongation at
the ribosomal-mRNA interface, one
specific amino acid at a time, in
assembly line fashion.
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60S
40S
Small (40S) and Large (60S) subunits
join to form a functional ribosome.
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Transfer RNA delivers amino acids to the ribosome
Amino acids are transported to the ribosomes by transfer-RNA (t-RNA).
There are approximately 45 different human t-RNAs. The cloverleaf
secondary structure is well conserved.
2⁰ Structure
3⁰ Structure
Anticodon
Used with permission: http://commons.wikimedia.org/wiki/User:Yikrazuul
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A Question of Logistics
Each mRNA codes for a specific amino acid sequence assembled by a ribosome.
Every amino acid is delivered to the ribosome by transfer RNA.
If the delivery of amino acids is disrupted…
…protein synthesis is disrupted.
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Disruption of t-RNA docking impedes protein synthesis
Docking of the t-RNA is required for amino acid transfer in initiation
and/or elongation of the polypeptide. Tranosyl™ is designed to interfere
with global protein synthesis within the target cell by disrupting t-RNA
docking in the ribosomal cleft. The mechanism is interference through
binding the D arm of transfer RNA.
t-RNA
t-RNA + Tranosyl™
Tranosyl™
Amino Acid
“D” arm
Anticodon
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Translation: a quick review
6. A “STOP” codon is reached; subunits
dissociate, the protein is released.
5. Subsequent t-RNAs are
cv
processed, elongating the peptide.
60S
The steps
marked in
magenta are
suppressed by
Tranosyl™.
2. Large (60S) ribosomal subunit
complexes with 40S subunit.
1. Small (40S) ribosomal
subunit binds mRNA.
40S
3. Ribosome is activated
and t-RNAs are evaluated.
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4. Successful binding of valid
cv
t-RNA initiates peptide
synthesis.
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Translation in the absence of Tranosyl™
A simple schematic of the transfer RNA (t-RNA) docking process.
t-RNA
Ribosomal Docking Site
Amino Acid
Protein
60S
Synthesis
mRNA
40S
“D” arm
of t-RNA
Up-regulation of t-RNA has been demonstrated in cancer cells.
Translation is up-regulated in response to increased metabolic
demand for global protein.
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Tranosyl™ impedes translation
Tranosyl™ interferes with global protein synthesis by disrupting t-RNA
docking in the ribosomal cleft. The mechanism is small hairpin RNA
interference (shRNAi) binding the “D” arm of transfer RNA, or the
ribosomal docking site.
t-RNA + Tranosyl™
Ribosomal Docking Site
Amino Acid
60S
Protein
Synthesis
mRNA
40S
“D” arm
of t-RNA
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Targeted Apoptosis
The effective action of Tranosyl™ is to reduce global protein production rate and
availability, leading to global protein deficit within the targeted cell. This will in turn
drive the target cell to apoptosis.
Apoptosis is an intrinsic mechanism to eliminate cells with decreased
viability. In a normal, healthy adult, it is estimated 50-70 billion cells a day
die through apoptosis. Apoptosis is a response by the cell to biochemical
stress which cannot be remediated.
Tranosyl
Block
Translation
Impede
Protein
Synthesis
Trigger
Apoptosis
Cells that undergo programmed cell death are naturally
degraded and eliminated from the body.
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Caspase-3 – An Apoptotic Initiator
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™
METHODOLOGY
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The Production Process
Tranosyl™ is a small peptide biologic, developed and produced using proven
bioengineering techniques:
 Custom complementary DNA strands are synthesized.
Strand 1
Strand 2
 Strands hybridize, forming the DNA template.
“Sticky” ends
 The double-stranded DNA (dsDNA) insert is
ready for ligation into vector.
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The dsDNA plasmid vector codes for the Protein Transport Capsule (PTC).
 The PTC vector is cut with specific restriction enzymes.
Restriction
Enzymes
 The PTC vector has complementary “sticky ends.”
dsDNA Templates
PTC
shRNA
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Ligation of the dsDNA template strand with the PTC vector yields a complete Tranosyl
plasmid.
 The dsDNA insert and PTC vector are joined.
Ligation
 The result is a complete dsDNA plasmid.
dsDNA Templates
PTC
shRNA
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The (shRNA + PTC) plasmid containing the “blueprint” for production of Tranosyl is
now transfected into bacteria.
 The plasmid is transfected into competent bacteria.
Transfect
 Only transfected bacteria grow on
selective media, forming clonal colonies.
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The (shRNA + PTC) plasmid containing the “blueprint” for production of Tranosyl is
now transfected into bacteria.
 The plasmid directs expression of the Protein Transport Capsule and the Tranosyl
shRNA.
Bacteria
PTC
shRNA
 The resultant bacteria contains Tranosyl
shRNA within the Protein Transport Capsule.
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Clonal colonies are derived from a single bacterium which has successfully integrated
the (shRNA +PTC) plasmid, which confers selective viability.
 Clones are harvested …
 …and cultured.
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Tranosyl™ is purified and is ready for administration.
 Bacteria are lysed and Tranosyl is purified using standard chromatography.
Lysis
Purification
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TRANOSYL™:
SELECTIVE TARGETING
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Selective Targeting and Therapeutic Administration
Tranosyl is targeted to induce apoptosis by selective binding to specific cell surface
receptors, which are over expressed on malignant cells.
 Surface cell receptors are recognized by the Protein Transport Carrier.
 Tranosyl binds to the surface in preparation for endocytosis.
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Selective Targeting and Therapeutic Administration
Tranosyl™ is brought into the cell in response to binding cell surface receptors.
Inside the cell, the Protein Transport Capsule releases the Tranosyl shRNA.
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RNA Interference: A Regulatory Mechanism
RNA interference effectively silences specific gene expression by binding
messenger RNA (mRNA). mRNA interference inhibits specific protein
RNA Interference Overview
production.
RNA Inhibition Targeting
mRNA = specific protein
t-RNA = global protein
Tranosyl™
http://www.uni-konstanz.de/FuF/chemie/jhartig/
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TRANOSYL™:
ATTRIBUTES
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Rational Design of Tranosyl
Tranosyl™ is rationally designed to leverage proven molecular biology tools
and innate physiological systems to develop a novel targeted therapeutic
against cancer. Features include:
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•
Synthesis of specific dsDNA sequence templates
•
Bioengineered protein encapsulation of dsDNA templates
•
Characterization of malignant cell surface receptor target
•
Built-in specificity of docking to malignant cell
•
Surface receptor activated endocytosis
•
Utilization of intrinsic RNA interference pathway
•
shRNA interference of novel target: t-RNA
•
Trigger apoptosis and destroy malignant cell
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Anticipated Benefits
Tranosyl is designed to provide an alternative to current therapeutic
intervention in cancer. Immunotrex envisions a number of possible
benefits:
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Utilization of intrinsic biological systems should minimize
unpleasant side effects of treatment.
•
Minimal expected toxicity. Tranosyl is a small molecule biologic.
•
Elimination from the body via natural excretory process
•
Non-infectious. Tranosyl is not self-replicating.
•
Immunotrex expects minimal immune response.
•
Tranosyl is flexible. Immunotrex has chosen Acute Myeloid
Leukemia as its investigational model. Adaptation to other
malignancies may be achieved by modification of the Protein
Transport Capsule targeting mechanism.
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Intellectual Property
• International Patent Application: WO 2008/131348 A2
• Title: COMPOSITIONS AND METHODS FOR TREATMENT OF
UNCONTROLLED CELL GROWTH
• M&G Ref: PCT/US2008/061038
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Acknowledgments
Dr. Michael Graves, Ph.D. Molecular Biology
University of Massachusetts - Lowell
Scientific Advisor
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Thank You
Immunotrex Biologics and Infinity Medical
Engineering appreciates the opportunity to
present our Tranosyl™ anti-cancer
therapeutic technology platform.
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Tranosyl - Infinity Medical Engineering

Transcription

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