COMPANY BACKGROUND INFORMATION

• Mirus Bio Corporation
  

o Biopharmaceutical Company that focused on discovering, developing and commercializing innovative nucleic acid based technologies and products.

o Gene therapy specialist, Jon Wolff, his colleagues James Hagstrom and Vladimir Budker founded the company in 1995, based on research they performed at the University of Wisconsin-Madison

o In 1990, Dr. Wolff and colleagues were the first to discover that muscle cells in the body could take up genes efficiently, an invention which helped launch direct injection approaches for gene therapy.

o Under development currently are RNAi gene silencing therapies based on Mirus’ proprietary Dynamic PolyConjugates™ delivery platform and gene therapies utilizing its Pathway IV™ delivery protocol.

o Other than that, Mirus was founded on the principle that non-viral (i.e.plasmid DNA based) methods offer distinct advantages over viral methods of gene transfer.

o Following this path, Mirus has pioneered the development of a wide range of non-viral delivery technologies using proteins, polymers, and lipids in conjunction with novel chemistries that provide unique nucleic acid delivery capabilities in vitro and in vivo.

RESEARCH & DEVELOPMENT

1996:
(1) Development of TransIT® LT (Low Toxicity) line of transfection products. The breakthrough resulted from the discovery that histone proteins, in conjunction with lipids, serve to increase transfection efficiency while reducing the cellular toxicity associated with cationic lipid mediated transfection.

(2) First demonstration that plasmid DNA could be delivered effectively into liver cells in vivo (mice) using rapid intravascular injections.

1997:
Development of Label IT® non-enzymatic nucleic acid labeling technology. This technology enabled fluorescent labeling and tracking of nucleic acids in cells and tissues.

1998:
First demonstration that plasmid DNA could be delivered effectively into skeletal muscle cells in vivo using rapid intravascular injections.

1999:
(1) First demonstration of high efficiency gene delivery to mouse liver following a rapid injection of plasmid DNA into the tail vein.

(2) First demonstration that “caged” DNA-containing nanoparticles are resistant to aggregation under physiologic salt conditions.

2001:
(1) Development and launch of the first high efficiency transfection reagent, TransIT-TKO® Reagent, designed specifically for siRNA delivery into mammalian cell.

(2) First demonstration of efficient non-viral delivery of genes (plasmid DNA) into primate skeletal muscle.

2002:
First demonstration of siRNA mediated knockdown of an endogenously expressed gene in mice.

2003:
Development of low toxicity, DNA-containing nanoparticles for gene delivery to lungs.

2004:
Development of clinically viable, high efficiency Pathway IV™ method for delivering plasmid DNA into mammalian skeletal muscle.

2005:
Development and launch of a one step, non-enzymatic labeling method for microRNA, the Label IT® miRNA Labeling Kit.

2006:
Development of genetic immunization method in research animals by intravenous delivery

COMMERCIALIZATION

Nucleic Acid research reagents:

o TransIT® Transfection Reagents

§ reagents of unique lipid and polymer formulations that achieve superior delivery efficiency of pDNA and siRNA with minimal cytotoxic effects

o Label IT® Labeling Reagents

§ employ proprietary chemistry to covalently attach labeling molecules to DNA and RNA.

§ faster and more robust than traditional multi-step enzymatic labeling, and can be used for intracellular tracking, chromosome analysis, and microarray labeling

Therapeutic products:

o MyoDys® Muscular Dystrophy Therapy

§ Stage: Phase I/II in preparation

§ treat disease by inserting a gene encoding active dystrophin into affected muscles to delay or prevent loss of muscle function

o Peripheral Ischemia Therapy

§ Stage: Research

§ treatment is based upon delivery of a gene encoding an angiogenic factor to the affected limb, which induces revascularization of the limb

PROPRIETARY TECHNOLOGY: Pathway IV™

• Discovered by the Mirus researchers and collaborators at the University of Wisconsin – Madison
• Relies upon elevating intravascular pressure to physically increase the permeability of the cellular membranes of the blood vessel
• When nucleic acids are injected intravascularly under normal physiological conditions, they are retained within the blood stream because these macromolecules are too large to be transported through the cell membranes.
• Quickly degraded by the nucleases in the blood stream & DNA can pass through the blood vessel wall into adjoining muscle cells before this occurs.
  

Restricting blood flow and elevating pressure are key in delivering plasmid DNA tissue sites

Intravenous (“IV”) delivery of plasmid DNA (“pDNA”) to targeted limb muscle:

• Blood flow in an arm or leg temporarily occluded by a tourniquet
• A pDNA solution is rapidly injected intravenously
• The elevated pressure within the occlusion zone, making the blood vessel wall more permeable and allowing the pDNA to migrate into the adjoining muscle cells
• Blood flow is then restored to normal within a few minutes, with no adverse affects to the vasculature.

Intravenous Nucleic Acid Delivery
to leg muscle.

• Once pDNA resident in muscle cells, the introduced DNA produces therapeutic proteins that can be active locally or secreted systemically. If the DNA encodes an antigenic protein, either antibody or cellular immune responses may be induced
• A single dose can result in long-term gene expression, and the ease of repeat administration makes this platform technology ideal for treating chronic illnesses.
• This versatile platform has potential utility to treat a wide range of diseases, including muscular dystrophy, peripheral vascular ischemia, arthritis, anemia, multiple sclerosis, and cancer.
• Preclinical work is ongoing to determine optimum dose levels, dosing frequency, plasmid construction, etc. for use in human clinical studies, the first of which are anticipated in 2005
• Mirus is actively exploring its wide range of uses, as well as investigating protocol modifications that would enable delivery to additional tissues such as joints and bone.

PRODUCTS IN DEVELOPMENT USING Pathway IV™

# Muscular Dystrophy (MyoDys®)
· A gene therapy product candidate for muscular dystrophy.

· MyoDys® is comprised of plasmid DNA encoding the full-length human dystrophin gene.Pathway IV™ delivery technology is used to administer the pDNA to a patient’s limb skeletal muscles.

· Clinical objective: to delay or prevent loss of muscle function, initially in the hand and forearm, thereby enhancing the patient’s quality of life.

· Being developed with Transgene S.A. of Strasbourg, France as part of a strategic alliance to develop gene therapies for muscular dystrophy.

· Development status: Phase I study has been completed (data released June 5, 2003 by Transgene) which has shown safety and dystrophin expression in skeletal muscle in Duchenne and Becker patients using an alternative delivery approach. A Phase I/II study is now in preparation using Pathway IV™ that will provide regional delivery to forearm muscles.

# Peripheral Ischemia (PAD)
· A gene therapy opportunity that utilize Pathway IV™.

· Inducing revascularization of the limb by delivery of a gene(s) encoding an angiogenic factor to the affected limb.

· Clinical objective: reduce ischemia and to delay or prevent loss of limb, thereby enhancing the patient’s quality of life.

· Development status: still in research; currently evaluating various candidate angiogenic genes in model systems.

# Anemia (EPO)
· Utilize Pathway IV™ protocol.

· Once the DNA is inside target muscle cells, it is anticipated that the patient’s own body will naturally produce sufficient EPO (erythropoietin) to regulate and maintain a stable volume of red blood cells in the bloodstream.

· Development status: current studies are supported by a SBIR Phase II grant from the NIH. The initial target group for such a gene therapy approach will most likely be end-stage renal failure patients.

# Cancer Vaccines
. One strategy to break tolerance and induce immunity involves genetic vaccination employing homologous TAA sequences from a different species.

. In proof-of-concept studies, Mirus Bio has demonstrated that Pathway IV ™ delivery of plasmid DNA encoding tumor antigens can:

.Generate a robust humoral response.
. Promote expansion of antigen-specific CD8+ T cells.
. Induce cytolytic T-lymphocyte (CTL) activity.
. Induce an effective anti-tumor response.

PROPRIETARY TECHNOLOGY:Dynamic PolyConjugates™

o Drs. Andrew Fire and Craig Mello discover gene silencing mechanism (RNA interference) in October 2006; jointly awarded “The Nobel Prize in Physiology or Medicine for 2006.”

o Gene silencing mechanism is the ability to suppress the production of disease causing proteins associates with the discover of the function and role of individual genes

o First to rationally designed synthetic formulation for nucleic acid delivery that mimics the natural viral targeting and disassembly process

o Chemically synthesized to include the following functional elements:

§ Endosomolytic Polymer

§ Charge Masking Agents

§ Environmental Responsive Linkage Chemistry

§ Targeting Ligand (specific to the target cell or tissue)

§ Short interfering RNA Sequence (siRNA specific to a disease target gene)

o Keys to the success of this formulation are:

(1) the endosomolytic potency of the polymer
(2) the unique ability of the complex to respond to location-specific environmental cues
(3) efficient cellular targeting.

o Various targeting molecules including sugar or peptide based receptor-targeting ligands, monoclonal antibodies, or heavy-chain antibody fragments can be incorporated into the DPCs to target specific cell types

o When injected into the bloodstream, unmodified and unprotected siRNAs are rapidly degraded.

o Modifications /strategies ( to increase circulation half-life and functionality):

§ incorporation of stabilizing chemical modifications

§ encapsulating the siRNA in liposomes

§ attachment of a cholesterol group or common polymers to siRNA

o Mirus Bio Researchers discovered and patented a new class of polymers that provide a unique dual benefit. These polymers both impart superior functionality to the nucleic acid while in the bloodstream and promote highly efficient transport of the siRNA from the endosome into the surrounding cytoplasm in the target cell.

o Special features of DPC:

-- combine efficient targeted delivery

-- low toxicity

-- effective gene silencing

-- able to generate the predicted physiologic effect

o Currently being optimized for liver and cancer tissues

o Actively being evaluated by select partners for research and clinical indications

PROPRIETARY TECHNOLOGY:Catheter-mediated Delivery

• allows for the efficient of non-viral vectors in delivery of genes to liver cells using by using intravascular delivery method
• high level gene expression in hepatocytes can be achieved via delivery to the bile duct, portal vein, or inferior vena cava/hepatic vein
• techniques for minimally invasive hydrodynamic gene delivery by Collaborators at Genzyme:-

a) hepatic lobes is isolated using a balloon occlusion balloon catheter to occlude selected hepatic veins

b) target a whole organ wherein the entire hepatic venous system was isolated and the pDNA solution injected hydrodynamically into the vena cava between two balloons used to block hepatic venous outflow

• these procedures shown significant levels of reporter gene expression and secretion were achieved, suggesting that with further refinement hydrodynamic delivery of pDNA to an isolated liver may be a useful method for human gene therapy
• the potential therapeutic application using catheter-mediated method are liver cancer and metabolic diseases

OTHER PROPRIETARY TECHNOLOGY

# TransIT® Transfection Reagents

Mirus Bio has developed the TransIT® Transfection Reagents for the delivery of all types of nucleic acids including DNA, siRNA, mRNA, viral RNA and oligonucleotides to mammalian cells. Each of the TransIT® Transfection Reagents exhibits low cellular toxicity enabling the acquisition of physiologically relevant data.

Additional significant benefits over competing products include:

· High Efficiency Transfection—Independent of the type of nucleic acid being transfected, all the TransIT® Reagents promote high efficiency transfection, increasing experimental success.

· Low Cellular Toxicity—The proprietary formulations and the serum compatibility of the TransIT® Reagents promotes the maintenance of cell density and health, reducing potential experimental biases due to reagent toxicity.

· Easy to Use—The TransIT® protocols are simple with easy optimization which means you spend less time optimizing and more time performing the critical transfection experiments.

· Serum Compatibility—All the TransIT® transfection reagents transfect cells growing in the presence of serum, both simplifying the transfection procedure, and helping maintain cells under normal growth conditions.

· Proprietary Formulations—Developed and manufactured by Mirus Bio scientists.

TransIT®-LT1



#Label IT® Labeling Reagents



The LabelIT® Technology is a non-enzymatic chemical labeling method using an efficient one-step chemical reaction and facilitates the direct covalent attachment of non-radioactive reporter molecules and functional groups to DNA and RNA.

Special features:

· Directly label native and synthetic RNA and DNA of any length from any species

· Eliminates enzymatic incorporation and replication bias

· Fast and simple reaction

· Cost effective—save up to 50% per reaction compared to enzymatic methods

# In Vivo Delivery

The primary delivery and expression site using the hydrodynamic tail vein (HTV) delivery method is to the liver. The rapid injection into a rodent’s tail vein with a sufficient volume of nucleic acid solution used to elevate the pressure within the blood vessel and enhance the vessel’s permeability, thereby enabling passage of large nucleic acid molecules to target cells outside the blood vessel. It is detectable, but significantly less delivery to other organs including the spleen, lung, heart and kidneys is also possible. The advantages of using this method are efficacy, ease-of-use, and have ability to allow long-term gene expression.

BUSINESS MODEL

Transgene initiated partnership to develop therapies using novel gene delivery technology, Mirus entered into a research and license agreement to develop gene therapy products using Mirus' muscle delivery technology for the delivery of Transgene's plasmid-based and adenoviral vectors to skeletal muscle.

Fund provided by the transgene for one year research program to Mirus to apply Mirus' technology to Transgene's gene therapy product candidates for muscular dystrophy and multiple sclerosis.

Mirus grants Transgene options to license its technology for the intra-vascular delivery of therapeutic genes to skeletal muscle. Transgene has developed a broad portfolio of viral and plasmid-based vectors for gene delivery and, through its partnership with Human Genome Sciences and its functional genomics approaches, has the capacity to identify novel genes for therapeutic use.

Mirus Corporation also responsible for the distribution of the TaKaRa Bio (Kyoto, Japan) products in North and South America since 2003. On 2004, Mirus Bio had been awarded a $1.26 million, multiyear Phase II SBIR grant from the National Institutes of Health to develop a gene therapy product to treat anemia.

Mirus also entered into an agreement with Genzyme Corporation covering research applications of Mirus’ proprietary hydrodynamic gene delivery technology. Under this agreement, Genzyme will have the non-exclusive right to utilize a range of Mirus’ technologies in its research programs, including its Pathway IV™ protocol, a novel method to efficiently deliver genes to muscle via the blood stream.


Mirus Bio Corporation has been awarded five federal grants and contracts since January of year 2007 totaling $5.4 million. They are:


*Two Phase II SBIR grants totaling $2.4 million to further develop the Company’s Dynamic PolyConjugates™ siRNA delivery platform and alternative nanoparticle technologies, focused primarily on improving potency for liver applications;

*One Phase II DOD contract for $1.8 million to optimize the Dynamic PolyConjugates™ siRNA delivery platform for lung delivery and to develop a treatment against respiratory infections;

*A Phase I SBIR grant for $0.3 million to improve gene expression technologies;

*A previously announced Phase II SBIR grant for $0.9 million to develop improved labeling technologies for microRNA, which is expected to lead to new research products.

These funds were critical in maintaining their leadership position in nucleic acid delivery technologies.

Bio Corporation had entered into a two year, multimillion dollar agreement with Pfizer Inc. to investigate and optimize gene silencing methods in animal models. The studies will utilize Mirus Bio’s nucleic acid delivery platforms to target and suppress the expression of genes of interest to Pfizer.

FUTURE PROSPECT

# Dynamic PolyConjugates™

This technology might solve the problem gene therapy because so far DPC technology can attach targeting molecules eventually attach to target cells of interest, and attach interaction-inhibiting agents to the polymer to prevent unwanted interactions with non-target cells

# Pathway IV™

This novel, clinically practical procedure might be the answer for the gene therapy as it is a simple, elegant method which has several special features such as overcome the delivery hurdle that lead to ease of repeat administration. It is able to target large numbers of cells within multiple muscle groups enables unprecedented levels of expression, and single dose can result in long term gene expression. All these features are keys to clinical utility for chronic diseases. By using this technology to answer problem in gene therapy, muscle will be the candidate of host site for gene therapies that induce secretion of therapeutic proteins to treat a range of diseases, including anemia, arthritis, ALS, and cancer immunotherapeutic.