Research & Development at Lippomix

LIPPOMIX's Opportunity Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, and hereditary diseases in which a defective mutant allele is replaced with a functional one. Although the technology is still in its infancy, it has been used with some success and promises to become one of the key biotechnologies of the future. To improve the delivery of the new DNA into the cell, the DNA must be protected from damage and its entry into the cell must be facilitated. To this end new molecules, liposomes have been created that have the ability to protect the DNA from undesirable degradation during the transfection process. Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome, this organized structure is then complexed with DNA .There are three types of lipids, anionic (negatively charged), neutral, or cationic (positively charged). Initially, anionic and neutral lipids were used for the construction of liposomes for synthetic vectors. There is little toxicity associated with them, they are compatible with body fluids and there is a possibility of adapting them to be tissue specific. The most common use of liposomes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. Recent studies have shown liposomes to be useful in transfecting respiratory epithelial cells, so they may be used for treatment of genetic respiratory diseases such as cystic fibrosis. When naked DNA comes into contact with the cell membrane, only a small amount will enter the cell, leading to relatively low gene-transfer efficiency. Therefore, a carrier or a virus vector is generally used to increase transfection efficiency and achieve adequate expression of the therapeutic molecule. Plasmid or liposomal complexes are the most commonly used carrier molecules. However, plasmid a small fraction of plasmid DNA enters the nucleus, where it persists in an episomal location (not integrated into the genome), resulting in limited duration transgene expression in both proliferating and non-proliferating cells. Although transgene expression has been reported to be as long as 3-4 weeks, in most cases the expression is far shorter in duration. The production and scale up of plasmid and liposomal complexes is relatively easy, but low transfection efficiency, short duration of and low levels of transgene expression limit this approach, which may not be sufficient to achieve therapeutic concentrations of the protein product. Phospholipid formulas such as 1, 2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-dodecanoyl/ 1, 2-dioleoyl-sn-glycero-3- phosphocholine and cationic polymers such as polyL-ornithine with galactose and the fusigenic peptide mHA2 (Galactose-polyLornithine-mHA2) can improve the transfection efficiency.In addition, EBV-based expression plasmids may prolong gene expression at therapeutic levels, and can efficiently and repeatedly re-transfect immunocompetent hosts. Lippomix is developing technologies and strategies to overcome these limitations and improve the efficiency of gene transfer. About 4,000 diseases have been traced to gene disorders. Current and possible candidates for gene therapy include cancer, AIDS, cystic fibrosis, Parkinson’s and Alzheimer’s diseases, amyotrophic lateral sclerosis (Lou Gehrig's disease), cardiovascular disease and arthritis. Lippomix is stratigacally positioned with unique and proprietary technology to take advantageof these market opportunities to develop a safe and effective gene delivery platform.

Lippomic Inc. has unique technology to deliver gene therapy products locally instead of systemically. Lippomix’s plan will center on the development of these proprietary technologies to provide superior gene delivery vehicles to specialized tissues. Localised application will minimize systemic delivery and distribution throughout the body. This technology will maximize gene delivery to the affected areas and decrease side effects. The ease of a topical treatment and administration will allow patients to forgo invasive procedures which may require hospitalization.

Lippomix will focus on the following three areas for gene delivery:

• Wound healing
• Cervical cancers
• Diseases of the eye

WOUND HEALING

Liposome-mediated DNA delivery has also been assessed in wound healing models. Topical application of lipofectamine-plasmid complexes containing the cDNA for FGF-1 (aFGF) results in an increased rate of wound closure, increased cellular density, and decreased scar formation in excisional wounds in diabetic mice. Subcutaneous injections of liposome-plasmid complexes containing the cDNA for KGF or IGF-1 have demonstrated dose-dependent improvements in reepithelization, collagen deposition, and angiogenesis in burn wounds in rats. Lipposome technology developed at lippomix can deliver these substances efficiently and cost effectively. Lippomix has extensive know-how and technology in the area of topical drug delivery and gene therapy.

OPHTHALMIC GENE DELIVERY

Lippomix can effectively produce gene delivery products targeted for ophthalmic tissues. Genes can be delivered to the retina, lens or cornea epithelium. The cells at the surface of the eye are easily accessible to liposomes carring DNA.

Conditions treatable by ophthalmic gene delivery:

• viral infections
• allograft rejection
• inherited retinal degradation
• cataract
• retinitus pigmentosa
• macular degeneration
• diabetic retinopathy
• glaucoma

GENE DELIVERY FOR GYNECOLOGICAL CANCERS

The three most common types of gynecological cancers are cervical, ovarian and endometrial.

Cervical Cancer:

Cancer of the cervix (also known as cervical cancer) begins in the lining of the cervix. Cervical cancers do not form suddenly. Normal cervical cells gradually develop pre-cancerous changes that turn into cancer. Doctors use several terms to describe these pre-cancerous changes, including cervical intraepithelial neoplasia (CIN), squamous intraepithelial lesion (SIL), and dysplasia.

There are 2 main types of cervical cancers: squamous cell carcinoma and adenocarcinoma. Cervical cancers and cervical precancers are classified by how they look under a microscope. About 80% to 90% of cervical cancers are squamous cell carcinomas, which are composed of cells that resemble the flat, thin cells called squamous cells that cover the surface of the endocervix. Squamous cell carcinomas most often begin where the ectocervix joins the endocervix.

The remaining 10% to 20% of cervical cancers are adenocarcinomas. Adenocarcinomas are becoming more common in women born in the last 20 to 30 years. Cervical adenocarcinoma develops from the mucus-producing gland cells of the endocervix. Less commonly, cervical cancers have features of both squamous cell carcinomas and adenocarcinomas. These are called adenosquamous carcinomas or mixed carcinomas.

Only some women with pre-cancerous changes of the cervix will develop cancer. This process usually takes several years but sometimes can happen in less than a year. For most women, pre-cancerous cells will remain unchanged and go away without any treatment. But if these precancers are treated, almost all true cancers can be prevented. Pre-cancerous changes and specific types of treatment for precancers are discussed in the section, "Can Cervical Cancer Be Prevented?"

Precancerous changes can be separated into different categories based on how the cells of the cervix look under a microscope. These categories are discussed in the section,

Ovarian Cancer:

According to the American Cancer Society, ovarian cancer ranks fifth in cancer deaths among women. It's estimated that about 20,000 women in the United States will develop ovarian cancer this year. About 15,000 deaths from ovarian cancer will occur in American women during that same time frame.

Ovarian cancer refers to a cancerous tumor that begins in a womans ovaries but may spread, or metastasize, to other parts of the body.

Although ovarian cancer affects far fewer women than many other cancers, it is troublesome because it is difficult to detect in its early stages, when treatment can be most effective. For this reason it is especially important for women to identify whether they are at particularly high risk, so they can have more rigorous screening than is available to the general public.

Lippomix’s gene carring liposomes can be directly applied into the peritoneal cavity and directly deliver their payload to the cancerous cells that are causing metastasis.

Endometrial Cancer:

Endometrial cancer , which originates in the inner lining of the uterus, accounts for about 90% of uterine cancers. Uterine sarcoma originates in the myometrium and accounts for less than 10% of cases.

According to the National Cancer Institute (NCI), uterine cancer is the most common type of gynecologic cancer. In the United States, approximately 37,000 cases are diagnosed and about 6000 women die from the disease each year. Incidence of uterine cancer increases after menopause and approximately 75% of cases are diagnosed in postmenopausal patients. The average age at diagnosis is about 60 years. In the United States, endometrial cancer is more common in Caucasian women and uterine sarcoma is more common in African American women.

Lippomix’s gene therapy technology may provide a breakthrough in treating this type of cancer.

ADVANTAGES OF LIPPOMIX’S TECHNOLOGY

Gene therapy potentially represents one of the most important developments to occur in medicine, but before this can be realised certain technical problems common to all methods of gene delivery must be overcome. In order to modify a specific cell type or tissue, the therapeutic gene must be efficiently delivered to the cell, in such a way that the gene can be expressed at the appropriate level & for a sufficient duration. Two broad approaches have been used to deliver DNA to cells, namely viral vectors & non-viral vectors, which have different advantages as regards efficiency, ease of production & safety.

Viruses are obligate intra-cellular parasites, designed through the course of evolution to infect cells, often with great specificity to a particular cell type. They tend to be very efficient at transfecting their own DNA into the host cell, which is expressed to produced new viral particles. By replacing genes that are needed for the replication phase of their life cycle (the non-essential genes) with foreign genes of interest, the recombinant viral vectors can transduce the cell type it would normally infect As viruses have evolved as parasites, they all elicit a host immune system response to some extent. In September 1999, an 18-year old patient died of complications caused by the introduction of a large amount of adenovirus in the portal vein of his liver as part of a gene therapy clinical trial at the University of Pennsylvania. His death was attributed to an immune response to the virus. This is an example of the insurmountable safety problems that adenoviral vectors face.

Non-viral delivery systems offer a very attractive alternative to viral systems. Although non-viral gene delivery vehicles are still relatively inefficient compared to viral vectors and the resulting gene expression is transient in nature, their non-immunogenic nature (safety) allows for repeated treatments. Lippomix can provide novel technology and expertise to develop non-viral liposomal delivery systems with greater safety.

Technology Comparisons Other liposomal gene therapy companies have encountered several barriers preventing them from commercialization, which will have the effect of minimizing their threat as viable competition to LIPPOMIX.

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