Beyond Gene Therapy: Broad Uses for Lentiviral Vectors

When hearing the term “lentiviral vector,” most researchers think of gene therapy. However, many are now realizing the broad potential applications of this powerful gene delivery tool. Beyond gene therapy, Lentiviral vectors (LVs) have a wide variety of applications, including:

• Drug discovery by creating cell lines used for drug screening
• Target validation by creating cell lines devoid of specific target proteins
• RNAi expressing cell lines – for the stable knockdown of gene expression
• Expression of cDNA and shRNA libraries for discovery
• Cell immortalization from primary cells
• High throughput protein production from mammalian cells for pre-clinical screening
• Rapid master cell line development for protein manufacture
• Rapid and cost effective method to manufacture vaccines from mammalian cells
• Efficient generation of transgenic animals and other animal models

Lentigen Corporation is a biopharmaceutical company focused on the manufacture and development of novel biologics through the use of LVs. In addition, Lentigen has already manufactured many unique lentiviral vector constructs for use by researchers in academic institutions, and biotechnology and pharmaceutical companies. These LV research products are now readily available, and have already been purchased by numerous universities and companies to both facilitate and accelerate their own pre-clinical research programs. The collaborations generated from these contracts will allow Lentigen to continue to grow and increase its opportunities for future product development. The increasing demand for these customized research products, along with positive customer feedback, validates the potential of LVs to be the pre-eminent gene delivery technology of the future.

LVs are viral-based gene delivery systems that can stably deliver genes or RNAi into primary cells or cell lines with up to 100% efficiency. LVs bind to target cells using an envelope protein that allows for the release of the LV RNA containing the gene or gene silencing sequence into the cell. The LV’s RNA is then converted into DNA through a process called reverse transcription. The DNA pre-integration complex then enters the nucleus and integrates into the target cell's chromosomal DNA.

Gene delivery is stable because the target gene or gene silencing sequence is integrated in the chromosome and is copied along with the DNA of the cell every time the cell divides. One of the defining features of LVs is their ability to integrate into non-dividing cells, in contrast to other vectors that either do not integrate efficiently into chromosomal DNA (e.g., non-viral, adenoviral and adenoviral-associated vectors), or can only integrate upon cell division (e.g., conventional retroviral vectors).

Other attributes also contribute to the usefulness of LentiMax as a research and development tool, including:

• A high through-put modular LV system that allows for the customization of gene and reporter, insertion of other elements, and choice of envelope pseudotype
• A variety of promoters, reporter genes and selection markers, including GFP, YFP, RFP, puromycin, and hygromycin, that facilitate research
• The ability to pseudotype LVs with several types of envelope proteins, allowing for greater customization and more specific cell targeting
• High titers in the range of 1x 108 - 1x109 per ml
• Stringent quality control to ensure high quality product; testing includes construct restriction map analysis, full sequence validation of your gene or gene silencing sequence of interest, sterility analysis, and titer determination by actual cell transduction and real-time DNA-PCR analysis
• Purest lentiviral vector particles available using Lentigen’s proprietary manufacturing process
• Clinical translatability – the vectors used for R&D are compatible for a wide variety of future applications
• Availability of reagents that allow for precise tittering of LV in transduced cells
• Scalable manufacturing process – LentiMax can be manufactured at scales from 100ul to multiple liter quantities of final concentrated and purified product
• Unique intellectual property that permits commercialization of products by Lentigen’s partners

Beyond the research setting, the use of LVs in clinical applications continues to advance. In terms of gene therapy, LVs are currently being evaluated in at least five clinical trials. LVs have been shown to be safe when introduced into humans, as no adverse events have been reported to date (Dropulic and June, Human Gene Therapy, June 2006). Lentigen believes that lentiviral vectors can provide the attributes needed to make gene therapy successful, including high transduction efficiencies and long-term stability in cells in the human body. Beyond gene therapy, LVs have other uses as well, including vaccine and therapeutic protein production as described below.

Vaccine Manufacturing

Lentigen has developed a virus-like particle (VLP) vaccine technology (LentiVLP™ technology) for the rapid and precise production of vaccines, such as pandemic and seasonal influenza. Lentigen has manufactured LVs that produce VLPs that comprise a core protein and various specific hemagglutinin (H) and neuraminidase (N) surface antigens of influenza. This VLP complex resembles a live virus to the immune system, and has already demonstrated that it can stimulate an immune response in ongoing animal studies. The same approach could be used for other infectious diseases.

Lentigen’s VLP-based vaccine has several advantages over current influenza vaccines, including:

• Rapid manufacture of the influenza VLP by PCR cloning of H and N surface antigens into LentiMax which efficiently transduce cell lines for VLP production in the cell culture medium, resulting in a dramatic reduction in time to market
• VLP vaccines are made precisely from the designated influenza strain since no viral adaptation in cell culture is required, increasing the potential potency of the vaccine
• Cells do not lyse during the manufacturing process, allowing for long harvest times and higher yields of vaccine from each batch of cells
• Lack of cell lysis during manufacturing dramatically decreases vaccine purification costs
• Flexibility of using any mammalian cell culture platform for vaccine production since LentiMax efficiently delivers genes into a wide variety of cell lines
• Mammalian cell glycosylation of vaccine can potentially increase the potency of the vaccine, in contrast to a non-mammalian system of vaccine production
• Since VLPs are genetically inactive, they are safer than live and live attenuated vaccines
• VLPs are particulates and are known to have high immunogenicity

While influenza is Lentigen’s first vaccine target, LentiVLP technology can be used to make vaccines for a wide variety of infectious and other diseases. By validating LentiVLP technology for influenza, a serious global problem, Lentigen intends to leverage its proprietary know-how to develop vaccines for other serious diseases.

Biogenerics

Lentigen and its partners are able to use its core technology to rapidly produce a broad array of biogeneric proteins and proprietary products, such as monoclonal antibodies. As lentiviral vectors insert genetic material in areas of open chromatin (areas that are consistently expressed), proteins produced by cell lines transduced with lentiviral vectors are likely to exhibit high levels of protein production. This process will lead to more efficient production of therapeutic proteins, including monoclonal antibodies, with higher yields, faster development times and decreased manufacturing costs.

Biopharmaceuticals have transformed the pharmaceutical industry since their origin in the 1970s. Biopharmaceuticals are generally large protein molecules derived from living cells, as opposed to smaller, chemical molecules derived from a chemical process that can be precisely controlled. Today, biotechnology drugs account for 10%-15% of sales of total pharmaceutical products. It is estimated that 50% of new approved pharmaceutical products will be derived from biotechnological methods.

Biogenerics, which are the generic versions of branded biopharmaceuticals, are expected to be a key growth area over the next several years because of the expiration of patents for biopharmaceuticals. With over 24 patent expirations expected through 2010 in the U.S., sales of biogenerics are expected to grow rapidly through 2010 and beyond, and may account for up to $12 billion in sales by 2010.

One of the significant advantages of using a LentiMax system is that protein production can be undertaken in mammalian cell lines, particularly human cell lines. This is important for the overwhelming number of proteins that are glycosylated, since producing such proteins from non-human or non-mammalian cells will result in glycosylation patterns that are very different to those found in humans, and abnormal glycosylation will result in proteins that may not function correctly or be antigenic when injected in humans. Proteins that are antigenic will have a very short half-life in the body; they will not be able to be re-administered and therefore have low utility in biomedicine. In contrast, lentiviral vectors are extremely efficient in delivering genes into human cell lines, and therefore proteins will be produced that have human glycosylation patterns resulting in high functionality, low or no antigenicity and, therefore, high utility in biomedicine.

The key advantages of using LentiMax for protein manufacturing are as follows:

• Rapid development of master cell lines for protein manufacturing
• High protein production due to instantaneous multiple integration events
• No selection required to generate high copy number cell lines
• Rapid transition from high throughput protein screening to master cell line
• Mammalian cell glycosylation of produced proteins

Genomics and Drug Discovery

Genomics has generated remarkable opportunities for the pharmaceutical, biotechnology and health services industries. The study of functional genomics facilitates the identification of genes within the genome along with the proteins they express – thereby helping to identify their various functions. However, translating the study of proteins into optimized drug targets poses substantial challenges. Thousands of potential new protein targets have been identified, but the resources to validate them effectively are lacking. A system to efficiently deliver these genes, or inhibitors of the expression of these genes, is needed to validate their function. Presently, there is no robust and reliable commercially available product to obtain stable long-term over-expression or “knock-down” of genes. Researchers have several options – but all of them suffer from serious limitations; as an example, plasmids have low delivery efficiency which is not suitable for sensitive cell analysis. In this context, LentiMax LVs offer significant benefits since they are capable of highly efficient and stable long-term gene expression in cells.
=

In the drug discovery and validation sector of the market, the type of cells used for the screening of drug libraries has become critical for the identification of new lead candidate compounds and early determination of their potential toxicity before they are administered to humans. Lentiviral vector technology rapidly and efficiently generates cell-lines of any desired phenotype, including ‘knocking out’ specific gene function. These cells can then be used in drug discovery for the identification of new compounds that will have higher potencies and lower toxicities than existing lead compounds. Also, LentiMax LVs can efficiently genetically modify primary cells of the body, providing a valuable cellular target for additional potency and toxicity testing. Therefore, LV technology can be used not only to accelerate drug discovery, but also to ensure that lead compounds are tested early for any signs of toxicity before significant resources are allocated to take the compound into clinical trials. The power of LentiMax LV technology is not limited to cell culture - the same vector can be used to create transgenic animals for toxicity validation at the animal level.

The key advantages of using LentiMax for genomics and drug discovery are as follows:

• Rapid development of cell lines expressing proteins or RNAi for high through-put drug screening
• The same LentiMax vector can then be used to efficiently genetically modify primary cells to evaluate drug potency and toxicity; primary cells can be optionally immortalized if desired
• The same LentiMax vector can then be used to generate transgenic animal models to evaluate drug potency or toxicity, or directly injected into animal tissues to generate a localized phenotype
• Cell lines developed with LentiMax can express multiple copies of a gene or RNAi, which may be important for optimal expression of certain genes or gene silencing sequences

Lentigen’s LentiMax system has been developed with clinical translation in mind. This means that companies using LentiMax can be confident that the vector they are using today in research is fully translatable for a wide variety of future applications such as protein manufacturing, animal toxicology and gene therapy. LentiMax is developed with unique intellectual property that provides Lentigen’s partners the freedom to commercialize products developed or derived from LentiMax.