Expression of human interferon in transgenic chloroplasts

Abstract

A plastid transformation vector for a stably transforming a plastid genome is provided. The vector includes, as operably-linked components, a first flanking sequence, a DNA sequence coding for a therapeutic human IFN, which is capable of expression in the plastid and a second flanking sequence. The invention also provides isolated and purified IFN, wherein the IFN is configured in a monomeric or multimeric form and is a structural equivalent to orally administered human IFN. Also provided are methods for variable-expressing biopharmaceutical proteins in plants suitable for mammal consumption. The method includes integrating a plastid transformation vector into a plastid genome of a plant cell; growing the plant cell to express a biopharmaceutical protein, such as therapeutic human interferon IFN. Also disclosed are plants transformed with the aforementioned vectors, and the progeny thereof. Also, disclosed is the IFN, which is IFNα2b.

Description

FIELD OF THE INVENTION [0001] This application relates to the field of genetic engineering of plant plastid genomes, particularly chloroplast, vectors for transforming plastids, transformed plants, progeny of transformed plants, and to methods for transforming plastid genomes of plants to generate Human Interferon (IFN). BACKGROUND [0002] Interferons are in a special class of antiviral proteins secreted in minute amounts from mammalian cells upon induction with viruses, double-stranded RNAs, immunotoxins, mitogenes, etc. There are two main types of interferon: type I represented by the interferons α (lymphocyte interferon) and β (fibroblasts interferon) and type II (or immune interferon) represented by the interferon γ (IFN). The interferon family has been extremely well characterized in the prior art, as can be seen in such publications as seen in. (Haus, L., Archivum Immunologiae et Therapiae Experimentalis, 2000, 48, 95-100.). [0003] The interferon (IFN) system is one of the majo...

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Problems solved by technology

Its disregulation may result in a greater tendency to infectious diseases and to the development of cancer.

However, the IFNα2b drugs that are being marketed are produced through an E. coli expression system and due to necessary in vitro processing and purification, the average cost of treatment is $26,000 per year.

Patients are normally injected with the drugs, Intron®A and PEG-Intron™, resulting in severe side effects which have been linked to route of administration.

However, many eukaryotic proteins cannot be expressed in prokaryotic hosts because their mRNAs contain introns that need to be removed in order for correct translation and E. coli is unable to process these transcripts (Glick and Pasternak, 1998).

Although numerous IFNα subtypes have been expressed in E. coli, special techniques that add to the cost of the drug have to be employed to produce the mature, biologically active interferon.

However, the treatment has many side effects and only 20% of patients who need treatment can actually afford to buy the drug (Harris-Stuart and Penny, 1997).

Although bacterial and fungal systems are the most predominant systems for commercial production of recombinant proteins, they have several important drawbacks when producing proteins from eukaryotes.

Proteins that require disulfide bonds or glycosylation are not well suited for expression in microorganisms (Glick and Pasternak, 1998).

However, recombinant protein expression in plants by nuclear transformation have been low, with most levels much less than the 1% of total soluble protein that is needed for commercial feasibility if the protein must be purified (Daniell et al., 2002).

Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which can result in very high levels of foreign gene expression.

Such gene transfers could potentially result in the emergence of “superweeds” able to resist certain herbicides thereby undermining the benefits of GM crops (Daniell, 2002).

Therefore, a foreign gene introduced by genetic engineering of the chloroplast genome could not transfer to genetically compatible weeds.

In contrast, nuclear transformation experiments in higher plants frequently suffer from epigenetic gene-silencing mechanisms resulting in inconsistent and unstable gene expression or complete loss of transgenic activity (Hager and Bock, 2000).

Good recombinant systems are still not available for many human proteins that are expensive to purify or highly susceptible to proteolytic degradation.

Proteolytic degradation is another serious concern for industrial bioprocessing.

To date no one has successfully transformed the plastid genome with IFN to create a delivery system that is easily administered and that stimulates both arms of the immune system without the severe side effects experienced by patients in current IFNα2b treatments.

. . ) have failed to provide a cost effective and functional IFN, which can be orally administered without the side effects, i.e., human pathogens that are associated with the current production vehicles.

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