The concept of an agroinfiltration kit for recombinant protein production for educational and commercial use-A journey through a forest of regulatory and legal implications.

Recombinant expression using Agrobacterium-mediated transient transformation (ATT) of plants has developed into a robust and versatile method to rapidly produce proteins. The capability of plants to efficiently synthesize even homo- and hetero-multimeric complex folded proteins featuring disulfide bonds and other post-translational modifications such as N-linked glycosylation makes them superior to most of the established microbial, especially prokaryotic expression hosts. Compared to production in mammalian cell cultures, ATT requires lower skills, simple technical equipment and cheaper media components. Taken together these features make the method optimally suited for R&D applications involving the development and engineering of recombinant proteins for various purposes ranging from vaccine candidates, therapeutic proteins, towards enzymes for different pharmaceutical and technical applications. Despite these advantages the technology is currently not being used outside the community of plant research. The design and realization of a kit containing all the information, instructions and ideally also the material required to perform recombinant protein production using ATT in an educational or commercial context was one of the objectives of the EU-funded Horizon 2020 project Pharma-Factory. While it is pretty straightforward to assemble a comprehensive instruction manual describing the procedure, the clarification of regulatory and legal aspects associated with the provision, dissemination and use of the different materials and organisms required to perform ATT is a complex matter. In this article, we describe the initial concept of an ATT kit for educational as well as research and development (R&D) purposes and the specific regulatory and legal implications associated with the various kit components. We cover aspects including intellectual property rights, freedom-to-operate (FTO), safety regulations for distributing genetically-modified organisms (GMOs), as well as export and import regulations. Our analysis reveals that important components of the ATT kit are freely available for research purposes but not or only with considerable effort for commercial use and distribution. We conclude with a number of considerations and requirements that need to be met in order to successfully disseminate such a kit in the future.

Freedom to Operate Analysis of Molecular Farming Projects.

Academic scientists are increasingly engaged in translational research oriented toward bringing products and processes to commercial markets. They need to diligently analyze the intellectual property (IP) rights of others to avoid infringement, and use their own IP strategically. For this it is useful to perform a freedom-to-operate (FTO) analysis which includes searching the prior art and patent databases. This chapter outlines the principles of FTO analysis with a special focus on plant biotechnology.

Freedom-to-operate analysis of a transgenic multivitamin corn variety.

In this article, we explore the intellectual property (IP) landscape relevant to the production and commercialization of Carolight(™) , a transgenic multivitamin corn variety created on humanitarian grounds to address micronutrient deficiencies in low-and-middle-income countries. The successful production of this variety requires IP rights risk management because there is a strong protection on inventions and processes via patent portfolios in both developing and industrialized countries. The IP framework is complex, and specialist patent lawyers are usually employed to perform such analysis, but the costs cannot always be met by small, publicly funded projects. We report an alternative strategy, a do-it-yourself patent analysis, to produce a review with limited legal value that can nevertheless lay the foundations for a subsequent more in-depth professional freedom-to-operate opinion.

Commercialization of new biotechnology: a systematic review of 16 commercial case studies in a novel manufacturing sector.

The 1980s and 1990s saw a major expansion of biotechnology into new areas of science including genomics and recombinant technologies. This was coupled to the widespread emergence of academics into the commercial sector as they were encouraged to spin out companies or commercialize their intellectual property. There were many opportunities to raise investment, and extraordinary success stories were prominent across many areas of technology. The field of plant biotechnology for manufacturing recombinant pharmaceuticals (molecular pharming) emerged and was developed in this period. Like other biotechnologies, this was an exciting new development which offered some very obvious benefits and commercial advantages. In particularly, plant molecular pharming represented a highly novel and potentially disruptive manufacturing technology for recombinant proteins. Twenty-five years on, a series of interviews with senior members of sixteen of the most prominent companies involved in the field provides insight into the original drivers for commercialization, strategic thinking and planning behind key commercial decisions and an insider view into the major reasons for commercial success or failure. These observations and recurring themes identified across a number of commercial ventures remain relevant today, as new biotech companies continue to spin out of the world of academia.

Learning from practice: compulsory licensing cases and access to medicines.

Compulsory license is one of the safeguards that international IP law provides to address the undesired effects of pharmaceutical patents on access to important medicines. This article looks into three important case examples to analyze the mechanism's effectiveness and feasibility: the first uses of the newer compulsory license regime established in 2003 under the WTO legislative framework to export medicines to third countries, which lack pharmaceutical manufacturing capacities; and further, the first compulsory license grant in India in March 2012. The case analyses are based on the historical, factual and legal background. They reveal the main challenges of the 2003 WTO regime, including the lack of economic incentives for the generic pharmaceutical companies' participation. In the case of India's compulsory license grant, the article takes as in depth look into possible reasons for the reluctance to use the safeguard until recently, and the important aspects and implications of the Indian authorization to manufacture and sell a generic version of a patented cancer drug.

Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies.

Molecular Pharming, the production of recombinant pharmaceuticals through plant biotechnology, has the potential to transform the biologics sector of the pharmaceutical industry. More fascinating however, is how it might be used to improve access to modern medicines, and improve health of the poor in developing countries and emerging economies. Although improving global health through molecular pharming has been discussed for at least two decades, little progress has actually been made. In this manuscript, a four point plan is described to maximise the opportunity for molecular pharming to provide solutions. These are (i) to identify and prioritise important drug targets that are relevant to the poor; (ii) to support research and development partners in low to middle income countries to develop local expertise, transfer technology and build capacity; (iii) to increase collaboration between regulatory bodies to enable national regulatory frameworks to be developed in low to middle income countries; and (iv) to promote intellectual property management approaches that include socially responsible licensing. An existing case study is described to illustrate how this might be achieved.

Cryptococcal meningitis: improving access to essential antifungal medicines in resource-poor countries.

Cryptococcal meningitis is the leading cause of adult meningitis in sub-Saharan Africa, and contributes up to 20% of AIDS-related mortality in low-income and middle-income countries every year. Antifungal treatment for cryptococcal meningitis relies on three old, off-patent antifungal drugs: amphotericin B deoxycholate, flucytosine, and fluconazole. Widely accepted treatment guidelines recommend amphotericin B and flucytosine as first-line induction treatment for cryptococcal meningitis. However, flucytosine is unavailable in Africa and most of Asia, and safe amphotericin B administration requires patient hospitalisation and careful laboratory monitoring to identify and treat common side-effects. Therefore, fluconazole monotherapy is widely used in low-income and middle-income countries for induction therapy, but treatment is associated with significantly increased rates of mortality. We review the antifungal drugs used to treat cryptococcal meningitis with respect to clinical effectiveness and access issues specific to low-income and middle-income countries. Each drug poses unique access challenges: amphotericin B through cost, toxic effects, and insufficiently coordinated distribution; flucytosine through cost and scarcity of registration; and fluconazole through challenges in maintenance of local stocks--eg, sustainability of donations or insufficient generic supplies. We advocate ten steps that need to be taken to improve access to safe and effective antifungal therapy for cryptococcal meningitis.

Analysing patent landscapes in plant biotechnology and new plant breeding techniques.

This article aims to inform the reader of the importance of searching patent landscapes in plant biotechnology and the use of basic tools to perform a patent search. The recommendations for a patent search strategy are illustrated with the specific example of zinc finger nuclease technology for genetic engineering in plants. Within this scope, we provide a general introduction to searching using two online and free-access patent databases esp@cenet and PatentScope. The essential features of the two databases, and their functionality is described, together with short descriptions to enable the reader to understand patents, searching, their content, patent families, and their territorial scope. We mostly stress the value of patent searching for mining scientific, rather than legal information. Search methods through the use of keywords and patent codes are elucidated together with suggestions about how to search with or combine codes with keywords and we also comment on limitations of each method. We stress the importance of patent literature to complement more mainstream scientific literature, and the relative complexities and difficulties in searching patents compared to the latter. A parallel online resource where we describe detailed search exercises is available through reference for those intending further exploration. In essence this is aimed at a novice patent searcher who may want to examine accessory patent literature to complement knowledge gained from mainstream journal resources.

Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice.

T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3'-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.

Molecular Pharming: future targets and aspirations.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George's, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

Molecular farming, patents and access to medicines.

Transgenic plants have several advantages over other expression systems for the production of recombinant medicines, including low costs, large-scale production and the ability to produce complex multimeric proteins with appropriate post-translational modifications. Several plant-made pharmaceuticals, including the enzyme glucocerebrosidase, insulin and IFN-alpha(2b), are approaching commercialization and these developments have been accompanied by considerable patenting activity. In the present article, we investigated plant-made pharmaceutical patents between the years 2002 and 2008. There was a clear downward trend in the number of patents filed between these years and a greater number of patents were filed by public sector institutions or inventors than by the private sector. The USA dominated patenting activity providing nearly 30% of inventors. The majority of patents were for vaccine candidates (55%), followed by therapeutics (38%) and antibodies (7%). The relationship of patenting to innovation and access to medicines, particularly in the developing world, will be discussed.

Social leverage of intellectual property: road to the development of better therapy for tuberculosis.

Current TB drug development is beset with many problems. There is a perceived lack of commercial return on investment, as the vast majority of TB patients come from impoverished areas of the world. Clinical trials for new TB drugs are complex, protracted and very expensive. Therefore, the development of new anti-tuberculosis drugs requires simultaneous forward planning of the design of the trials that will be required for licensing purposes. In this article we briefly review the current state of new TB drug development and discuss issues related to intellectual property (IP), with a special emphasis on how IP can facilitate rather than hinder the development of better TB drugs. We also list and discuss the major patent applications that underpin TB drugs that have entered prominent clinical trials and additional applications that were filed over the last five years for drugs resulting from basic upstream research.

Emerging aspects of Buruli ulcer.

Buruli ulcer, caused by the pathogen Mycobacterium ulcerans, is a major mycobacteriosis that affects people in scattered foci in the third world. It is amongst the most neglected of diseases in terms of primary healthcare strategies. However, this is changing as the World Health Organization launches a number of major global initiatives. Recent progress includes the unraveling of the genetic structure of the pathogen, examination of the mechanisms of virulence and the role of chemotherapy in disease treatment and prevention of recurrence, together with strategies aimed at reducing the economic burdens placed upon healthcare budgets of poorer nations. This review focuses upon the recent developments and the understanding of the disease, with particular focus on potential chemotherapy.

Buruli ulcer.

Buruli ulcer (Mycobacterium ulcerans) is an emerging disease. The mode of transmission is still unknown. Mycobacterium ulcerans has been detected (by polymerase chain reaction) in water and water insects. Extensive surgery is still the main treatment. Recognition and excision - of the early nodular stage - is effective. The toxin, a polyketide, causes immunosuppression with potent inhibition of monocytes, T cells and nuclear factor kappa-B activation.