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Transgenic Plants in Medicine and Vaccines

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Daria Ruginis

on 14 December 2015

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Transcript of Transgenic Plants in Medicine and Vaccines

Agrobacterium Plasmid
Agrobacteria Tumefaciens
Soil phytopathogen
Crown gal disease
T-DNA region
Virulence region
How do we protect the intellecutal property of plant based vaccines?
Transgenic Plants in Medicine and Vaccines
Use of transgenic plants to produce pharmaceutical protiens
Pharmaceutical proteins used to make vaccines and drugs
Plant-made Pharmaceuticals
Potential for plant based
edible vaccines
Potential for production of new pharmaceuticals to target disease diagnosis and treatment
Possibility of developing large quantities of drugs and vaccines for a fraction of the price
Pharmaceutical proteins obtained from plants are stable
Plant proteins are easier to purify and less labor intensive than animal based pharmaceuticals
Plants pharmaceuticals can be grown on an agricultural scale
In 2014 more than 19 000 people were infected with
the Ebola virus
As of December 19, 2014 – 7388 confirmed deaths due
to EVD
ZMapp is an antibody cocktail in which includes humanized mAbs
ZMapp production occurred mainly in 2 steps:
Mice were inoculated with Ebola virus and immunological memory cells of the mouse lymphocytes were harvested
Cloned genes containing memory cells related to Ebola were transferred to tobacco plants via
bacterial carrier
Ebola mAbs were cultured and purified
Possibilities are endless – can potentially be
applied to many diseases and viral infections
Anti-viral vaccines expressed in plants
Patent number: 5,612,487
Filing date: March 4, 1993
Oral Immunization by transgenic plants
Patent number: 5,679,880
Filing date: June 1st 1995
- invention directed to

transgenic plants
that contain

DNA sequences
which code for a colonization antigen, a virulence antigen, antigenic determinants thereof or fusion proteins thereof of pathogenic mircoganisms.
-these plants are produced by transforming plants with a
plant transformation vector
which contains a DNA sequence which codes for an antigen of a specific pathogenic microrganism
- the purpose of this invention is for elicting a
secretory immune response
and thus immunizing humans or animals against certain pathogens
Although there are many benefits, there are even more controversies
Details of Invention
Health Controversies
Process cont.
Effects cannot be accuratrely predicted
Possible allergic reactions
Possible contamination from food supplies
Possibility of transferring antibiotic resistance into human microbiota
increasing suseptibility to diseases
Possiblity of introducing a gene into a plant that causes the plant to produce toxins
Legal and Regulatory Implications
A transgenic plant
, comprising and expressing a
DNA sequence for an antigen
of a pathogenic microogranism or an antigenic determinant therof, said antigen or antigenic determinant thereof
eliciting a secretory immune response in a human
or other animal upon
oral administration of tissue of said plant.
Why we shouldn't worry
Likelihood that consumers will eat contaminated food crops is very low
techniques have evolved to reduce contamination
Chances of active drug compounds being found in final food product at high levels is very small
Pharmaceuticals are pooled into a large harvest – dilutes pharmaceuticals
Chances of harmful effects occurring is not zero – but odds are low
Benefit of producing pharmaceuticals to target diseases and symptoms outweigh the costs
Environmental Controversies
Effects cannot be accurately predicted
Fear of spreading unwanted compounds into the air, water, soil, and food
Potential bioaccumulation of toxins in the environment
Contamination of food crops may result in increased pesticide and herbicide use
Possibility for the introduction of an invasive species into the environment
Possibility of affecting non target organisms
Invasive species
KAILUA – KONA, Hawaii, August 3, 2005 – residents suing for environmental effects caused by genetically modified algae
Kailua has many popular camping grounds, surfing locations, beaches and is home to many
endangered species
U.S department of Agriculture (USDA)
permitted Monsanto, ProdiGene, Garst Seed and the Hawai’i Agricultural research center to develop experimental drug products
Was determined that regulatory oversight was inadequate
USDA failed to record locations of field trial sites and did not conduct inspections
Improper documentation of storage locations and concealed information
Ethical Controversies
Some believe genetic modification goes against the natural workings of nature
“Genetic modifications takes mankind into realms that belongs to God and God alone”
People are not willing to accept genetic modifications without knowing all the possible effects
Genetic manipulation of plants and crops has been happening for decades
Shift in thinking needs to occur for plant based pharmaceuticals to advance
5.The plant of claim 1 wherein the antigen is selected from a group consisting of
SpaA, GtfB, dextranase, K88, K99, LT-B or an antigen determinant thereof
The present invention includes...
plant seeds and plant tissue
capable of expressing an antigen or determinant of a pathogen
that can stimulate a secretory immune response in humans or animals
methods for stimulating a secretory immune response
in humans and other animals so as to inhibit conlonzation through muscosal surfaces by pathogens
unique vectors containing DNA sequence
coding for conlization or virulence of antigens
a method
of producing colonization or virulence antigens of pathogenic mircoorganisms in plants

Plant transformation vectors
- the vector produced will depend on which type of plant and type of plant tissue
- a Ti plasmid-derived vectors are one possibilty
- a chimeric DNA sequence is constructed with the proper promoter and antigen coding DNA sequence
Media Perceptions
Vector construction
Description of Invention
Oral Immunization by transgenic Plants
Patent number: CA 1339307
Filing date: September 5th 1989
A method of making a composition suitable for eliciting a secretory immune response
in a human or other animal which comprises a transgenic plant or tisse obtained from a
transgenic plant
, and a suitbale carrier, wherein said transgenic plant expresses a DNA sequence coding for an antigen of a pathogenic microorganism or virus; comprising mixing said
transgenic plant tissue with a food subtance
What stakeholders are involved in the regulation and safety of GMO plants for medicinal purposes?
World Health Organization
- held informal consultation on Scientific basis for regulatory evaluation of candidate human vaccines from plants (January 24-25, 2005)

- concluded that GMO plants used for medicinal purposes should:
1. Be produced under the
national regulatory authority
of the country in which it is produced
Be genetically stable,
be consistently produced and have proper monitoring and control
3. Be
produced under ISO guidelines
following standard good manufaturng practices (GMP), with the addition of standard farming practices
4. Should follow the
same approval process of other vaccines
including pre-clinical trials ,inverstgation new drug applications, clinical trial, licensing and post-marketing survelliance.
Canadian Food Inspection Agency
- has a Plant Biosafety Office (PBO)
-regulates plants with novel traits used for the production of pharamceuticals
- before a plant with novel traits can be grown in Canada, the developer must present it to the PBO
-the PBO does a science-based environmental safety assessment before giving approval or denial
Health Canada
United States Department of Agriculture
Federal Drug Administration
Guidance document - Plant Molecular Farming Applications
1. Plant-derived biologic drugs are subject to the
same general submission requirements
obtaining market authorization as other biologic drugs.

2. Health Canada's evaluation of plant-derived biologic drugs is dependent on
information that addresses risks unique to PMF production platforms

3.Plant-derived biologic drugs shall meet the s
ame quality and safety standards
as conventional drugs

4.Health Canada is committed to strive for
regulatory convergence with other regulatory authorities internationally
Regulation of plant-derived biologic drugs in Canada
Health Canada
- oversees safety, efficacy and quality of drug under the Food and Drug Act
- interested in the
drug derived from the plant
Environment Canada
-responsible for t
he New Substance Program
, that oversees the environmental and indirect human health assessment of the drug
-interested in the
production organism
(plant cell line)
Canadian Food Inspection Agency
oversees the plant with novel traits
that are planted in the environment. Enforced the Seeds Act and Seed Regulations or the Plant Protection Act and Plant Protection Regulations
- interested in
the whole plant
17. The use of claim 14 wherein the pathogenic microorganism or virus is selected from the group consisting of Actinomyces...Bacillus...Bordetella...Samonelle...Giardia...
- US: only 26 claims, only looks at Streptoccocus mutan or Escherichia coli.
- Canada: 64 claims, multiple pathogenic microogranisms mentioned
- the present invention is directed to a respiratory syncytial virus (RSV) vaccine that overcomes some of the deficiencies of conventional RSV vaccines.

- the present invention is directed to plant-dervied RSV vaccines developed by introducing RSV nucleotide sequences into transgenic plants

1. A method for
eliciting a Th1 immune response
in a subject comprising
orally administering to a subject
in need thereof an edible portion of a plant comprising a chimeric nucleic acid construct comprising a nucleic acid molecule encoding
an antigenic respiratory syncytial virus (RSV)-F protein
that induces RSV-F-specific serum IgG and IgA and RSV-F-specific mucosal IgA,
- It is possible to prevent the initial infection of mucosal surfaces by stimulating production of mucosal sIgA levels directed against an infecting organism

- Traditional vaccines elicite protective serum IgG and delayed type hypersensitivity reactions against organisms

- However, traditional vaccines are not effective at eliciting mucosal sIgA responses.
- the expressed RSV-F protein is recognized specifically by the monoclonal antibodies raised against authentic F-protein.
-leaves from 3 week old apple shoots were used to isolate protoplasts

- Protoplasts were transfected with chimeric nucleic acid construct DNA

- an ELISA assay was used to detect if a muscosal response was ellicited

- monoclonal antibody against the RSV-F protein was added to each ELISA well

- chimeric nucleic acid constructs containing the
RSV-F coding sequence
were generated to produce F-protein expression in
apple leaf mesophyllprotoplasts

- the DNA of interest was transferred into the plant using an
medium to the target plant cell where it integrates into the plant genome.
Process Cont.
The Animal and Plant Health Inspection Service
is responsible for protecting agriculture

- they have control over (handling, import, release) organisms that have been genetically altered

- individuals must provide a petition to the APHIS department to have a genetically engineering plant approved

In laboratory
- all agencies stress the importance of GMP and GLP in all medical laboratories

- ISO/IEC 17025 – General requirements for the competence of testing and calibration laboratories

- ISO 15189 Medical Laboratories

- pSUN341 and pSUN343 vectors used to successfully transform plants with DNA sequences encoding for antigen
- vectors were inserted through either direct gene transfer or an agrobacterium intermediate
Antigen selection
-surface protien antigen A (SpaA) is a major protein antigen found on S.mutans
- humans and animals immunized against S.mutans produce IgA against SpaA protein
Process cont.
Heritability of the ability to produce SpaA
- SpaA producing tobacco plants were made and permitted to form seeds
- SpaA positive plants were determined to be homozygous or heterozygous for the SpaA producing trait
Processing of transgenic plant material for use in animal feed
- leaves from SpaA plants were cut, and dried at 37 degree for two days
- plant mixture was inserted into mouse meal at different dosages
Immunogenicity of SpaA Protein Expressed in Plants
- This diet is fed to female mice, 9 to 10 weeks old. Mice were recorded visually to determine their health status
- Saliva samples are collected weekly.
- Serum anti-SpaA IgG and salivary IgA are detected by ELISA
- plant based medicines are under much the same regulation as regular medicines
- Invesigational New Drug Applications
- Biologic License Applications
- pre-clinical trials, clincial trials
- New Drug Applications
- Plant Breeders’ Rights Act protects species of plant varieties. A plant variety must be “
distinguishable from other varieties, stable in its essential characteristics and sufficiently homogenous.”

-A plant breeders’ right-holder is granted the exclusive rights to to sell and produce propagating material of a plant variety in Canada
- created in response to the 1989 Pioneer Hi-Bred Ltd. vs. Canada decision of the Supreme Court of Canada which
decided plants are not patentable in Canada
Legal Issues
- being a new technology there is little in terms of plant dervied medicinal cases to date
-however anything with transgenic plants is subject to similar legal implications
Monsanto Canada Inc. v. Schmeiser, [2004]
- Roundup Ready Canola, a Monsanto Invention was introduced to Canada in 1996
- Percy Schmeiser never bought Round up Ready Canola seeds however through contamination he found that some of his plants were Roundup-resistant
- He cultivated these seeds, but soon was discovered by Monsanto

"The patent is valid. The respondents did not claim protection for the genetically modified plant itself, but rather for the genes and the modified cells that make up the plant. "
Risk Assessment
Pioneer Hi-Bred Ltd. v. Canada (Commissioner of Patents), [1989]
- "An application was filed to obtain a patent for a new soybean variety developed from artificial cross-breeding but cultivated naturally."
- The Patent Office Examiner
rejected the application

There was no indication of the genetic engineering involved so that even someone skilled in the science of the invention could not arrive at the same result as the inventor without further explanation. "
- since plant based medicines are a fairly new technology, there is little data on the long term effects
- however these plant based vaccines and medicines share many of the same concerns as GMO crops
FDA has been hesitant to approve plant based pharmaceuticals
Some believe plant based pharmaceuticals can be the next big thing
Most scientists agree genetically modified organisms and plants in particular are safe
Others believe benefits and over exaggerated and effects are under exaggerated
Transgenic Plants
Genetically modified plants
Ability to express recombinant proteins such as antigens and antibodies
Biomonitors/ Bioreactors
Current Uses
Microparticle Bombardment
Other Direct Methods

Method of using electric fields to permeabilize the plasma membrane, which allows macroparticles too diffuse through
Potential for Exposure
Routes of exposure
over-exposure or under-exposure through improper administered dose, or through second hand exposure (contaminated food supply or handling)
GMP requirements upheld in laboratories
Production of Transgenic Plants
unintentional exposure through oral, nasal or dermal contact causing adverse effects
assessment of the LOEC and all companies must follow GMP or face fines
Risk Managment
- plant based medicines have both pros and cons
-proper management and production can ensure safety of these substances:
precautionary principle
must be applied
- application of
GLP and GMP practices
- reworking of
of regulatory agencies to better address food grade manufacturing and pharamceuticals
Finding Balance
- for a new technology to move forward the pros must outweigh the risks
Current vaccines
- contaminated syringes
- fear of injection
- mercury based- preservatives
Plant-based medicine
- could contaminate food crops
- could be expressed in non-food plants
-heat stability
1) Agrobacterium binds to the surface receptors located on the host cell
2) Signal Transduction occurs
3) Transcription Activation occurs
4) DNA metabolism
5) Intercellular transport occurs
6) T-DNA complex gets imported into nucleus
7) T-DNA gets integrated into the plant cell genome

Gene Gun: Biolistic PDS- 1000/ HeParticle Delivery System
Biolistic Method
Microprojectiles: particles covered with DNA coating
Tungsten or Gold most used
Driven by acceleration
Via pressure of gas or gunpowder explosion
Used to differentiate between real transgenic plants and mutant transgenic plants
Observations of "gene specific band"
Purpose: Determining effectiveness and functionality of the transgene
GUS assay

Southern Blot Analysis
Used to confirm the presence of transgenes by determining copy number and degree of homoplasmy of the transgene
DNA digested by restriction enzymes
Nictrocellulose or Nylon filter

Analysis of Gene Expression

PMP ... An ongoing research
Example of successful PMP
Taliglucerase alfa (Elelyso)
Transgenic Carrots
Ability to express enzyme :Glucocerebrosidase
Drug used to treat Gaucher’s disease
Company: Protalix and Pfizer
Approved by FDA and in the market since 2012
Health Canada approved drug in 2014
Marker-Free Transgenic Plants
Absence of selective genes of antibody and herbicide resistance
Increasing biosafety of edible vaccine

Solanum tuberosum  Transgenic potato marker-free plant
Plant transformation via Agrobacterial transformation
PCR screening for HBsAg gene
Monoclonal ELISA used for quantification of HBsAgs in the transgenic potato
Blot analysis using electrophoresis and MiniTrans-blot apparatus
Marker-Free Transgenic Plants (Cont.)
Immunization of mice
Two groups of 10 mice
Group 1: experimental group that was fed transgenic potato plant (Days 1, 7, 14)
Group 2: Control group that was fed untransformed potato
After 71 days, group 1 were injected with the hepatitis B vaccine
Blood samples were taken of all mice to analyze the presence of antibodies against HBsAg using ELISA

Pipeline: Breast Cancer Drug
Trastuzumab: monoclonal antibody which disrupts the growth of breast cancer
Current drug: Herceptin
Transgenic Tobacco
University of Guelph
2013 - conducted phase 1 clinical trials
2014 - 2015 - currently conducting phase 2 clinical trials
2016 - Conduct phase 3 clinical trials
3 patent families filed
Clinical trial application submitted after phase 3
Aimed to release drug by 2018
Breast Cancer Drug
Helios Gene Gun
Tritech Research microinjector
Plant Form
"A Canadian biotech company with an innovative, low cost manufacturing system for a wide range of plant-made pharmaceuticals"
Federally funded
Research pipe line includes:
breast-cancer drugs
antibodies for HIV/AIDS
antibodies for Ebola virus
protection against nerve agents
"Focused on developing recombinant therapeutic proteins for large, established pharmaceutical markets."
Producers of ELELYSO
Partnered with Pfizer
Research pipeline includes producing transgenic proteins and antibodies addressing:
Fabry disease
Immune and inflammatory diseases
Cystic fibrosis
Gaucher disease

MappBio (USA)
"Develops novel pharmaceuticals for the prevention and treatment of infectious diseases, focusing on unmet needs in global health and biodefence."
Producers of Zmapp
Antibodies produced in
Used to treat Ebola
Has been granted Fast Track Status by the FDA

Other Projects
University Studies
University of Arizona
University of Washington
Privately Owned Biotechnology Companies
Planet Biotechnology (USA)
Defyrus Inc. (CA)

The industry is just scratching the surface of potential projects!
MLDC Tech's Blue Banana
Agrobacterium mediated transformation
Expression of hepatitis B surface antigen
Presence identified and confirmed by PCR and Southern Blot Analysis
ELISA assay used to detect and confirm antigen activity
Patented gene sequence and growth techniques in the name of MLDC Technologies
Well-researched safety precautions
Green house growth conditions
In 2003, ProdiGene went under because of two separate cases of cross contamination
Antimicrobial-free markers
Ready for Clinical Trials!

Production of the Blue Banana
Clinical Trials
Greenhouse Expansion
2 Marketing Schemes
1) Targets those with aversion to needles
Oral vaccine reduces anxiety
2) Targets those afraid of GMOs
Advertising the trials and tribulations of the production process
Press releases at each stage of clinical trials
Educating consumers

Where your Money is Going
Dragons! Will you invest?
Full transcript