Biotechnology and Biotechnology and Bioinformatics: Bioinformatics: MedicineMedicineEssential Idea:Essential Idea: Biotechnology can be Biotechnology can be used in the diagnosis and treatment of used in the diagnosis and treatment of disease.disease.

TOKTOK

There have been cases around the world where There have been cases around the world where subjects have died as a consequence of subjects have died as a consequence of participating in a gene therapy research participating in a gene therapy research protocol. How is the decision to proceed with protocol. How is the decision to proceed with risky procedures made?risky procedures made?

What constitutes an acceptable level of risk for What constitutes an acceptable level of risk for allowing humans to be involved in scientific allowing humans to be involved in scientific research?research?

ELISA TestsELISA TestsPathogens can be detected Pathogens can be detected using a variety of molecular using a variety of molecular techniques. techniques.

One popular method is to One popular method is to use an ELISA test. “ELISA” use an ELISA test. “ELISA” stands for stands for EEnzyme nzyme LLinked inked IImmunommunossorbant orbant AAssay.ssay.

This method allows for rapid This method allows for rapid detection of a specific detection of a specific disease that allows for quick disease that allows for quick treatment of an affected treatment of an affected individual.individual.

www.bio-rad.com

ELISA TestELISA Test

ELISA tests involve ELISA tests involve multiple steps:multiple steps:

In the first step, In the first step, the sample in the sample in question is placed question is placed into a sample well into a sample well plate and then plate and then washed out. Some washed out. Some of the sample will of the sample will adhere to the sides adhere to the sides of the well plate.of the well plate.

http://technologyinscience.blogspot.com/2011/12/elisa-protocol-types-of-elisa.html#.VgtFsrRVhBc

A primary antibody A primary antibody is then added to is then added to the well plate the well plate which will bind to which will bind to any of the antigens any of the antigens in the original in the original sample.sample.

ELISA TestELISA Test

http://technologyinscience.blogspot.com/2011/12/elisa-protocol-types-of-elisa.html#.VgtFsrRVhBc

ELISA TestELISA TestThe wells are then The wells are then washed again.washed again.

A secondary antibody A secondary antibody which has an enzyme which has an enzyme attached to it is then attached to it is then added to the well added to the well plate. plate.

The secondary The secondary antibody (along with antibody (along with the enzyme) will then the enzyme) will then bind to the primary bind to the primary antibody which was antibody which was bound to the antigen.bound to the antigen.

http://technologyinscience.blogspot.com/2011/12/elisa-protocol-types-of-elisa.html#.VgtFsrRVhBc

An enzyme An enzyme substrate is then substrate is then added to the added to the wells. This wells. This substrate will substrate will react with any react with any enzyme present enzyme present and indicate a and indicate a positive result positive result from the original from the original sample.sample.

ELISA TestELISA Test

http://technologyinscience.blogspot.com/2011/12/elisa-protocol-types-of-elisa.html#.VgtFsrRVhBc

MetabolomicsMetabolomics

Metabolomics is a Metabolomics is a rapidly expanding field rapidly expanding field of study dedicated to of study dedicated to the detection of the detection of metabolites in metabolites in biological systems.biological systems.

Metabolites that Metabolites that indicate disease can indicate disease can be detected in blood be detected in blood and urine.and urine.

http://www.intechopen.com/books/calcific-aortic-valve-disease/proteomics-and-metabolomics-in-aortic-stenosis-studying-healthy-valves-for-a-better-understanding-of

MetabolomicsMetabolomics

For instance, urine samples can be used to For instance, urine samples can be used to detect breast and ovarian cancer.detect breast and ovarian cancer.

If these cancers are detected at an early stage, If these cancers are detected at an early stage, the 5-year survivability rates exceed 90%.the 5-year survivability rates exceed 90%.

http://www.metabolon.com/technology/about-metabolomics.aspx

MetabolomicsMetabolomics

PKU is a metabolic disorder that results in PKU is a metabolic disorder that results in severe intellectual disability caused by severe intellectual disability caused by elevated serum phenylalanine levels.elevated serum phenylalanine levels.

The primary cause is phenylalanine The primary cause is phenylalanine hydroxylase deficiency.hydroxylase deficiency.

Blood tests can be used to detect Blood tests can be used to detect phenylalanine levels, and appropriate clinical phenylalanine levels, and appropriate clinical steps can then be taken to manage the steps can then be taken to manage the disease.disease.

Marker GenesMarker Genes

Some people are predisposed to a genetic Some people are predisposed to a genetic disease based upon the whether or not a disease based upon the whether or not a family member has been afflicted with the family member has been afflicted with the disease.disease.

Tests can be run to determine whether or not Tests can be run to determine whether or not you will inherit the disease.you will inherit the disease.

These tests look for genetic markers.These tests look for genetic markers.

Marker GenesMarker Genes

For instance, Huntington’s disease is a For instance, Huntington’s disease is a neurodegenerative disorder that affects some neurodegenerative disorder that affects some people in mid-life.people in mid-life.

Those who get it usually die within a few years Those who get it usually die within a few years of diagnosis.of diagnosis.

It is a dominant gene. Thus, if a mother or It is a dominant gene. Thus, if a mother or father have it, you have a 50/50 chance of father have it, you have a 50/50 chance of inheriting it.inheriting it.

Marker GenesMarker Genes

Tests can be run to look for markers in the Tests can be run to look for markers in the genome to determine the likelihood of genome to determine the likelihood of developing the disease.developing the disease.

DNA MicroarraysDNA Microarrays

DNA microarrays DNA microarrays enable scientists enable scientists to study multiple to study multiple genes in one genes in one experiment.experiment.

This is very useful This is very useful due to the due to the tremendous tremendous numbers of genes numbers of genes found within found within organisms.organisms.

https://commons.wikimedia.org/wiki/File:DNA_microarray.svg

DNA MicroarraysDNA Microarrays

DNA microarrays have many DNA microarrays have many uses, but some of the uses, but some of the common ones include: common ones include:

Measuring changes in the Measuring changes in the expression levels of expression levels of genes (expression genes (expression analysis).analysis).

Detecting single Detecting single nucleotide nucleotide polymorphisms.polymorphisms.

Determining the Determining the genotype of an organism.genotype of an organism.

https://mla-lima.wikispaces.com/Southern+Blotting+and+DNA+Microarray

DNA MicroarraysDNA MicroarraysFor example, when gene For example, when gene expression is being expression is being monitored with a monitored with a microarray, thousands of microarray, thousands of genes can be genes can be simultaneously monitored.simultaneously monitored.

The results can be used to The results can be used to compare the level of gene compare the level of gene expression in diseased expression in diseased tissue to that of a normal tissue to that of a normal tissue.tissue.

This can help to improve This can help to improve treatment methods/options.treatment methods/options.

http://cisncancer.org/research/how_cancer_is_studied/background/research_technology_02.html

DNA MicroarraysDNA Microarrays

DNA microarrays can also be used to detect DNA microarrays can also be used to detect predisposition to disease. predisposition to disease.

The information gathered can be used to The information gathered can be used to inform individuals about environmental factors inform individuals about environmental factors that may trigger certain genetic diseases.that may trigger certain genetic diseases.

PCR UsesPCR Uses

PCR can be used to detect PCR can be used to detect a wide variety of a wide variety of biomolecules.biomolecules.

It can be used in crime It can be used in crime scene analysis, tissue scene analysis, tissue typing, and disease typing, and disease outbreak/diagnosis, outbreak/diagnosis, among many other things.among many other things.

We used it in class to We used it in class to detect the presence of detect the presence of GMOs in foods.GMOs in foods. www.thermofisher.com

PCR and Influenza PCR and Influenza DetectionDetection

PCR is widely used to detect and track new PCR is widely used to detect and track new strains of influenza.strains of influenza.

Due to the rapid, sensitive, and specific assay Due to the rapid, sensitive, and specific assay that is followed, governmental agencies are that is followed, governmental agencies are given the opportunity to quickly respond to given the opportunity to quickly respond to emerging threats, and take the appropriate emerging threats, and take the appropriate measures to protect citizens.measures to protect citizens.

http://path.upmc.edu/virology/s-01.htm

Tumor Cell TrackingTumor Cell TrackingTracking experiments are used Tracking experiments are used by researchers to gain by researchers to gain information about proteins, information about proteins, their interaction within the their interaction within the cellular environment, and what cellular environment, and what the localization of these the localization of these proteins within the cell can tell proteins within the cell can tell them.them.

Advances in biotechnology have Advances in biotechnology have also enabled researchers to use also enabled researchers to use tracking experiments to track tracking experiments to track tumor cells using transferrin tumor cells using transferrin linked to luminescent probes.linked to luminescent probes.

https://www.mskcc.org/blog/new-imaging-agent-could-improve-prostate-diagnosis-and-treatment

Tumor Cell TrackingTumor Cell Tracking

Transferrin is an iron-Transferrin is an iron-binding blood plasma binding blood plasma glycoprotein normally glycoprotein normally present in the present in the bloodstream and binds bloodstream and binds to fast-growing cells to fast-growing cells such as tumor cells (as such as tumor cells (as well as fast dividing well as fast dividing normal cells). normal cells).

Its main job is to control Its main job is to control the level of free iron in the level of free iron in body fluids.body fluids. http://biologie.ens-lyon.fr/ressources/bibliographies/m1-12-13-biosci-reviews-curis-c-1c-

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Tumor Cell TrackingTumor Cell TrackingScientists are using PET Scientists are using PET scans to track transferrin scans to track transferrin uptake in tumor cells.uptake in tumor cells.

With this strategy, they can With this strategy, they can assess the speed at which assess the speed at which the iron is taken up by the the iron is taken up by the cells to monitor how rapidly cells to monitor how rapidly the tumor may be growing.the tumor may be growing.

This allows for early disease This allows for early disease detection before any tumor detection before any tumor growth can be seen with growth can be seen with conventional imaging conventional imaging techniques.techniques.

https://www.mskcc.org/blog/new-imaging-agent-could-improve-prostate-diagnosis-and-treatment

Tumor Cell TrackingTumor Cell TrackingHere’s how it works:Here’s how it works:

Scientists have developed a way to couple Scientists have developed a way to couple transferrin to a radionuclide called zirconium-89.transferrin to a radionuclide called zirconium-89.

Transferrin is rapidly taken up by actively Transferrin is rapidly taken up by actively dividing cells, and zirconium-89 is easily dividing cells, and zirconium-89 is easily detected by a PET scan.detected by a PET scan.

http://www.nature.com/nm/journal/v18/n10/full/nm.2935.html?WT.ec_id=NM-201210

Tumor Cell TrackingTumor Cell Tracking

These two tools produce These two tools produce very high quality images very high quality images that enable researchers that enable researchers to better understand and to better understand and design treatments for design treatments for cancer patients.cancer patients.

QuickTime™ and aGIF decompressor

are needed to see this picture.

https://en.wikipedia.org/wiki/Positron_emission_tomography

Gene Therapy and Viral Gene Therapy and Viral VectorsVectors

Viral vectors are used in gene therapy.Viral vectors are used in gene therapy.

Gene therapy is a method of inserting a gene into Gene therapy is a method of inserting a gene into an organism that does not have it.an organism that does not have it.

The gene must be inserted into a spot on the The gene must be inserted into a spot on the chromosome where it will be actively expressed.chromosome where it will be actively expressed.

The gene must also be inserted into a cell that can The gene must also be inserted into a cell that can be transplanted into the organism where it can be transplanted into the organism where it can proliferate and take up long term residence. proliferate and take up long term residence.

SCID (severe combined SCID (severe combined immunodeficiency) is a immunodeficiency) is a disease in which the disease in which the patient cannot make patient cannot make antibodies nor can antibodies nor can mount a cell-mediated mount a cell-mediated response to an antigen.response to an antigen.

It is a disease of young It is a disease of young children because most children because most children get infected children get infected with an illness that kills with an illness that kills them.them.

SCIDSCID

http://labs.pathology.med.nyu.edu/feske-lab/research/immunodeficiency

SCIDSCIDAbout 25% of all About 25% of all patients are the result patients are the result of being homozygous of being homozygous recessive for a recessive for a defective gene coding defective gene coding for adenosine for adenosine deaminase.deaminase.

This faulty purine This faulty purine metabolism creates a metabolism creates a situation that is toxic to situation that is toxic to T and B cells--cells T and B cells--cells needed for proper needed for proper immune system immune system function.function.

http://primaryimmune.org/about-primary-immunodeficiencies/relevant-info/inheritance/

SCID Treatment OptionsSCID Treatment Options

Raise the child in a Raise the child in a bubble.bubble.

Regular bone marrow Regular bone marrow transplants.transplants.

This can pose problems This can pose problems if the donor is infected.if the donor is infected.

Regular injections of ADA Regular injections of ADA (adenosine deaminase).(adenosine deaminase).

Insertion of ADA genes Insertion of ADA genes with a retrovirus as a with a retrovirus as a form of gene therapy.form of gene therapy.

http://www.worldmag.com/2014/12/with_breakthrough_parents_can_finally_hug_and_kiss_their_bubble_babies

Gene Therapy for SCID--Gene Therapy for SCID--How it Works.How it Works.

First you need to identify and clone a normal copy First you need to identify and clone a normal copy of the ADA gene.of the ADA gene.

The gene then needs to be inserted into the DNA The gene then needs to be inserted into the DNA of one of the patient’s own cells. This is done with of one of the patient’s own cells. This is done with a retrovirus and reverse transcriptase. a retrovirus and reverse transcriptase.

The gene needs to be inserted into a spot where it will be The gene needs to be inserted into a spot where it will be adequately expressed (transcribed and translated) thus adequately expressed (transcribed and translated) thus producing reasonable amounts of enzyme.producing reasonable amounts of enzyme.

Treating the cells in tissue culture so they Treating the cells in tissue culture so they multiply, and then returning them to the patient.multiply, and then returning them to the patient.

Gene Therapy for SCIDGene Therapy for SCIDTo get the DNA into the To get the DNA into the cell, a retrovirus is used cell, a retrovirus is used as the vector. This as the vector. This makes use of an makes use of an envelope protein envelope protein enabling the virus to enabling the virus to infect the human cell.infect the human cell.

RNA copies of the RNA copies of the human ADA gene can human ADA gene can then be incorporated then be incorporated into the retroviral into the retroviral genome using a genome using a packaging cell.packaging cell.

http://learn.genetics.utah.edu/content/genetherapy/gtsuccess/

Gene Therapy for SCIDGene Therapy for SCIDPackaging cells are: Packaging cells are:

cells that express an RNA copy of the (ADA) cells that express an RNA copy of the (ADA) gene, gene,

cells that have an RNA copy of the retroviral cells that have an RNA copy of the retroviral genes to assist in the copying of the genes genes to assist in the copying of the genes which need to be expressed,which need to be expressed,

cells that contain inverted repeats to assist cells that contain inverted repeats to assist with the insertion of the DNA into the DNA of with the insertion of the DNA into the DNA of the target cell.the target cell.

Packaging cells lack the genes Packaging cells lack the genes necessary for critical viral necessary for critical viral replication.replication.

They become proviruses that They become proviruses that replicate as the cell divides.replicate as the cell divides.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GeneTherapy.html

Gene Therapy for SCIDGene Therapy for SCID

Retroviruses are often altered Retroviruses are often altered and used for gene therapy.and used for gene therapy.

Researchers insert the Researchers insert the desired gene(s) into the virus desired gene(s) into the virus and then remove the coding and then remove the coding regions for the genes needed regions for the genes needed by the virus to replicate itself. by the virus to replicate itself.

Thus, these viruses are Thus, these viruses are capable of infecting their capable of infecting their target cell, but are unable to target cell, but are unable to continue their destructive life continue their destructive life cycle.cycle.

http://learn.genetics.utah.edu/content/genetherapy/gtsuccess/

Gene Therapy for SCIDGene Therapy for SCIDIf the gene is If the gene is inserted correctly, inserted correctly, in this case the in this case the ADA gene, the ADA gene, the cells begin cells begin synthesizing the synthesizing the enzyme needed to enzyme needed to produce adenosine produce adenosine deaminase and the deaminase and the patient’s immune patient’s immune system begins to system begins to function normally.function normally.

http://www.mun.ca/biology/scarr/Somatic_Therapy_for_SCID.htm

Gene Therapy for SCIDGene Therapy for SCID

Gene therapy for SCID involves some risks:Gene therapy for SCID involves some risks:

4 of the 10 patients undergoing SCID gene therapy 4 of the 10 patients undergoing SCID gene therapy developed leukemia as a result of the ADA gene being developed leukemia as a result of the ADA gene being inserted near an oncogene.inserted near an oncogene.

2 of the 4 patients were successfully treated.2 of the 4 patients were successfully treated.

Other methods of gene insertion have also Other methods of gene insertion have also been explored.been explored.

Using adeno-Using adeno-associated viruses, associated viruses, for example, have for example, have been shown to be been shown to be much safer because much safer because they always integrate they always integrate in the same spot on a in the same spot on a chromosome and are chromosome and are designed to insert in designed to insert in spots not known to spots not known to cause problems. cause problems.

Gene Therapy for SCIDGene Therapy for SCID

https://en.wikipedia.org/wiki/Gene_therapyhttps://en.wikipedia.org/wiki/Gene_therapy