PHAR 813 Exam 4 Analytical methods biomarkers, cost-effectiveness, FDA regulations and role of PharmD

DNA isolation

Removal and separation of DNA by

1. Cell Lysis

2. Precipitation

3. Wash

4. Resuspension

Denaturation

Separation of DNA strands (94-96 ₒC)

Annealing

Primers will bind to the DNA (68 ₒC)

Elongation

Taq polymperase synthesizes new DNA (this polymerase is able to "work" at high temperatures) (72 ₒC)

Polymerase chain reaction (PCR)/Genotyping

Goal: Amplify specific fragments of DNA, This technique is used for: Genetic testing (Genotyping), Tissue typing, Detection of infectious disease organisms, DNA cloning

Limitation of genotyping?

We need primers to amplify a specific part of the DNA....what would happen if your mutation is on the place that the primer is binding? The primer won't be able to help amplifying that part, and obtain a clear genotype

DNA sequencing

Bases are labeled with fluorescent dye, color coded for each base, and polymerase reaction will produce the products after introduction of primers (to produce more DNA product)

Restriction enzyme digestion Goal

To study selective regions of the DNA, Restriction enzymes will recognize specific sequences and cut.

Restriction Enzyme Purpose

Genetic marker, DNA fingerprinting

Electrophoresis

Gel, Run after digestion, samples are run through. (Remember DNA has phosphate groups) Small fragments will run longer, while long fragments will run less quickly

DNA cloning

Goal: Make identical copies of DNA and introduce them into a bacteria

Transformation

The mechanism of introducing foreign DNA into a bacteria

How can bacteria be used in DNA Cloning?

Produce the protein encoded by the inserted gene

Applications of DNA Cloning

Generation of recombinant proteins (growth hormone, insulin...), Gene therapy, Gene analysis (study the effect of a mutation on the phenotype)

RT-PCR

real time polymerase chain reaction, Goal: What if we cannot get the DNA but we could isolate the RNA?, or if we want to know which genes are actually expressed (not that you have the gene or not)?

RT-PCR Applications

Measure gene expression (example, effect of a drug on DNA expression over different time points), Diagnosis, Biomarker

RT-PCR Limitation

From mRNA we can only get information about the exosome (exons) not introns/promoters..

Microarrays / chip hybridization

Goal: To compare many genes at the same time.

Sometimes will also help to compare genes among different populations. Different samples are labeled differenlty and we can compare which genes were over-expressed vs. down-regulated. (Imagine doing many PCRs at the same time using different primers looking at particular SNPs/genes)

Microarray/Chip Hybridization Limitation

What do you consider control??

Purpose Doctor prescribed PGx

Ordered by a prescriber- to guide therapy

Purpose Direct to Consumer PGx

Requested by patient w/o provider for personal curiosity, ancestry or wellness

Clinical utility Doctor prescribed PGx

The findings of the test will be actionable - adjust treatments accordingly

Clinical utility Direct to Consumer PGx

The patient will need to show the results to the prescriber - interpretation

Validity Doctor prescribed PGx

Tests have to be validated- FDA approved.

Validity Direct to Consumer PGx

May include tests for polymorphisms that do not have any guidelines or action

Clinical integration Doctor prescribed PGx

EHR

Scope Doctor prescribed PGx

Dug-gene pairs

Scope Direct to Consumer PGx

Broad information

What is gene therapy?

Genes to treat or prevent disease.

Several approaches to gene therapy, includes

1. Replacing a mutated gene that causes disease with a healthy copy of the gene.

2. Inactivating, or "knocking out," a mutated gene that is functioning improperly.

3. Introducing a new gene into the body to help fight a disease.

Transgene

Therapeutic gene (gene w/o the mutation).

Episome

Non-integrated extrachromosomal DNA (circular) that may be replicated in the nucleus. (ex. Viruses-Herpes/EB and cancer)

"Sanctuary" or immune privileged tissues

Tolerate antigens w/o triggering inflammatory and immune response. These are: eyes, articular cartilage, placenta, fetus, testicles, CNS.

Modes of Gene Therapy

1. Germ Line 2. Somatic

Germ Line Gene Therapy

Germ cells are modified by the introduction of functional genes, which are integrated into their genome

Somatic Gene Therapy

Genes are transferred into the somatic cells of a patient,

Is somatic cell gene therapy changes inheritable?

No

Is germ line gene therapy changes inheritable?

Yes

Techniques of gene therapy

1. Genetically modified cell-based immunotherapies

2. Viral Vectors

3. Gene editing

4. Non-viral vectors

What cells to gamma-retroviruses work on?

Dividing cells

What cells to lentiviruses work on?

Non-dividing cells, and dividing cells

Types of gene therapy

Ex vivo/In vivo

CAR T-Cell Therapy

Remove blood from the patient to get T cells, Make CAR T cells in lab, Grow millions of CAR T Cells, Infuse CAR T cells into patient, CAR T cells bind to cancer cells and kill them

The ethical questions surrounding gene therapy include

How can "good" and "bad" uses of gene therapy be distinguished? Who decides which traits are normal and which constitute a disability or disorder? Will the high costs of gene therapy make it available only to the wealthy? Could the widespread use of gene therapy make society less accepting of people who are different? Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?

CRISPR-Cas9

In USA, only allowed to be used for gene therapy, ONLY in somatic cells, not germ-line.

Monogenic Disease

Associated with the mutation of one specific gene/chromosome

Complex Disease

Multiple genes involved and Non-genetic factors (life style, environment)

Candidate gene approach

Choose genes that have a function that could be related to the phenotype of interest. Polymorphisms in and/or near the gene that may alter the protein or its expression were chosen. The variants were genotyped in a population and allele frequencies were compared. This method is more successful for single-gene diseases.

What drawbacks do you think of for a complex disease?

Failure to replicate in different populations.Lack of knowledge to know which gene is associated with the disease.

Genome-Wide Linkage Analysis

Genetic mapping, uncovering genes and genomic regions not previously known to be related to the disease in question. 200-400 polymorphic markers are fairly evenly spaced across the genome are selected. The markers are genotyped in different families with this disease. Statistical analysis and linkage analysis -> locus that may contain genes contributing to the disease.

How to identify potential SNPs linked with drug response?

Using extreme discordant phenotype approach

FDA regulations

Ensuring that doctors and patients have access to safe, accurate and reliable diagnostic tests to help guide treatment decisions is a priority for the FDA

"CLIA certified" (clinical laboratory improvement amendments)

Categories are based on the level of complexity on the techniques run in the lab and low or high risk for a false result.

CLIA Score 1

Indicates the lowest level of complexity

CLIA Score 3

Indicated the highest level of complexity

CLIA Total Scores Added

The 7 scores are added together and the tests with a score of 12 or less are categorized as moderate complexity, and those with a score above 12 are categorized as high complexity. The FDA will notify the sponsor—usually within two weeks of the marketing clearance or approval of their CLIA categorization.

CLIA Categorization

1. Knowledge

2. Training and experience

3. Reagents and materials preparation

4. Characteristics of operational steps

5. Calibration, quality control, and proficiency testing materials

6. Test system troubleshooting and equipment maintenance

7. Interpretation and judgment

Biomarkers Functions

Diagnosis, Prognosis, Prediction, Response

Biomarkers Utilities

Dose selection, patient selection, monitoring

Biomarkers Challenge

Limited size sample (few participants in first stages of the clinical trial) and they could be "false positive".

Sensitivity

The ability to detect those patients that truly have the condition.

Specificity

The ability to not falsely diagnose those who don't have the condition.

How to evaluate the potential cost-effectiveness of PGx testing?

Accuracy, Cost, Timliness, Prevalence, Penetrance, Prevalence and risk, Outcomes and economic impacts

Accuracy PGx testing cost-effectiveness

What are the specificity and sensitivity of the test for detecting the genetic variant of interest?

Cost PGx testing cost-effectiveness

Of the test and counseling

Timeliness PGx testing cost-effectiveness

What is the time frame for obtaining test results?

Gene

Prevalence PGx testing cost-effectiveness

How common is the genetic variant? How many patients would have to be tested to identify a patient with a variant? What are the positive and negative predictive powers of the test in a patient population?

Penetrance PGx testing cost-effectiveness

What is the relationship between genetic variant and drug response? What is the relative risk of an adverse event in a patient with a variant genotype vs. those without? What is the probability of drug response in patients with a variant genotype vs. those without?

Type 1 Error

False Positive

Type 2 Error

False Negative

Prevalence and risk PGx testing cost-effectiveness

How common is the drug-related AER that should be avoided? What is the difference in absolute risk? How common is drug nonresponse? What is the difference in variants vs. non variants?

Outcomes and economic impacts PGx testing cost-effectiveness (Outcomes)

How expensive is the AER or drug nonresponse? What is the impact of the AER or disease on quality of life?

Outcomes and economic impacts PGx testing cost-effectiveness (Treatment)

Is there a clear intervention based on the result of the PGx test? How effective is the intervention? What risks are associated with the intervention? What is the cost? What alternatives to individualized therapy are available other than PGx testing? What is the likelihood that treatment decisions suggested by test will be followed?

FDA Regulations PGx in package insert

1. Technology and knowledge

2. Drug development is more targeted

3. Drug labeling of Pharmacogenomic information

FDA Regulations: Technology and knowledge

Drug approvals are based on response in the specific population, but inter-individual variability exists leading to unwanted side effects in the form of both toxicity and/or lack of efficacy.

FDA Regulations: Drug development is more targeted

Drugs will be designed for a particular molecular target or populations with a particular metabolic status.

When is "targeted therapy" design more useful?

1. The target of the drug is variably expressed.

2. The disease has certain specific driving mutations.

3. A subgroup of patients has a different prognosis.

4. The metabolism of the drug is known to be via polymorphic pathway.

5. Diminished activity is observed in certain subjects in early phase studies.

How do they discover PGx markers after the drug is being approved/marketed?

Through retrospective approaches: 1. Studies indicate differential efficacy in a subgroup that is mechanistically supported by the drug's mechanism of action. 2. Substantial toxicity is observed in a certain subgroup.

FDA Regulations: Drug labeling of Pharmacogenomic information

The first genomic-based information to appear in drug labeling was in 1949 regarding glucose-6-phosphate dehydrogenase deficiency with the use of chloroquine.

The therapeutic areas with the most labeling containing genomic information

Oncology, Infectious diseases, Psychiatric disorders

Industry Pharmacogenomics Working Group (I-PWG)

Closely follows a variety of regulatory agencies and policy makers engaging and exchanging information about PGx

Preclinical Drug Discovery and PGx

Using cell lines and transgenic animals that lack a specific gene or have extra copies of the gene. ADME. In vitro and in vivo models to obtain insights into the drug metabolism and PK.

Disease research 1. Identifying subtypes of disease based on gene expression or imaging. 2. Identify biomarkers of response.

Clinical Drug Discovery and PGx

Identify the intrinsic (age, gender, body weight, renal function...) and extrinsic (food, medications...) factors that could contribute to the drug variability.

Preclinical: 1.Preparation for human clinical studies

SNP Discovery in genes thought to be relevant, Additional SNPs discovery in genes previously shown to influence the disease of interest

Preclinical: 2.Exploratory hypothesis

GWAS of patients with disease without drug treatment to identify biomarkers. RNA microarrays studies of the disease

Phase I

PK/PD exploration in healthy volunteers Search for polymorphisms/biomarkers in: 1. Drug metabolizing enzymes (CYPs, UGTs..) 2. Drug transporters (P-glycoproteins, OCT...) 3.Drug targets (Protein G-coupled receptors, ion channels...)

Phase II/III

1.Confirmation of healthy volunteers PK observations in patients. 2.PK/PD modeling supplemented with PGx data 3.Drug response 4. Exploratory hypothesis