Chapter 9 Genetic Approaches to Treating Diseases

What are the three different broad approaches to treating genetic disorders

- Based on genetic deficiency - Based on disease phenotype due to a positively harmful effect - Those that reduce susceptibility to disease

Describe how genotypes are multiple loci in patients are important in drug treatment

- Delivering the optimal warfarin dosage is clinically very important because there is a narrow therapeutic window. The final warfarin dose is critical, but because of genetic variation, the optimal dose varies enormously between individuals - Variations in VKORC1 and CYP2C9 remain the largest genetic determinants, accounting for about 40% of the variation in the final warfarin dose

Describe how genetic variation of the CYP2D6 enzyme affects drug metabolism

- Due to severe inactivating mutations or deletions in both CYP2D6 alleles some people have a very low activity of the enzyme and when treated with certain drugs that are normally metabolized by this enzyme alone, they fail to metabolize and excrete the drug. They are classified as poor metabolizers - people can also be classified into three additional groups: intermediate metabolizers; extensive metabolizers (with one or two activeCYP2D6 alleles); and ultrafast metabolizers. - Ultrafast metabolizers may also get little benefit from drugs that are principally metabolized by CYP2D6 (because the drug is metabolized and detoxified so quickly)

How can somatic gene therapy modify diseased cells?

- If the problem is loss of function, a simple solution is to add functioning copies of the relevant gene - If the problem is some harmful or toxic gene product within cells. The approach might be to selectively inhibit the expression of the harmful gene product

Genetic factors are implicated in the variation between individuals in drug metabolism at what levels

- absorption - activation - target response

What are the two broad strategies of somatic gene therapy

- disease cells are simply modified in some way so as to alleviate disease - they are selectively killed

What are some contributors to how fast a drug is metabolized?

- genes - Environment - enzymes acting on the drug

What are some of the concerns with using viral vectors to get therapeutic gene constructs into cells?

- there has been little control over where they will insert into the genomic DNA of patient cells - They might insert by accident into an endogenous gene and block its function - They might accidentally activate an oncogene, causing tumor formation - the patient might mount a strong immune or inflammatory response

What are some examples clinically significant genetic variation in drug receptors that lead to altered responses to drugs?

- variants of beta-adrenergic receptors, cell surface receptors that have central roles in the sympathetic nervous system (ADRB1 and ADRB2) - H2RA serotonin receptor and the RYR1 ryanodine receptor

How many cytochrome P450 enzymes catalyze 90% of the phase I reactions on commonly used drugs

6

Describe what occurs during phase II of drug metabolism

A transferase adds a chemical group that facilitates excretion

Define prodrug and what phase of drug metabolism does it occur in?

An inactive compound which can be converted into an active drug. Phase I

Which of the following statements, if any, is false? a) Gene therapy involves the direct genetic modification of the cells of a person (or animal model) to achieve a therapeutic goal. b) Current gene therapy is directed at modifying somatic cells. c) The only successful gene therapies are those in which cells are removed from a patient, genetically modified, and then returned to the patient. d) Gene therapy successes have largely involved treatment of recessively inherited disorders.

C) The only successful gene therapies are those in which cells are removed from a patient, genetically modified, and then returned to the patient. There have been some successes with in vivo gene therapy as well as ex vivo gene therapy.

_______ deficiency shows marked differences between different ethnic groups, as revealed by the frequency of poor metabolizers; poor metabolizers require a lower dose of certain drugs such as clopidogrel, an antiplatelet agent that is used to inhibit blood clotting in some diseases such as coronary artery disease

CYP2C19

_______ deficiency is important in metabolizing the anticoagulant warfarin (as described below), and also results in an exaggerated response to tolbutamide, a hypoglycemic agent used in treating type 2 diabetes.

CYP2C9

What cells are said to be totipotent

Cells from the very early mammalian embryo

What is the main cause of the difference in performance of small molecule drugs between people

Genetic variation

What is the concern in pharmogenetics

How the actions of drugs and the reactions to them vary according to variation in the patient's genes

Monooxygenases are found in what reaction phase of drug metabolism

I

(NAT1/NAT2) is polymorphic in a wide range of human populations, with rapid acetylators (who eliminate drugs rapidly) and slow acetylators

NAT2

Concerning transport of genes into human (or animal) cells, which, if any, of the following statements is false? a) Transduction means using viruses to transfer DNA into human (or other animal) cells. b) Tropism refers to the ability of certain viruses to transduce only certain types of cell, such as hepatocytes, but not neurons. c) Tropism depends on a virus being able to recognize a specific receptor molecule on the surface of the cell. d) Transfection means transferring DNA into the cells by any means other than using viruses.

None

Which, if any, of the following statements is true? a) Autologous cell transplantation is involved in in vivo gene therapies: cells from an individual are genetically modified and then returned to that individual. b) Adenovirus vectors have the advantage that they offer very high level expression and are well suited to gene therapy for blood disorders. c) Adenovirus vectors have a better safety profile than adeno-associated virus vectors and have a larger insert size capacity. d) Adeno-associated virus vectors are well suited to gene therapy for blood disorders but have a low insert size capacity

None all are false Autologous cell transplantation is involved in ex vivo gene therapy. Both adenovirus vectors and adeno-associated virus vectors are not well suited to gene therapy for blood disorders because neither of them are integrating vectors (because blood cells have short lives some kind of retroviral integrating vectors are needed for gene therapy for blood disorders - the hope is to insert the gene constructs into the chromosomal DNA of hematopoietic stem cells). Adenovirus vectors have a poor safety record because they often induce powerful inflammatory responses in the recipient of therapy.

With regard to drug metabolism, which, if any, of the following statements, is true? a) The therapeutic window is simply the range of plasma drug concentrations in which the drug has therapeutic benefit. b) Each individual drug molecule is metabolized by a specific drug-metabolizing enzyme that is dedicated to the metabolism of that drug. c) An ultrafast metabolizer is a person who metabolizes a drug too quickly and so is at risk of an overdose d) A poor metabolizer is a person who cannot metabolize a drug properly and is at risk of anunderdose.

None. All are false. a) The therapeutic window is the range of plasma drug concentrations in which the drug has therapeutic benefit without causing extra safety risks due to drug toxicity. b) Individual drug molecules can sometimes be metabolized by one of several drugmetabolizing enzymes, and many drug-metabolizing enzymes metabolize multiple different drugs. c) An ultrafast metabolizer is a person who metabolizes a drug too quickly and so is at risk of an underdose. d) A poor metabolizer is a person who cannot metabolize a drug properly and is at risk of an overdose.

Describe what occurs during phase I of drug metabolism

Oxidation reactions as a result of monooxgenases that produce a polar drug derivative with a reactive group. Prodrugs can be created

What is the most actively used method for treating disease by gene silencing therapy?

RNA interference

What is thought to be the significant contributor to the variability of CYP3A4 between individuals?

Regulatory mutations due to it being inducible

What are the two types of gene therapy

Somatic; germ-line

Define augmentation therapy

Something given to the patient that supplements a depleted or missing factor to overcome the deficiency and restore function

Define pharmacogenetics

Study of the interrelationship of genetic differences and drug effects

T/F Germ-line gene therapy that involves modifying nuclear DNA is widely banned in humans for ethical reasons

T

T/F drug response phenotypes can be multifactorial

T

T/F genetic variation between individuals has an important influence on both the efficacy and the safety of drugs

T

Why can't augmentation therapy be used for diseases caused by a positive harmful effect?

The disease can't be corrected by administering normal genes, products are cells to the patient.

Define transfection

The nonviral transfer of DNA, RNA, or oligonucleotides into human or other animal cells

What is gene therapy

The the direct genetic modification of cells to achieve a therapeutic goal

Concerning the efficacy of small molecule drugs, which, if any, of the following statements is true? a) At the level of clinical trials drugs can vary widely in how effective they are. b) Once a drug has received regulatory approval, we can be sure that it will be effective in all patients, although some people will receive more benefit from it than others. c) Drugs used to treat psychiatric disorders are particularly effective. d) Stains and beta blockers that were meant to reduce the risk of heart disease are goodexamples of drugs that are largely ineffective.

a) At the level of clinical trials drugs can vary widely in how effective they are

With regard to treatment of inborn errors of metabolism (IEM), which of the following statements, if any, is false? a) IEMs are all single gene disorders that have been studied for many decades, leading to the development of successful treatment in all cases. b) IEMs can be treated by augmentation therapy, treatment to inhibit positively harmful effects, or by prevention therapy. c) Treatment for some individual IEMs can involve both augmentation therapy plus treatment to inhibit positively harmful effects. d) Treatment of some IEMs involves artificially forcing an increase in a minor metabolic pathway to counteract a build-up in a toxic metabolite produced by a metabolic block in a major metabolic pathway.

a) IEMs are all single gene disorders that have been studied for many decades, leading to the development of successful treatment in all cases. (For some IEMs there remains no suitable treatment.)

Concerning stem cells, which of the following statements is incorrect? a) Stem cells occur frequently in our bodies. b) A stem cell can divide asymmetrically to give a daughter stem cell plus a daughter transit amplifying cell that can undergo a series of differentiation steps to give rise to a differentiated cell. c) If for any reason, stem cells are depleted, a stem cell can divide symmetrically to regenerate the stem cell population. d) In an adult person, stem cells are normally multipotent or unipotent.

a) Stem cells occur frequently in our bodies.

Which of the following descriptions, if any, is false? A person's ability to absorb or metabolize a drug that is intended to treat a genetic disorder a) is entirely due to genetic factors. b) depends on a person's lifestyle. c) is not modified by having a bacterial infection. d) is independent of a person's diet

a) is entirely due to genetic factors. c) is not modified by having a bacterial infection. d) is independent of a person's diet.

Which, if any, of the following can be classified as a type of augmentation therapy? a) Treatment using a small molecule drug to bind a target protein and prevent it working. b) A bone marrow transplant. c) Corrective surgery for cleft lip and palate. d) Insulin treatment in diabetes.

b) A bone marrow transplant. d) Insulin treatment in diabetes.

Concerning gene transfer into human cells, which, if any, of the following statements is false? a) Integrating viruses can insert genes into the chromosomes of a host cell. b) Most integrating viruses insert their DNA into a specific location within the genome. c) The great value of integrating viruses is that they allow foreign (and therefore, therapeutic) DNA to be stably inherited so that it passes to all descendant cells of the transduced cell. d) Because non-integrating viruses cannot insert their DNA into the chromosomes of a cell, the transduced DNA is quickly destroyed by enzymes within the host cell.

b) Most integrating viruses insert their DNA into a specific location within the genome. d) Because non-integrating viruses cannot insert their DNA into the chromosomes of a cell, the transduced DNA is quickly destroyed by enzymes within the host cell.

Concerning making animal models of human disease, which, if any, of the following statements is false? a) Pronuclear microinjection is a general way of making a transgenic animal and involves microinjection of foreign DNA into a fertilized egg cell. b) Pronuclear microinjection is best suited to modelling recessively inherited single gene disorders. c) Gene targeting using embryonic stem cells depends on having well-characterized embryonic stem cell lines that can readily allow transmission through the germ line. d) Gene targeting using embryonic stem cells in mice is a popular way of modelling disease phenotypes that result from a gain-of-function.

b) Pronuclear microinjection is best suited to modelling recessively inherited single gene disorders. d) Gene targeting using embryonic stem cells in mice is a popular way of modelling disease phenotypes that result from a gain-of-function. (Pronuclear microinjection is not suited to modelling recessively inherited single gene disorders, but has often been used to model disease phenotypes that result from a gain-of-function. Gene targeting using embryonic stem cells in mice is not well suited to modelling disease phenotypes that result from a gain-of-function but is a popular way of modelling the loss-of-function in recessively inherited single gene disorders.)

Concerning animal models of human disease, which, if any, of the following statements is false? a) Primates should be the best animal models, but for mostly practical reasons, rodent models have been preferred. b) Rats have been the preferred disease models because they offer the best balance between rapid breeding, size and the cost of maintaining colonies. c) Rodent models are especially suited to modelling neuropsychiatric disorders. d) All animal models have limitations regarding how far we can make inferences to help understand human disease.

b) Rats have been the preferred disease models because they offer the best balance between rapid breeding, size and the cost of maintaining colonies. c) Rodent models are especially suited to modelling neuropsychiatric disorders. Mice have been the preferred disease models, but rodent models are poorly suited to modelling neuropsychiatric disorders (in part, because they are not good models of cognitive capacity).

Which, if any, of the following statements is false? a) The great majority of clinical gene therapy trials have had limited success b) The only successful gene therapies have been for recessive blood disorders. c) The only successful gene therapies have been ex vivo gene therapies. d) Gene therapy for inherited disorders represents a minority of clinical gene therapy trials.

b) The only successful gene therapies have been for recessive blood disorders. c) The only successful gene therapies have been ex vivo gene therapies. (There have been examples of other successful gene therapies that involve brain disorders and in vivo gene therapy for eye disorders, for example. )

Which, if any, of the following statements is false? a) Genome editing means making a predetermined change to the nucleotide sequence at just one locus within an intact cell. b) The specificity of genome editing depends on an initial site-specific cleavage of doublestranded DNA following base pairing with specifically designed nucleotide sequences. c) Genome editing has the potential to permit specific "gene correction" in which a mutant sequence in a cell is restored to the normal sequence. d) Genome editing might also have therapeutic potential by specifically inactivating a gene in some cases.

b) The specificity of genome editing depends on an initial site-specific cleavage of doublestranded DNA following base pairing with specifically designed nucleotide sequences. (The site-specific cleavage is also often carried out following recognition of the sequence at the target locus by a combination of zinc fingers within zinc finger nuclease proteins.)

Which, if any, of the following descriptions is false? a) Zinc fingers are elements of protein secondary structure in which the polypeptide chain folds back upon itself after co-ordination of a Zn2+ ion with selected amino acids, often a pair of cysteines and a pair of histidines. b) Zinc finger nucleases are natural proteins containing a sequence of zinc fingers that can bind to specific sequences in DNA. c) After zinc finger nucleases bind to both DNA strands at a specific DNA sequence they attract cellular DNA cleavage enzymes, inducing them to make a double-stranded break at just that one position in the genome. d) The CRISPR-Cas system also allows genome editing but in this case the target DNA sequences are recognized by guide RNA sequences rather than proteins.

b) Zinc finger nucleases are natural proteins containing a sequence of zinc fingers that can bind to specific sequences in DNA. c) After zinc finger nucleases bind to both DNA strands at a specific DNA sequence they attract cellular DNA cleavage enzymes, inducing them to make a double stranded break at just that one position in the genome. (Zinc finger nucleases are not natural: they are proteins produced after genetic engineering to covalently join DNA sequences that can specify a series of zinc finger modules to a bacterial DNA sequence that can specify a DNA cleavage domain.)

Which of the following statements, if any, is false? a) Monoclonal antibodies are made by identical immune cells and so will recognize and bind just one specific epitope on a target molecule. b) Monoclonal antibodies of rodent origin are far from ideal therapeutic agents because of their short half-life in human serum and the potential for immune responses by the recipient. c) Humanized antibodies are hybrid antibodies that have constant regions of human origin but variable regions of rodent origin. d) An intrabody is an artificial constructs with just a single chain that is linked to variable domains and, unlike regular antibodies with four polypeptide chains, has the potential to work inside cells.

c) Humanized antibodies are hybrid antibodies that have constant regions of human origin but variable regions of rodent origin. The variable domains in humanized antibodies are of human origin, except for the complementarity-determining regions, which are of rodent origin.

Which, if any, of the following statements is false? a) Hematopoietic stem cells are multipotent because they can give rise to a variety of different cell types. b) Mammalian embryonic stem cell lines are pluripotent because they can give rise to all types of cell in the body. c) Transdifferentiation is a type of epigenetic reprogramming in which a differentiated cell is induced to become pluripotent. d) A transit amplifying cell is a cell produced b asymmetric division of a stem cell and has the potential to give rise to differentiated cells.

c) Transdifferentiation is a type of epigenetic reprogramming in which a differentiated cell is induced to become pluripotent.

What a the reason natural selection appears to have driven an increase in frequency of the slow-acetylator phenotype in some populations?

certain chemicals in well-cooked meat are converted by NAT2 into carcinogens; individuals with slowacetylator phenotypes would be comparatively protected in populations that have had a long tradition of eating well-cooked meat

Which, if any, of the following statements is false? a) Transdifferentiation means reprogramming of a differentiated cell so that it acquires the characteristics of another type of differentiated cell. b) In dedifferentiation a differentiated cell is artificially reprogrammed so that it behaves as a pluripotent cell. c) Human induced pluripotent stem (iPS) cell lines are usually generated by artificial dedifferentiation of readily accessible human cells, such as skin cells. d) Human iPS cell technologies do not offer clinical applications but they are of value for studying pathways of cellular differentiation

d) Human iPS cell technologies do not offer clinical applications but they are of value for studying pathways of cellular differentiation (Human iPS cell technologies have the potential for valuable clinical applications, most readily in creating cellular disease models and possibly in extending ex vivo gene therapy.)

Which, if any, of the following statements is false? a) RNA interference (RNAi) is a cellular defense mechanism that is triggered by the presence in cells of unnatural double-stranded RNA, as can occur after viral infections. b) RNAi therapy is a type of RNA-targeted therapy in which specific double-stranded RNA constructs are engineered to appear in diseased cells in order to incite the cells to destroy any RNA that contains the same sequence. c) By destroying RNAs that are related to a specifically introduced genetic construct, artificial RNAi is effectively a type of gene-specific silencing. d) RNAi therapy is best suited to silencing genes so as to replicate a phenotype caused by loss-of-function mutations.

d) RNAi therapy is best suited to silencing genes so as to replicate a phenotype caused by loss-of-function mutations. (RNAi is a convenient way of silencing a gene in cultured cells as a way of understanding its function but RNAi therapy is better suited to specific silencing of a positively harmful gene in diseased cells than it is to replicating loss-of-function phenotypes.)

What type of disorders has ex vivo gene therapy been used to treat?

disorders by genetic modification of impure populations of hematopoietic stem cells that give rise to blood cells or some types of tissue immune system cell

(Somatic/germ line) therapy is achieved by modifying the DNA of a gamete, zygote, or early embryo

germ line

(Somatic/germ line) therapy produce a permanent modification that can be transmitted to descendants

germ line

Define pharmacodynamics

how the person is affected by the drug

Define transgene

nucleic acid molecule introduced as a cloned gene, but sometimes RNA or oligonucleotides into a patient

(Recessive/dominant) disorders are more suited for molecular augmentation. Why?

recessive because with dominant disorders, one normal allele is still present in the cells which would require precise augmentation therapy due to the gene being dosage sensitive

What does ex vivo gene therapy involve

removing cells from a patient, genetically modifying them in culture and returning the genetically modified autologous cells to the patient

(Somatic/germ line) therapy seeks to modify specific cells or tissues of the patient in a way that is confined to that patient

somatic

Why is drug "repurposing" valuable"?

the drug has already been through lengthy and expensive clinical trials to assess its safety profile.

Why does natural selection foster genetic variation in genes encoding drug handling enzymes?

the principal natural role of these enzymes is to deal with unusual exogenous chemicals (xenobiotics) in our diet and environment.

Define pharmacokinetics

the study of what the body does with, and to, the drug

Polymorphism in _______ is a particular clinical concern when using certain immunosuppressant drugs such as 6 mercaptopurine, which is commonly used to treat childhood leukemia

thiopurine methyltransferase

What cells are targeted in somatic cell gene therapy

those directly involved in the pathogenic process

Is transfection or viral transduction more efficient

viral transduction

____ are commonly used to get therapeutic gene constructs into cells at high efficiency, and they often allow high level expression of the therapeutic transgenes

viral vectors