Exam VI - Gene Therapy

Question 1

Diseases with Mendelian Inheritance

Answer 1

Myotonic and Duchenne muscular dystrophies
Cystic fibrosis
Neurofibromatosis type 1
Sickle cell anemia
Huntington chorea

Question 2

Diseases with Multifactorial Inheritance

Answer 2

Breast, ovarian, and colon cancers

Question 3

Neurofibromatosis Type 1

Answer 3

Autosomal dominant inheritance
Gene locus on chromosome 17q11
Mutation in the NF1 gene 
Gene product neurofibromin
Café-au-lait spots- patches of hyperpigmentation
Cutaneous and subcutaneous tumors

Neurofibromas: nerve sheath neoplasms that develop along the peripheral nerve fibers, and enlarge causing major disfigurement and bone erosion
Peripheral nerve structures damaged
Lisch nodule: small hamartoma in the iris of majority of patients

Question 4

Neurofibromatosis Type 2

Answer 4

Central neurofibromatosis 
Few skin manifestations
Primarily bilateral acoustic neuromas: deafness and intracranial and paraspinal neoplasms (meningiomas and gliomas)
Autosomal dominant inheritance
Gene locus- 22q11-13
Mutation in the NF2 gene 
Gene product merlin

Some people think it is responsible for elephant man’s disease: Proteus syndrome

Question 5

Huntington chorea

Answer 5

A neurodegenerative disorder
Onset in third or fourth decade (20s/30s)
Characterized by: chorea (involuntary movements) and dementia
Autosomal dominant inheritance with complete penetrance= all who have the gene have the phenotype
Cause: mutation associated with trinucleotide repeat expansion in the Huntington gene (HD) on chromosome 4p

Question 6

Genomics, Genetics, and Proteomics

Answer 6

The genome is the gene complement of an organism
Genome sequence= comprises the information of the entire genetic material of an organism

Genetics studies single genes and their role in heredity
Genomics= determination of the complete DNA sequence for all the genes of an organism (DNA,RNA, and protein level)
Functional genomics = functional proteomics
Aims for determination of proteome function
Proteome: all proteins encoded by the genome

Question 7

Structural Genomics

Answer 7

Its goal: Determination of complete structure of entire proteome aka structure of proteins
Tools: X-ray crystallography, NMR spectroscopy
Indirect by interpreting DNA/mRNA sequence

Question 8

Pharmacogenomics

Answer 8

Move away from “One-Size-Fits-All” therapeutics
Find correlation between DNA variants and individual responses to medication
Customize drugs for groups of patients that have similar response to medical treatment
Adverse responses to drugs kill over 100,000 yearly
Over 2 million people: have side effects or have no benefit at all
More effective, specific drugs require correlation between patient’s genetic make-up and response to drug
Drug-protein binding can have genetic correlation
Pharmacogenomics application: human gene therapy, protein therapy, drug targets combining chemistry and genomic, drug design

Question 9

Ab and Cellular Immunotherapeutics

Answer 9

Certain gene families present themselves as targets for new drugs based on sequenced human genome
Protease- protease inhibitors
Small-molecule drugs
Associating targets with disease processes and pathways aims at producing specifically designed small molecule therapeutics

Antibody immunotherapeutics- monoclonal Abs and magic bullet (Ab + drug = target only bad cells)

Cellular immunotherapeutics: transfected WBC, dendritic cells

Question 10

SNPs

Answer 10

Single nucleotide polymorphisms
One amino acid encoded by multiple codons
Do these differences matter in drug response?
This could offer an opportunity for drug adaptation to fit the individual’s genetic makeup
Personalized drugs supposed to work effectively and safely without side effects

Question 11

Herceptin

Answer 11

Herceptin is the oldest Ab “personalized” drug
shrank tumors and prolonged lives
Prescribed for: metastatic breast cancer
Only 30% of patients have HER2 + tumors
Herceptin does not work for HER2- breast cancer patients
Monoclonal antibody specific for HER2 antigen
It binds to HER2 and slows tumor growth

Question 12

Powerful Medicines

Answer 12

Drugs based on knowledge of disease association with:
Proteins (receptors and enzymes) and RNA molecules (genes)
Targeting of drugs to specific diseases
Improve therapeutic effect
Less collateral damage of healthy cells
result of pharmacogenomics

Question 13

Higher Drug Safety

Answer 13

Matching drug therapy and disease to patient’s genetic profile leads to improved drug choice, individualized or almost
Safer drug: it works from the first time, adverse side effects less likely, faster recovery
due to pharmacogenomics

Question 14

Drug Dosage Accuracy

Answer 14

Dosage is based now on: weight, age, gender
Adding patient’s genetics as a major factor for determining drug and dosage makes overdose less likely by taking into consideration:
Specific drug processing in the body of the individual and speed of drug metabolism
due to pharmacogenomics

Question 15

Improved Disease Screening and Prevention

Answer 15

Genetics based prediction of disease susceptibility
Establishing MHC (HLA) ID of diseases that might affect an individual: diabetes, ankylosing spondylitis
Can facilitate: lifestyle fine tuning to genetic makeup, improved monitoring, better treatment, personal environment based on genetics aides in disease prevention or making it milder at least
due to pharmacogenomics

Question 16

Recombinant Vaccines

Answer 16

Current vaccines often depend on whole killed or attenuated live (OPV polio vaccine; Sabin) pathogen
Unnecessarily challenge the immune system with too many antigens
DNA vaccines: elicit immune response specifically for exposed antigens with no infection, no disease from vaccine
Cover multiple strains of the infectious agent by combining genes on one plasmid
DNA storage: easy, stable and economic
Up to 6 months at rm. Temp.
due to pharmacogenomics

Question 17

Drug Discovery and Approval Processes

Answer 17

Drugs that target genome
Retesting and allotting drugs that did not pass in the past for smaller, yet responding population
Drug approval process becomes faster
Clinical trial shorter, cost lower
Testing drug on/for targeted group instead of ad random
due to pharmacogenomics

Question 18

Breast Cancer Prevention via Pharmacogenomics

Answer 18

Understanding the role of genes in cancer helps prevention
Tamoxifen reduces risk of breast cancer in women with BRCA1 (breast cancer susceptibility allele) and BRCA2 gene variants that increase cancer risk
Antagonist of estrogen receptor in breast tissue
Agonist in endometrium (Uterus) - estrogen-receptor modulator

Question 19

Cytochrome P450: Personalized Drugs

Answer 19

Cytochrome P450 multigene family has several DNA variants and involved in drug metabolism and detoxification of the body

Psychiatric, neurological, cardiovascular drugs
Warfarin- cardiovascular drug
Drug and dosage can be determined and adjusted to the specific P450 gene of the patient because the drug reacts differently to individual P450 genes that can cause adverse/unbeneficial reactions
Genotype enable reduction of adverse reactions

Question 20

Gene Therapy

Answer 20

Gene therapy applications:

1. Tx of disease here defective genes can be replaced or supplemented by healthy genes
2. Supporting normal function- immune response to cancer or infection
3. Tx of genetic disorders is practiced and still have promising future -cancer (Transfection) and metabolic disorders

Trials focus on cancer, multigenic multifactorial diseases, single gene defects, infectious diseases (AIDS), cardiovascular diseases
Safety of gene-delivery is a stumbling block- viruses used as vehicles might revert to pathogenicity

Question 21

Recurrence Risk

Answer 21

The probability that an affected child will be produced in a family that already has produced at least one affected child
Recurrence risk can be given with confidence for mendelian traits
50% for autosomal dominant: Affected children are born to a heterozygote (Aa) and unaffected homozygote (aa) parent
25% for autosomal recessive

Question 22

Sickle Cell Anemia and Malaria

Answer 22

A genetic disease with beneficial effect on survival
Sickle-cell trait protects carrier from falciparum malaria
Individuals with 2 copies of the sickle-cell gene  encode defective hemoglobin and suffer from sickle-cell anemia
With one sickle-cell gene and one normal gene  unaffected and able to resist Plasmodium falciparum
Advantage for people in malaria endemic West Africa
Evolution
Malaria was selective for sickle-cell trait

Question 23

Lesch-Nyhan

Answer 23

Focus on genetic metabolic diseases
Defective gene= loss of enzyme or its function
Gene therapy- faulty gene is replaced by a working gene

Lesch-Nyhan syndrome possible good candidate
Patients are deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRTase)
Metabolizes hypoxanthine and guanine to uric acid
Bizarre impulse for self-mutilation- very severe biting of the lips and fingers
Wild-type gene of the mutated gene has been cloned
Adequate tool to introduce the gene into human cell is needed
Modified non-virulent virus

Question 24

PKU

Answer 24

Phenylketonuria (PKU)
Normal gene needs to be introduced into only one organ
Defect in phenylalanine hydroxylase gene in the liver
PKU affects 1/12,000 white children
No early treatment = severe mental retardation
Only available option: early detection
Phenylalanine-free diet

Question 25

Germline Therapy

Answer 25

From ethical point of view, somatic genetic defects can be treated and germline gene replacement cannot
Such manipulation affects genetic heritage of the progeny and the gene pool of the species would be changed
Technical difficulties + ethical issues = germline therapy unlikely

Question 26

Oligonucleotides Experiment

Answer 26

Experiment: Oligonucleotide was delivered into melanocytes by injecting the oligos and topical application to start the oligo induced gene repair process
This changed the color of the mice fur
Melanocytes produced a changed color due to the changed gene

Question 27

Antisense vs. Sense

Answer 27

Antisense strand = template
Sense strand = complement DNA = mRNA with U instead of T
mRNA sequence is “sense” RNA because it encodes a gene product (protein)
Unpaired nucleotides are read by transfer RNA anticodons during ribosomal translation of message

RNA can form duplexes
Complementary RNA strand needed is a second
5´ C A U G 3´ mRNA 3´ G U A C 5´ Antisense RNA
Its nucleotide sequence is the complement of message sense
Duplex formation between sense mRNA and antisense RNA sequence to block translation because ribosome cannot gain access to the nucleotides in the mRNA
Duplex RNA fast degradation by cellular RNases

Question 28

Antisense Therapy: BCL-2

Answer 28

Antisense RNA complementary to proto-oncogene BCL-2 on chromosome 18
Possible therapy for B-cell lymphoma and leukemia
Gene product: integral membrane protein (Bcl-2)
Located in the membranes of ER, nuclear envelope, and outer membranes of the mitochondria
BCL-2 locus on chromosome 18 underwent reciprocal translocation with the portion of chromosome 14 containing the antibody heavy chain locus
t(14;18) translocation places the BCL-2 gene close to the heavy chain gene enhancer 
Bcl-2 protein is expressed at high levels in t(14;18) cells
Using the antisense, it can block the translation of the BCL-2 mRNA to not produce the protein to inhibit the disease; antisense is used in many other treatments of diseases currently

Question 29

Antisense Oligodeoxynucleotides

Answer 29

Antisense ODNs are synthetic polymers:
Deoxynucleotides: monomers; DNA building blocks
ODN, oligo, has only 15-20 deoxynucleotides
Oligo sequence (3´ to 5´) is antisense complementary to part of the sense sequence of mRNA
Essentially the antisense binds the sense mRNA, then translation is blocked and the peptide is not produced

Question 30

Antisense ODN Strategy

Answer 30

Antisense ODNs are therapeutic agents that block particular protein synthesis to inhibit disease process

Mechanism: ODN binds mRNA that carries the message for the synthesis of that protein and forms a duplex that physically blocks ribosome movement along the messenger RNA
Increase rate of mRNA degradation in the cell

Question 31

ODN Requirements

Answer 31

Requirements to be useful in medical treatment

1. Ability to enter the target cells
2. Escape DNAse digestion
3. No unpleasant side-effects

Approaches to fulfil these requirements include:

1. Chemical modification to make ODN DNAse resistant
2. Attachment to targeting device using receptor ligand that usually binds receptors on target cells or antibodies/monoclonal Ab specific for surface proteins of target cells

Question 32

ODN Uptake

Answer 32

Endocytosis is the main mechanism of ODN uptake, which is not very efficient

Uptake improvement:

1. Add large hydrophobic groups during ODNs synthesis to increase adsorption to cell membrane to increase endocytosis
2. Couple ODNs to a ligand that has a receptor on the target cell to cause endocytosis with ligand
   ex. Transferrin as a ligand causes endocytosis of ODNs through transferrin receptors

Question 33

ODN Structural Modification

Answer 33

Phosphate-sugar backbone of antisense DNA molecule can be modified by replacing:
One non-bridging oxygen by phosphodiester = monothiophosphate
Two non-bridging oxygen = dithiophosphate

Question 34

Antisense ODN as Drugs

Answer 34

Experimental or in use to treat
HIV/AIDS and other viral diseases – even Ebola

Cytomegalovirus (CMV)= frequent opportunistic infectious agent of AIDS patients; Fomivirsen (VITRAVENE) is FDA approved for use against CMV eye infections, retinitis

Hemorrhagic fever viruses= Ebola and Marburg

Cancer= Glioma tumor of brain or spine (CNS)

Familial hypercholesterolemia (FH)
High cholesterol with genetic component
FDA approved drug for the homozygous FH

Question 35

Imprinted Genes

Answer 35

Imprinted genes: expression determined by the contributing parent
Both alleles in a heterozygote are NOT equally expressed
Usually both alleles are equally expressed like in blood group inheritance
A from father + B from mother = child AB

Mechanism: Imprinting starts in the gametes
Allele in one parent will be marked to be inactive in the future embryo
Marking by methylation of the DNA in the promoter(s) of the gene
CH3 added to C in CG islands to prevent transcription factors from binding promoters so no transcription
Methylation is the imprinting signal

Question 36

Igf2r Gene Imprinting

Answer 36

Maternal copy of the gene is upstream promoter, unmethylated
Active= bind transcription factors to cause transcription of the sense strand and produce Igf2r messenger RNA
Downstream set of CpG islands methylated

Paternal copy of Igf2r gene is imprinted and upstream promoter methylated therefore inactive
Downstream promoter= unmethylated
Active =bind transcription factors, transcription of the antisense strand to produce antisense RNA then shuts the gene down or at least it cannot be translated
Downstream set of CpG islands methylated

*You need the gene from the mother because the gene in the father is imprinted aka inactive

Question 37

Wilms Tumor

Answer 37

Wilms tumor= pediatric kidney tumor
Tumor cells inherit active paternal and maternal copies of Igf2 gene
Only paternal allele is needed to be normal because if you have the mother’s, the father and mother genes will form a duplex and result in the tumor
If imprinting in somatic cells fails it results in cancer

Question 38

Prader Willie

Answer 38

Congenital disorder
No paternal copy inherited of a gene locus on chromosome #15 because you NEED paternal copy

Short stature, mental retardation, polyphagia, obesity, sexual infantilism, severe muscular hypotonia
Poor responsiveness to external stimuli, which decreases with age
A small deletion in the paternal-derived chromosome 15q11–13
Some cases are due to maternal uniparental disomy (child receives both chromosomes 15 from the mother)

Question 39

Angelman Syndrome

Answer 39

Congenital disorder
No maternal copy inherited of a gene locus on chromosome #15 because you need the maternal copy

Microdeletion of maternal 15q-13
Mental retardation
Ataxia
Paroxysms of laughter
Seizures
Characteristic happy faces
Minimal speech

Question 40

Cystic Fibrosis

Answer 40

Congenital metabolic disorder -autosomal recessive
Abnormal secretions of exocrine glands
Passageways obstruction results of excessive viscous sticky mucus
Meconium ileus results in poor growth
Malabsorption & bulky foul stools
Recurrent pneumonia (P. aeruginosa)
Emphysema, club fingers, salt depletion

Characteristic changes of end-stage CF include:
Bronchial wall thickening
Bronchiectasis
Persistent atelectasis

Question 41

Strabismus

Answer 41

A manifest lack of parallelism of the visual axes of the eyes - crossed eyes, heterotropia, squint
A manifest lack of parallelism of the visual axes of the eyes