4. Molecular Therapeutics

Question 1

List the important considerations that must be taken into account in using molecular therapeutic approaches (objective)

Answer 1

Answer later

Question 2

Discuss the pros and cons of the common vector classes for gene replacement therapy: adenoviral, adeno-associated, and lentiviral vectors and the risks involved in each approach for the patient (objective)

Answer 2

Answer later

Question 3

Discuss the circumstances and the main lessons from the Jesse Gelsinger case (objective)

Answer 3

Answer later

Question 4

Discuss the use antisense RNA and siRNA to inhibit pathological gene expression (objective)

Answer 4

Answer later

Question 5

Describe the basic premise behind the new gene editing technique CRISPR-CAS and discuss the ethical questions that could arise from future wide-spread use of this technique (objective)

Answer 5

Answer later

Question 6

Molecular Therapeutics (why)

Answer 6

To improve life expectancy
To improve quality of life
To change a genetic trait (unethical)

Question 7

Augmentation Therapy

Answer 7

Synthesize missing gene product in vitro and return it to the patient (blood clotting factors, insulin, thyroid hormone)

Non-permanent (augmentation for life)

Question 8

Molecular Therapeutics (what)

Answer 8

Gene product administration (augmentation): provide missing gene product

Gene replacement: addition of a new gene in the presence of defective gene

Gene expression correction: restoration of normal gene expression or reduction of abnormal RNA and protein expression levels

Gene correction (editing): substitution of a new gene for a defective gene to restore normal genotype

Question 9

Molecular Therapeutics (how)

Answer 9

Replacement- normal genes cloned into viral or non-viral vectors under control of the vector’s gene regulatory signals and inserted in patient cells

Alteration of expression- small molecule drugs or RNAs (antisense and siRNAs) used to decrease expression of pathological genes

Gene editing- bacterial CRISP-Cas9 enzyme system used to repair a mutant gene with a normal gene under control of natural regulatory elements

Question 10

Molecular Therapeutics (where)

Answer 10

Ex vivo- cells removed, CAS9 protein, therapeutic modified cells returned to patients

In situ- inject gene in specific place

In vivo-CRISPCAS9 in delivery vehicle (lipid nanoparticles), therapeutic delivery directly into patients

Question 11

Molecular Therapeutics Where (continued)

Answer 11

Whole organism (in vivo)- introduction into bloodstream

Specific organ or tissue (in situ)- interventional delivery to organ or tissue

Cells (ex vivo)- removal of cells from patients, modification of cells in culture, and returned

Question 12

Molecular Therapeutics (when)

Answer 12

Germ cells and gametes (not good at this yet)
Early embryo and fetus (ethical problems)
Infant, Adolescent, and Adult

Question 13

Increasing Complexity of Methods

Answer 13

1. Least difficult: unregulated intracellular proteins made by cells transfected ex vivo (ie adenosine deaminase transfected into hematopoietic stem cells in culture)
2. Unregulated secreted proteins made by cells transfected in situ (ie expression of VEGF in muscle cells
3. Unregulated intracellular proteins made by cells transfected in situ (ie CFTR transfected into airway epithelial cells)
4. Most difficult: Regulated, tissue-specific intracellular proteins made by cells transfected in situ (ie transfection of insulin into pancreatic islet cells)

Question 14

Gene Replacement Therapy

Answer 14

Need to know relationship between genotype and phenotype

Assumption is that correct gene can be delivered to and expressed in appropriate tissue in controlled manner

Question 15

Successful Gene Replacement Therapy

Answer 15

Are target tissues accessible and will there be efficient transfer?
Can DNA be stably integrated in nuclear DNA and appropriate expression maintained?
Do target cells have long lifespan?
Will gene in target cells be properly regulated?

Question 16

Examples of Gene Replacement Therapy for Intracellular Proteins

Answer 16

SCIDs due to adenosine deaminase and blocks in interleukin receptors
A and B-globin to treat thalassemias (bone marrow cells)
Mini-dystrophin and utrophin for Duchenne and Becker muscular dystrophies
CFTR for CF

Question 17

Most Successful Viral-Based Gene Therapy

Answer 17

SCIDs

Question 18

Common Gene Therapy Viral Vectors

Answer 18

Vector- engineered piece of DNA derived from natural occurring human virus that can carry the replacement gene and transfect desired cell/tissue

1. Adenovirus (upper respiratory infections)
2. Adeno-associated virus (unknown clinical)
3. Lentivirus (retroviruses like HIV)

Question 19

Viral Vector Infection Can be Carried Out ex vivo

Answer 19

Patient’s somatic or stem cells can be removed, infected and then transplanted back in the patient

Question 20

In situ Tissue-Specific Delivery by Surgery

Answer 20

Surgically-mediated localized delivery of transfected cells into specific tissues for partial restoration of function
-Pancreas, Liver, Heart, Brain

Question 21

Adenovirus Vectors

Answer 21

Advantages: infects respiratory epithelia (infection can be spray or virus can be infused), carry large payload, high infection efficiency

Disadvantages: high rate of immune response (most people have this infection sometimes in lives), not stably integrated into genome (can replicate extrachromosomally) and can be lost from the cell resulting in only transient expression of therapeutic gene (need to do this many times over lifetime)

Question 22

Adenovirus-Associated Virus Vectors

Small gene delivery

Answer 22

Advantages: site-specific (chromosome 19) stable integration in some cell type but not all (chances of mutagenesis are low), non-pathogenic, infects non-dividing cells, most people won’t build immune response

Disadvantages: small payload, recognized by CD8+ T cells as compromised and killed

Question 23

Lentiviral (Retroviral) Vectors

Answer 23

Advantages: stable integration, independent enhancer and promoter, infect wide range of cell types (dividing and non-dividing), good sized payload

Disadvantages: potential for activating cellular genes leading to oncogenesis, insertional mutagenesis potential

*Divide retrovirus into three parts to promote packaging of gene construct and maximize patient safety

Question 24

Non-Viral-Vector

Answer 24

Lipid Delivery of Nucleic Acids

Question 25

RNA-Based Therapies to Knockdown or Modify Mutant Gene Expression

Answer 25

Antisense RNAs RNA Interference (microRNAs siRNAs)

Question 26

Antisense RNA Therapy (in vivo)

Answer 26

Oligoribonucleotides that base pair to exons or introns in mRNA Can block translation or RNA splicing Made with modified nucleotides (protective groups) to make them resistant to cellular RNases

Question 27

Duchenne Muscular Dystrophy

Answer 27

1/3500 male births Progressive loss of muscle strength 2-6 age diagnosis Large muscles in pelvic girdle are first to show weakness. Calf and forearm become enlarged (fat replaces atrophied muscle) Paralysis and dilated cardiomyopathy (wheelchair-bound) Death in early twenties from cardiac/pulmonary M: mutations in gene coding for dystophin

Question 28

Dystrophin Gene

Answer 28

X chromosome Largest gene in human genome 2.5 million bps Expressed primarily in skeletal and cardiac muscle Alternative promoters and splice sites produce variants *Utrophin- located on chromosome 6, general membrane function, found in fetal muscle but disappears later in development

Question 29

DMD

Answer 29

Mutations eliminate expression of dystrophin (nonsense, frame-shift, splicing, large deletions) Symptoms 3-6 and are more severe than BMD. Die between 15-25 years of age

Question 30

BMD (Becker Muscular Dystrophy)

Answer 30

Mutations cause partially defective dystrophin mutation (point, in-frame duplications/deletions, rearrangements and small deletions) Symptoms later onset (>6 years and slower to progress). Can remain ambulatory into late adolescence, but live to 50s DMD/BMD die from dilate cardiomyopathy

Question 31

DMD Splicing Mutations (how corrected)

Answer 31

Antisense RNA-induced exon skipping Now in frame but skips some exons

Question 32

RNA Interference

Answer 32

RNA molecules inhibit or destroy specific mRNAS by using RNA Interference silence complex pathway (RISC) 1. Small interfering RNA (siRNA) 2. Micro RNA (miRNA) Both share the RISC pathway

Question 33

Small Interference RNA (siRNA)

Answer 33

SiRNA dicer duplexes, activate RISC with single-strand siRNA, seek out and bind any homologous complementary RNA, cleaves and degrades RNA Transfection of siRNAs with Liposomes to promote RNA interference in vivo

Question 34

Applications of RNA Therapeutics

Answer 34

Knockdown an mRNA to inhibit expression of toxic protein or a protein whose over-expression causes disease Knockdown of the expression of mutant regulatory or structural protein that has dominant negative phenotype Inhibit mutations in splice site that cause dominant phenotype

Question 35

Crispr-Cas9 (cut and paste in E. coli)

Answer 35

Bacteria use Crisprs as immune system to ward off phage viruses-scavenge viral DNAs by cutting segments out and inserting them into own genome using nuclease enzyme Cas9 system Inserted segments are transcribed into "guide RNAs) used to specifically recognize the virus if it reinfects the cell Crispr-Cas9 + guide RNAs recognize/cleave virus genome

Question 36

B-thalassemias (caused by splice site mutations in B-globin gene)

Answer 36

Corrected through CRISPR/Cas9: Take patient fibroblasts then convert to induced pluripotent stem cells (iPSCs) Corrected beta-globin gene with system using guide RNAs, and differentiate the iPSCS into hematopoietic cells Cells produced normal beta-globin Implant hematopoietic cells back to patient

Question 37

Hypertrophic Cardiomyopathy (HCM)

Answer 37

S:Heart enlarged, portion of myocardium dilated and cannot pump blood to lungs and tissues efficiently Cause of sudden cardiac death M: Autosomal dominant Mutations in genes for myosin heavy chain protein, myosin binding protein C, cardiac troponin T, troponin 1 and tropmyosin