4 - Gene Therapy & Oligonucleotides

Question 1

What are the 2 types of gene therapy?

Answer 1

• Germ-line gene therapy and somatic gene therapy
• • Germ-line gene therapy aims for the intro of therapeutic genes into germ-cells or omnipotent embryonal cells (at the 4-8 cellular stage)
• • Somatic gene therapy is the intro of genes into somatic cells

Question 2

Ex vivo vs. in vivo gene therapy

Answer 2

• Ex vivo gene therapy – cells from a number of organs and tissues (ex: skins, hematopoietic system, liver) or from tumours can be removed from the pt and cultured ex-vivo in the lab
• • Therapeutic gene may be introduced during further culture of such cells; this is then followed by re-infusion or re-implantation of these transduced cells into the pt
• • In the majority of cases, retroviral vectors are used to insert the therapeutic gene into the recipient’s cells
• In vivo gene therapy – organs (lung, brain, heart) are less suitable for ex vivo gene therapy, as culture of the cells or re-implantation isn’t feasible
• • When this is the case, somatic gene therapy can only be attempted by in vivo gene transfer, aka by administering the gene of interest either locally or systemically

Question 3

Process of ex vivo gene therapy

Answer 3

1. Cells are removed from pt
2. In the lab, a virus is altered so that it can’t reproduce
3. Gene is inserted into the virus
4. Altered virus is mixed w/ cells from the pt
5. Cells from the pt become genetically altered
6. Altered cells are injected into the pt
7. Genetically altered cells produce the desired protein or hormone

Question 4

Stable vs. transient gene transfer

Answer 4

• Stable = genetically altering original cells and it is passed on to daughter cells (this is what we want)
• Transient = genetically altering original cells and it isn’t passed on to daughter cells

Question 5

Potential target diseases for gene therapy

Answer 5

• Inherited disorders (ex: cystic fibrosis, adenosine deaminase, Duchenne’s muscular dystrophy, familial hypercholesterolemia, hemophilia)
• Cancer
• • Cytokine genes (ex: GM-CSF, IFN-gamma, and interleukins)
• • “Suicide genes” (ex: HSV-tk)
• • Tumour suppressor genes (ex: p53)
• • Protecting hematopoietic stem cells from toxic effects of chemotherapy by inserting a drug-resistant gene (ex: multiple drug resistance gene MDR-1)

Question 6

Function of cytokine genes

Answer 6

• Cytokines induce local inflammatory reaction in the tumour, which destroys a lot of the treated tumour
• Inflammation then induces an anti-tumour cell immune reaction, which destroys any surviving malignant cells in the primary tumour as well as the distant metastases

Question 7

Give an example of a suicide gene

Answer 7

• HSV-tk (herpes simplex virus thymidine kinase gene) is known to phosphorylate ganciclovir (systemically administered pro-drug)
• Phosphorylated ganciclovir is incorporated into the DNA of dividing cells, which leads to the termination of DNA-chain elongation => cell death
• Ganciclovir (GCV) – HSV-tk –> GCV-P – cellular kinases –> GVP-P(3)

Question 8

Give an example of the bystander effect

Answer 8

• When ganciclovir (GCV) is added to a person w/ HSV-tk, GCV is phosphorylated into the toxic product GCV-P
• The phosphorylated GCV is transported to neighbouring cells through gap junctions, killing all the surrounding cells

Question 9

Example of tumor suppressor gene

Answer 9

• p53 mutation occurs in a large number of cancers
• Delivery of native p53 gene into these cancers will have anti-proliferative effects on cancer cells
• Antisense genes targeted at oncogenes to reduce or abolish their expression, achieving anti-proliferative effects on these cells

Question 10

Give an example of protection of hematopoietic stem cells

Answer 10

• Multiple drug resistance gene (MDR-1) is isolated from drug resistant tumour cells, where MDR-1 pumps anticancer drugs out of the cells
• Transfecting MDR-1 into hematopoietic stem cells will protect these cells from the toxic effect of chemotherapy

Question 11

Gene therapy of cancer

Answer 11

• Cytokine gene therapy is intended for tx of both the primary tumour and distant metastases
• Suicide and tumour suppressor genes are designed to mediate direct cytotoxic or anti-proliferative effects on the tumour cells and are only effective for the tx of localized tumours
• MDR-1 gene therapy is expected to allow cancer px to tolerate higher doses of chemotherapy, thereby increasing the efficacy of therapy

Question 12

Gene transfer methods

Answer 12

• Can be generally divided into non-viral gene transfer or viral gene transfer
• Gene transfer in non-dividing cells – can be done for adenovirus, AAV, naked DNA, and liposome-mediated but not retrovirus
• Limitation of retrovirus = cell division required

Question 13

Examples of non-viral gene transfer

Answer 13

• Injection of naked DNA
• Particle bombardment
• Entrapping DNA in liposomes

Question 14

Describe injection of naked DNA

Answer 14

• Most straightforward procedure
• Direct injection of naked plasmid DNA into tissue allow scells of that tissue to take up DNA
• This method works w/ reasonable efficiency in muscle and skin
• Advantages = easy, safe, suitable for transfer of large gene constructs

Question 15

Describe particle bombardment

Answer 15

• Gene-gun is a high pressure or electrical discharge device, which forces microscopic gold or tungsten particles coated w/ DNA into tissues
• Electroporation makes use of the change of permeability of the cell membrane that is induced when a strong electrical field is applied; as a result, large molecules including DNA molecules are taken up by the cells

Question 16

Liposome-mediated DNA transfer

Answer 16

• Liposome/DNA complexes bind to the cellular membranes and are internalized into endosomes
• Relatively few DNA/plasmid molecules enter the nucleus and are expressed (0.1%) – due to the breakdown of DNA in acidic environment of endosomes
• Specific target of a particular tissue or cell type (Ab or proteins attach to the complex)

Question 17

Application of non-viral gene transfer methods

Answer 17

• Direct injection of naked DNA has the potential use in tx of skin disorders, and virus and cancer vaccination
• Gene-gun has application for tx of skin, liver, and tumours
• Liposome-mediated gene transfer is a common method of non-viral gene transfer and is currently being investigated in clinical trials w/ CF and cancer px

Question 18

Is non-viral gene transfer or viral gene transfer preferred and why?

Answer 18

• Viruses have natural capacity to infect cells and deliver their genes very efficiently to the nucleus of target cells
• In general, viruses have shown much more efficient gene transfer rates than non-viral gene delivery methods

Question 19

Retrovirus vectors – most commonly used one and what does it cause?

Answer 19

• Most commonly employed retroviral vectors are derived from Murine Leukemia Virus (MuLV)
• When these viruses are injected into newborn rodents, leukemia develops after a latency period
• Not associated w/ any known pathology in humans

Question 20

Describe the retroviral genome

Answer 20

5’-LTR pi+ gag pol env 3’-LTR

• 5’ LTR = long terminal repeat
• pi + (psi) = non-coding region required for encapsidation of RNA during virus particle formation (must have pi + to be able to replicate **very important)
• gag = internal capsid structural proteins
• pol = reverse transcriptase and integrase
• env = envelope protein
• 3’ LTR = long terminal repeat

Question 21

Features of retrovirus

Answer 21

• Can infect a wide variety of cell types w/ high efficiency
• Proviral copy integrated in a stable manner into the chromosomal DNA of the cell, thereby warranting life-long correction of the target cell types and its descendants
• Sequences required for viral replication can be physically separated into cis and trans acting elements – enables generation of replication defective recombinant retroviruses
• Cell division is needed for integration of retroviral DNA into host cell

Question 22

Generation of retroviral vectors – what occurs, what is needed, and when are they used?

Answer 22

• In vitro recombination of 1 retroviral genome w/ encapsidation signal (pi+ psi) w/ gene of interest
• Need a packaging cell line, which contains retroviral genome w/o encapsidation signal
• Recombinant retrovirus will be generated but isn’t stable, so it is difficult to purify viruses from medium
• Retroviruses used predominantly for ex vivo gene therapy
• Retrovirus vector needs cell division for stable gene expression
• Retrovirus vector won’t transduce terminally differentiated cells and other non-dividing cells

Question 23

Define lentivirus and give an example

Answer 23

• Lentivirus: any member of a genus of retroviruses that have long incubation periods and cause chronic, progressive, usually fatal diseases in humans and other animals
• • Species include the types of human immunodeficiency virus

Question 24

What is needed for generation of lentivirus?

Answer 24

• Need to perform in vitro recombination of 3 lentiviral vectors:
• • Packaging vector
• • Transducing vector w/ encapsidation signal
• • Envelope vector
• Need packaging cells w/o lentivirus genome

Question 25

Lentivirus application

Answer 25

• To express short-hairpin RNA (shRNA) to reduce expression of a specific gene
• To introduce a new gene into human or animal cells (ex: a model of mouse hemophilia is corrected by expressing wild-type coagulation factor VIII, which gene is mutated in human hemophilia)
• Lentiviruses have also been successfully used for transfection of diabetic mice w/ the gene encoding PDGF (platelet-derived growth factor) – a therapy being considered for use in humans

Question 26

What do adenoviruses normally infect? What is their genome like?

Answer 26

• Normally infect respiratory tract, eye, GI tract, and bladder
• In most cases, these infections are subclinical
• Adenovirus genomes are linear, double-stranded DNA, ranging from 36-38 kilo-base pairs
• Viral genomic DNA is flanked by 100-140 bp inverted terminal repeats (ITRs) which serve as replication origins

Question 27

Which genes are transcribed by the adenoviral genome during the immediate early phase?

Answer 27

E1a

Question 28

Which genes are transcribed by the adenoviral genome during the early phase?

Answer 28

E1b, E2a, E2b, E3, E4, and some virion proteins

Question 29

Which genes are transcribed by the adenoviral genome during the late phase?

Answer 29

Late genes, mostly virion proteins

Question 30

Generation of adenovirus vector

Answer 30

• Clone your gene of interest into a shuttle vector
• Transfect shuttle vector into packaging cells (such as HEK293 cells) which contains adenovirus genome
• In vivo recombination of shuttle vector w/ adenovirus genome in HEK293 cells
• Amplify and purify recombinant adenovirus
• Titration of the virus

Question 31

Features and safety of adenoviral vectors

Answer 31

• Causes benign infection
• Safety – lack of association w/ oncogenicity
• Well characterized and easily manipulated
• Stability and high titers of recombinant vectors
• Ability to infect a broad range of cell types, including dividing and nondividing cells
• High efficiency of cellular uptake of insert capacity (up to 37 kb)
• Little risk of random chromosomal integration

Question 32

What are oligonucleotides? What is their function?

Answer 32

• Short chains of chemically modified ribo or deoxyribonucleotides
• Have ability to bind to chromosomal DNA and mRNA through Watson-Crick base-pairing
• Can specifically alter gene transcription, translation, repair, and recombination
• It is possible to design therapeutic ONs that specifically target DNA sequences of transcription factor, or DNA sequences that are necessary for intramolecular folding

Question 33

Triple helix-forming oligonucleotides – what are they and what is their function?

Answer 33

• Specific type of ONs that bind to DNA and prevent transcription of mRNA
• Polypurine or polypyrimidine sequences of TFO can bind to a polypurine/ polypyrimidine region of genomic DNA, especially in the major groove of DNA region to form Hoogsteen H bonds
• As a result of this binding, TFO can prevent transcription initiation and elongation by prevention of promoter binding to specific DNA region
• *Triple helix blocks transcription

Question 34

Transcription factor decoys

Answer 34

• ONs that match the attachment site for the transcription factor
• The sequences that bind to transcription factors are known as consensus sequence or cis element
• Transcription factor decoys can lure the transcription factor away from its natural target and alter gene expression
• Limitation to control gene expression = many transcription factors are involved in regulation of a certain gene and many genes are controlled by a single transcription factor

Question 35

Antisense oligonucleotides

Answer 35

• “Classical” = single-stranded DNA or RNA molecules that are complementary to a sense mRNA sequences
• Can bind to its complementary sequence through Watson-Crick base-pairing

Question 36

Mechanism of antisense ONs

Answer 36

• mRNA-blocking – physically prevent or inhibit the progression of splicing or translation through binding of complementary mRNA sequence
• mRNA cleaving:
• • Induce degradation of mRNA by binding complementary mRNA sequences and recruiting the cytoplasmic nuclease (RNase H)
• • Induce degradation of mRNA by recruiting nuclear RNase P
• • Induce degradation of mRNA by inducing nuclease activity of the nucleic acid itself (ribozymes/ DNAzymes)

Question 37

RNA interfering

Answer 37

• Appears to be conserved in all eukaryotes and may play a role in prokaryotic cells
• Important endogenous process in regulation of gene expression/ translation

Question 38

Mechanism of RNAi

Answer 38

• dsRNA or shRNA are synthetic RNA molecules and can be introduced into cells
• • After digestion by Dicer, they will form siRNA duplex and form RISC w/ other enzymes or proteins
• After RISC binds to mRNA, it can slice mRNA
• Micro RNA is an endogenous RNA sequence and encoded by genes
• miRNA is transcribed by pol II to pri-miRNA and then form pre-miRNA
• • After digestion by Dicer, it forms miRNA duplex and then miRNA
• miRNA can bind other enzymes and proteins to form miRISC to silence gene expression

Question 39

miRNA vs. siRNA

Answer 39

• miRNA encoded by endogenous genes and recognize multiple targets
• siRNA derived from exogenous dsRNA or shRNA and can be specific to single target

Question 40

What are some endosomal escape strategies?

Answer 40

• Disrupt endosomes of target cells
• Addition of specific endosome destabilizing peptides
• Virus shells to DNA/liposome complex

Question 41

When viruses are exploited as vehicles for therapeutic genes, what criteria must they meet?

Answer 41

• Must be replication defective to prevent uncontrolled spreading of virus in vivo (unlike their wild-type variant)
• Virus itself should not possess undesirable properties
• Viral genome must be able to accommodate the therapeutic gene (size constraints)

Question 42

What are the main genes coding for the viral proteins of a lentivirus? What are the genes involved in?

Answer 42

• 5’-gag-pol-env-3’
• Other accessory genes depending on the virus (ex: HIV-1: vif, vpr, vpu, tat, rev, nef)
• • These genes involved in regulation of synthesis, processing viral RNA, and other replicative functions

Question 43

Advantages of lentivirus vector vs. other virus vectors?

Answer 43

• High-efficiency infection of dividing and non-dividing cells
• Long-term stable expression of a transgene
• Low immunogenicity