4 - Gene Therapy Flashcards
(95 cards)
1
Q
Gene therapy may be divided into what types ?
A
- Germ-line gene therapy
- Somatic gene therapy
2
Q
Describe germ-line gene therapy
A
aims for the introduction of therapeutic genes into germ-cells or omnipotent embryonal cells (at the 4-8 cellular stage)
3
Q
Describe somatic gene therapy
A
is the introduction of genes into somatic cells
4
Q
For a genetic gene disease, what therapy is it best to use?
A
germ-line therapy
5
Q
Ex-vivo gene therapy:
Cells from a number of _____ and ______ (skin, liver) or from tutors can be removed from the patient and cultured ex-vivo in the lab.
A
organs and tissues
6
Q
Ex-vivo gene therapy:
A therapeutic gene may be introduced during further culture of such cells. This introduction is them followed by ??
A
re-infusion or re-implantation of these transduced cells into the patients.
7
Q
Ex-vivo gene therapy:
In the majority of cases ____ ______ are used to insert the therapeutic gene into the recipient’s cells.
A
retroviral vectors
8
Q
In-vivo gene therapy:
Organs such as ___, ___, and _____ are less suitable for ex-vivo gene therapy, as culture of the cells or re-implantation is not feasible.
A
lung, brain, and heart
9
Q
In-vivo gene therapy:
Cells in the lung, brain, and heart, ____ gene therapy can only be attempted in vivo gene transfer, in other words by administering the gene of interest either locally or systemically.
A
somatic
10
Q
The cell that we use has to be able to ______ and _____ the gene.
A
isolate and culture
11
Q
List the two types of gene transfer
A
stable and transient
12
Q
Describe Stable Gene Transfer
A
-Should be able to express how the cell divides
Two ways you can do this:
1-Incorporate your vector into a chromosome (so when the chromosome replicates and the cell divides, your gene is carried onto the daughter cell)
2-Another way is you can introduce an origin of replication so it can replicate in the cell and then can also carry on into the daughter cell
13
Q
Describe Transient Gene Transfer
A
If the gene doesn’t have an origin of replication, it will not integrate into the chromosome so then it won’t replicate; it will only be expressed in one generation of the cell and after this generation the gene will be lost
14
Q
List some inherited disorders that are potential target diseases for gene therapy
A
- Cystic fibrosis
- Adenosine deaminase
- Duchenne’s muscular dystrophy
- Familial hypercholesterolemia
- Hemophilia
15
Q
____ is another target for gene therapy
A
cancer
16
Q
**EXAM Q
What are the 4 strategies for gene therapy for cancer
A
- Cytokine genes, such as GM-CSF, IFN-y and interleukins
- “Suicide genes” such as the herpes simplex virus thymidine kinase gene (HSV-tk)
- Tumor suppressor genes such as p53
- Protection of hematopoietic stem cells from toxic effects of chemotherapy by inserting a gene that confers drug resistance, such as multiple drug resistance gene MDR-1
17
Q
What do cytokines do?
A
Induce a local inflammatory reaction in the tumor, which destroys a significant fraction of the treated tumor.
The inflammation in turn induces an anti-timor cell immune reaction, which destroys any surviving malignant cells in the primary tumor as well as the distant metastases
18
Q
If your cancer is metastatic (moved to other areas of the body), what is the best way to treat it and why?
A
cytokine gene therapy
because cytokines can kill your local tumor cells and get to circulate throughout your body so if your cancer is metastatic this is the best way to treat the metastatic cancer
19
Q
HSV-tk is known to phosphorylate the systemically administered pro-drug _______
A
ganciclovir (a nucleotide analogue)
20
Q
Phosphorylated gangciclovir is then incorporated into the DNA of _____ cells which leads to the termination of DNA-chain elongation, resulting in cell death.
A
dividing
21
Q
Cells surrounding a cell that expresses _____ are also killed after ganciclovir treatment (the bystander effect)
A
HSV-tk
22
Q
What phosphorylates the ganciclovir?
A
tyrosine kinase (tk) genes
this is what we want to integrate into our cancer cells using gene therapy
23
Q
Once ganciclovir has been phosphorylated __ times, it is very toxic to the cancer cells.
A
3
24
Q
In the Bystander effect:
The phosphorylated GCV is transported to neighbouring cells through ___ ______.
A
gap junctions
25
Its best to use __ _____ gene therapy with the bystander effect.
ex vivo
26
_____ _________ genes are mutated in cancers (more frequently in liver cancer).
Because we know this gene is mutated in cancers and is a large cause of cancer developing, we want to put more of the original gene (NOT mutated) into the cancer cells so it can treat the cancer and kill the cancer cells
tumor supressor
27
___ mutation occurs in a large number of cancers on tumor suppressor genes.
p53
28
Delivery of native p53 gene into these cancer will have _______-_________ effects on the cancer cells.
anti-proliferative
29
______ genes targeted at oncogenes to reduce or abolish their expression, achieving anti-proliferative effects on these cells.
Antisense
30
Antisense genes inhibit ______ expression (these genes can cause cancer)
oncogene
31
Describe the protection of hematopoietic stem cells
You expel drugs from the inside of the cell to the outside of the cell to protect the normal cells from the anticancer toxic effects
32
Give an example of protection of hematopoietic stem cells
- Multiple drug resistance gene (MDR-1 is isolated from drug resistant tumor cells, where DMR-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
33
Cytokine gene therapy is intended for treatment of both the primary tumor and distant _________
metastases
34
Suicide and tumor suppressor genes are designed to mediate direct ______ or _____-_______ effects on the tumor cells and are only effective for the treatment of localized tumors
cytotoxic
anti-proliferative
35
_____ gene therapy is expected to allow cancer patients to tolerate higher doses of ______, thereby increasing the efficacy of the therapy.
MDR-1
chemotherapy
36
A variety of gene transfer systems are currently employe to insert therapeutic genes into ______ cells.
somatic
37
List 2 types of gene transfer methods
- Non-viral gene transfer
| - Viral gene transfer
38
What does non-viral gene transfer systems include?
- The injection of naked DNA
- Particle bombardment
- Entrapping DNA in liposomes
39
Describe the injection of naked DNA
-This is the most straightforward procedure
-The direction injection of naked plasmid DNA into tissue allow cells of that tissue to take up the DNA
-This method work with reasonable efficiency in muscle and skin
The advantages are clear: it is easy, safe and suitable for the transfer of large gene constructs
40
Describe particle bombardment
- The gene-gun is a high pressure or electrical discharge device, which forces microscopic gold or tungsten particles coated with 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
41
Once your cell membrane opens up due to electrical discharge, your ___ _____ can go inside your cell.
gene particle
42
Describe liposome-mediated DNA transfer
- The liposome/DNA complexes bind to the cellular membranes and are internalized into endosomes
- Relative few DNA/plasmid molecules enter the nucleus and are expressed (0.1%), due to the breakdown of DNA in acidic environment of endosomes.
- Endosomal escape strategies (disrupt endosomes of target cells, addition of specific endosome destabilizing peptides, virus shells to DNA/liposome complex)
- Specific target of a particular tissue or cell type (antibodies or proteins attach to the complex)
43
Describe liposome-mediated DNA complexes
- Trap your DNA inside liposome particles; and because liposomes fuse with the phospholipids of the cell membrane and then they are trapped and carried into the endoscope and can deliver the DNA into your nucleus.
- Only a few DNA molecules enter the nucleus because the endoscope is acidic and can destroy your DNA, therefore only a very small amount can reach the nucleus and get expressed.
44
Describe the application of non-viral gene transfer methods
- The direct injection of naked DNA has the potential to the treatment of skin disorders, and virus and cancer vaccination.
- The gene-gun has the application for the treatment of skin, liver and tumors.
- Liposome-mediated gene transfer is a common method of non-viral gene transfer and is currently being investigated in clinical trials with cystic fibrosis patients and cancer patients.
45
Describe gene transfer using recombinant viruses.
- Viruses has a natural capacity to infect cells and deliver their genes very efficiently to the nucleus of the target cells.
- In general, viruses have shown much more efficient gene transfer rates than non-viral gene delivery methods !!
46
When viruses are exploited as vehicles for therapeutic genes, they must meet the following criteria:
-what is the criteria ?
- They must be replication defective to prevent uncontrolled spreading of virus in vivo (unlike their wild-type variant)
- The virus itself should not possess undesirable properties
- The viral genome must be able to accommodate the therapeutic gene (size constraints)
47
The most commonly employed retroviral vectors are derived from what virus?
Murine Leukemia Virus (MuLV)
48
What happens when retrovirus vectors are injected into newborn rodents?
leukemia develops after a latency period
49
When retrovirus vectors are injected into humans, what happens?
not associated with any known pathology - safe for us to use
50
Describe what a retrovirus is
means that it uses an RNA gene; has to go through the retro replication; so it is RNA converted to DNA
51
List the parts of a retroviral genome
- 5' LTR
- psi
- gag
- pol
- env
- 3' LTR
52
Parts of a retroviral genome:
| Describe the 5' LTR
long terminal repeat
53
Parts of a retroviral genome:
| Describe the psi
non-coding region required for encapsidation of the RNA during virus particle formation
**doesn't code for anything but is required for the encapsidation of the RNA into the virus particle; this is a very important component of the genome
54
Parts of a retroviral genome:
| Describe the gag
internal capsid structural proteins
55
Parts of a retroviral genome:
| Describe the pol
reverse transcriptase and integrase
56
Parts of a retroviral genome:
| Describe the env
envelope protein
57
Parts of a retroviral genome:
| Describe the 3' LTR
long terminal repeat
58
List some features of a retrovirus
- They can infect a wide variety of cell types with high efficiency
- The proviral copy integrated in a stable manner into the chromosomal DNA of the cell, thereby warranting lifelong correction of the target cell type and its descendants
- The sequences required for viral replication can be physically separated into cis and trans acting elements. This enables the generation of replication defective recombinant retroviruses
- Cell division is needed for the integration of retroviral DNA into the host cell
59
Describe the generation of retroviral vectors
-In vitro recombination of one retroviral genome with encapsidation signal (psi) with gene of interest.
60
Generation of retroviral vectors:
| -Need a ______ ____ line, which contains retroviral genome without encapsidation signal
packaging cell
61
Generation of retroviral vectors:
| Recombinant retrovirus will be generated but it is not _____. So it is difficult to purify viruses from medium.
stable
62
Generation of retroviral vectors:
| Retroviruses are used predominantly for __ _____ gene therapy
ex vivo
63
Generation of retroviral vectors:
| Retrovirus vector needs cell division for ____ gene expression
stable
64
Generation of retroviral vectors:
| Retrovirus vector will not transduce what type of cells?
terminally differentiated cells and other non-dividing cells
*lung, heart, would not be able to use this vector in these genes
65
What is a 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.
66
Lentivirus Vector:
There are 3 main genes coding for the ____ proteins
5'-gag-pol-env-3'
viral
67
Lentivirus Vector:
| There are other accessory genes that are involved in ?
- regulation of synthesis
- processing viral RNA
- other replicative functions
68
The generation of a Lentivirus needs to be done in vitro recombination of 3 lentiviral vectors. What are the 3 lentiviral vectors?
- Packaging vector
- Transducing vector with encapsidation signal
- Envelop vector
69
List 3 advantages of lentivirus vector versus other virus vectors
- high-efficiency infection of dividing and non-dividing cells
- long-term stable expression of a transgene
- low immunogenicity
70
What are some purposes of Lentivirus application?
- To express short-hairpin RNA (shRNA) to reduce the expression of a specific gene
- To introduce a new gene into human or animal cells
- Lentiviruses have also been successfully used for transfection of diabetic mice with the gene encoding PDGF (platelet-derived growth factor). This is a therapy being considered for use in human.
71
Give an example of lentivirus introducing a new a gene into human or animal cells.
A model of mouse hemophilia is corrected by expressing wild-type coagulation factor VIII, which gene is mutated in human hemophilia.
72
Adenoviruses are a large part of a family of ___ viruses.
DNA
73
What do Adenoviruses normally infect?
The respiratory tract, eye, gastrointestinal tract and bladder. In most cases, those infections are subclinical (not very severe).
74
Describe the generation of an adenovirus vector
- Cloning your gene of interest into a shuttle vector
- Transfection of shuttle vector into packaging cells such as HEK293 cells, which contains adenovirus genome
- In vivo recombination of shuttle vector with adenovirus genome in HEK293 cells
- Amplification and purification of recombinant adenovirus
- Titration of the virus
75
What are some features of adenoviral vectors?
- Causes benign infection
- Safety - lack of association with 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 (will not integrate into your own chromosomes; this is an advantage)
76
What are Oligonucleotides?
short chains of chemically modified ribo or deoxyribonucleotides
77
What do the Oligonucleotides have the ability to do?
bind to chromosomal DNA and mRNA through Watson-Crick base-pairing
78
What can Oligonucleotides specifically intervene?
gene transcription, translation, repair and recombination
79
It is possible to design therapeutic ONs, that specifically target DNA sequences of transcription factor or DNA sequences that are necessary for ________ __________
intramolecular folding
80
What are triple helix-forming oligonucleotides?
TFO's are a specific type of oligonucleotide that binds to DNA and prevents transcription of mRNA.
81
TFOs can bind to specific spots, and then what happens?
TFO can prevent transcription initiation and elongation by prevention of promoter binding to specific DNA region.
Triple helix blocks the double stranded DNA from opening up to transcribe it into mRNA, therefore you cannot replicate the DNA or have gene expression
82
What are transcription factor decoys?
* they match the attachment site for the transcription factor
* normally transcription factors need to bind to the cis-element (CAT box); we synthesize the TFD's to bind where the transcription factor normally binds which inhibits your gene expression
83
What are antisense oligonucleotides?
They are the oldest oligonucleotide-based therapeutic approach.
Bind to your mRNA and can block the translation or it can cause mRNA degradation altogether.
They bind to it's complementary sequence through Watson-Crick base-pairing.
84
What are the 2 mechanisms of antisense ON's?
- mRNA blocking
| - mRNA cleaving
85
Describe mRNA-blocking mechanism of antisense ON
Physically prevent or inhibit the progression of splicing or translation through binding of complementary mRNA sequence
86
Describe mRNA cleaving mechanism of antisense ON
- 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)
87
What is Fomivirsen sodium?
- It is a phosphorethioate oligonucleotide
- It has twenty one nucleotides
- Indicated for local treatment of cytomegalovirus (CMV) retinitis in patients with AIDS who are intolerant of or have contraindication to other treatments or who hare insufficiently responsive to previous treatment of CMV retinitis
88
RNAi
RNA interfering
89
RNAi was first described in the _______
nematode
90
The silencing phenomenon of RNA interfering also occurs in ??
plants, protozoa, fungi and animals
91
RNAi is conserved in ________ and may play a role in prokaryotic cells
eukaryotes
92
What is RNAi?
It is an important endogenous process in regulation of gene expression/translation
93
Describe the mechanisms of RNAi (dsRNA or shRNA)
- dsRNA or shRNA are synthetic RNA molecule
- these molecules can be introduced into cells
- after digestion by Dicer, they will form siRNA duplex and form RISC with other enzymes or proteins
- after RISC binds to mRNA, it can slice mRNA
*once it has a dicer, it can cut the mRNA into small fractions called siRNA (small-interfering RNA)
94
Describe the mechanisms of RNAi (MicroRNA)
- microRNA is an endogenous RNA sequence and encoded by genes
- miRNA is transcripts by Pol 2 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
95
Describe the difference between miRNA verses siRNA
miRNA:
- encoded by endogenous genes
- recognize multiple targets
siRNA:
- derived from exogenous dsRNA or shRNA
- can be specific to single target
