TOOLS FOR GENE THERAPY Flashcards
1
Q
What are the three main mechanisms of gene therapies
A
- replacing a disease-causing gene with a healthy copy of the gene
- inactivating a disease-causing gene that is not properly functioning
- introducing a new/modified gene
2
Q
what are the most commonly used tools for neurological disorders
A
adeno-assoicated viral vector and lentivirus vectors
3
Q
AAV characteristics (5)
A
- non-envleoped, ssDNA parvoviruses
- non-pathogenic
- 4.7kB DNA packaging capacity
- can transudce both dividing and non-dividing cells
-protein shell more defined based on serotypes
4
Q
Lentivirus vectors properties (6):
A
- commonly used for ex vivo gene therapy
- enveloped, ssDNA viruses
- packaging capacity of 10kb
- broad cell tropism (e.g. neurons and glia)
- ability to transduce both dividing and nondividing cells
- integration into host genome leads to stable transgene expression
5
Q
Life cycle of AAV
A
- binds to surface repector - enters cell via clathrin-coated pit
- vector particles accumulate around perinuclear space
- enter the nucleus via nuclear pore complexes
- inside nucleus - the vector genome released and transcribed
- resulting in expression of transgene
6
Q
Life cycle of LVs:
A
- derived from HIV-1
- LVs integrate preferentially in introns of transcriptionally active genes
7
Q
Ex vivo gene therapy
A
- remove bone marrow - transduce HSC with viral vector carrying gene
- condition patients and replace transduced HSCs
- all progeny cell lineages express the gene incl. microglia
-microglia progenitor cell can mirgate and cross BBB - expression in the brain
8
Q
Advantages of Ex vivo gene therapy (3)
A
- widespread therapy throughout brain
- other affected organs can be targeted
- protocol for bone marrow transplants well established.
9
Q
Disadvantages of Ex vivo gene therapy (3)
A
- Only suitable for certain diseases
- Risks of insertional mutagenesis
- Risks associated with bone marrow engraftment.
10
Q
Retroviral vectors
A
- Derived from moloney murine leukimia virus
- large packing capacity (7-9kB)
- produce at relatively low yields
11
Q
Adenoviral vectors
A
- large packaging capacity (up to 36kB)
- trasngene does not integrate into the host genome and instead remains episomal
- leads to stable and sustained in brain
- direct infusion into brain results in gene transfer to broad range of cell populations
- in vivo delivery at high dose - cytotoxicity and neuroinflammation
12
Q
What is biodistribution
A
the spreading of the vector in tissue/organs following administration
13
Q
In vivo gene delivery to CNS options
A
- Direct intracranial (intraparenchymal) injection - bypass BBB
- Delivery and distribution through the CIV intracerebroventricular, IT intrathecal, CM cisterna magna
- systemic administration of vectors that cross BBB.
14
Q
Battens disease and parenchymal administration
A
- rare autosomal recessive lysosomal storage disorder
- caused by loss of TPP1 enzyme, causing severe neurodegeneration and vision loss.
- targetted using AAV2-hCLN1
- drilled six holes -12 injections
- slowed disease progression
15
Q
Advantages of Parenchymal administration (4)
A
- Direct delivery of vector to the brain
- Vector expression is restricted to a limited region of brain
- Ideal if very specific region affected (e.g. PD or HD)
- Minimal dosage with reduced risks
