TOOLS FOR GENE THERAPY Flashcards
What are the three main mechanisms of gene therapies
- 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
what are the most commonly used tools for neurological disorders
adeno-assoicated viral vector and lentivirus vectors
AAV characteristics (5)
- 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
Lentivirus vectors properties (6):
- 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
Life cycle of AAV
- 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
Life cycle of LVs:
- derived from HIV-1
- LVs integrate preferentially in introns of transcriptionally active genes
Ex vivo gene therapy
- 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
Advantages of Ex vivo gene therapy (3)
- widespread therapy throughout brain
- other affected organs can be targeted
- protocol for bone marrow transplants well established.
Disadvantages of Ex vivo gene therapy (3)
- Only suitable for certain diseases
- Risks of insertional mutagenesis
- Risks associated with bone marrow engraftment.
Retroviral vectors
- Derived from moloney murine leukimia virus
- large packing capacity (7-9kB)
- produce at relatively low yields
Adenoviral vectors
- 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
What is biodistribution
the spreading of the vector in tissue/organs following administration
In vivo gene delivery to CNS options
- 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.
Battens disease and parenchymal administration
- 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
Advantages of Parenchymal administration (4)
- 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
Disadvantages of Parenchymal administration (3)
- Spread of virus is limited
- Not always feasible for whole brain pathology
- Surgically invasive.
what is ICV delivery
intracerebroventricular - an intrathecal method.
what is CM delivery
- intrathecal
cisterna magna delivery - one of three principal openings in the subarachnoid space. Problematic due to sensitive vasculature.
what is IT delivery
intrathecal delivery.
- non-invasive and rapid delivery to the CSF.
intrathecal delivery advantages (4)
- direct delivery of vector to the CNS
- administration of larger doses
- widespread delivery throughout the CNS
- clinically translatable
intrathecal delivery Disadvantages (2)
- invasive procedure - especially ICV/CM
- penertation to deeper/distal areas remains a question
systemic delivery
- achieves body-wide cellular targets
- but off-target? can be an advantage but…
- AAV are optimal for this
- currently used as a one-off as patients develop immune responses to protein capsid, vector genome and transgene product.
- PLUS, most of Pop has pre-existing ABs against WT AAV.
- LIVER toxicity so worrying.
What is Capsid Engineering?
- novel synthetic capsids with improved neuronal tropisms express a hundred times more than standard AAV9
- Not always translatable to humans.
Advantages of systemic administration (3)
- Minimally invasive.
- Can target the entire nervous system.
- Can also target other affected organs (global delivery).
Disadvantages of systemic administration (3)
- High dose correlated with higher costs and risks
- Immunogenicity and possible toxicity
- Early intervention may be essential in certain diseases.
what is mutagenesis
Mutagenesis is the process by which an organism’s deoxyribonucleic acids (DNA) change, resulting in a gene mutation
