TOOLS FOR GENE THERAPY

Question 1

What are the three main mechanisms of gene therapies

Answer 1

1. replacing a disease-causing gene with a healthy copy of the gene
2. inactivating a disease-causing gene that is not properly functioning
3. introducing a new/modified gene

Question 2

what are the most commonly used tools for neurological disorders

Answer 2

adeno-assoicated viral vector and lentivirus vectors

Question 3

AAV characteristics (5)

Answer 3

• 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

Question 4

Lentivirus vectors properties (6):

Answer 4

• 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

Question 5

Life cycle of AAV

Answer 5

• 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

Question 6

Life cycle of LVs:

Answer 6

• derived from HIV-1
• LVs integrate preferentially in introns of transcriptionally active genes

Question 7

Ex vivo gene therapy

Answer 7

• 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

Question 8

Advantages of Ex vivo gene therapy (3)

Answer 8

1. widespread therapy throughout brain
2. other affected organs can be targeted
3. protocol for bone marrow transplants well established.

Question 9

Disadvantages of Ex vivo gene therapy (3)

Answer 9

1. Only suitable for certain diseases
2. Risks of insertional mutagenesis
3. Risks associated with bone marrow engraftment.

Question 10

Retroviral vectors

Answer 10

• Derived from moloney murine leukimia virus
• large packing capacity (7-9kB)
• produce at relatively low yields

Question 11

Adenoviral vectors

Answer 11

• 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

Question 12

What is biodistribution

Answer 12

the spreading of the vector in tissue/organs following administration

Question 13

In vivo gene delivery to CNS options

Answer 13

1. Direct intracranial (intraparenchymal) injection - bypass BBB
2. Delivery and distribution through the CIV intracerebroventricular, IT intrathecal, CM cisterna magna
3. systemic administration of vectors that cross BBB.

Question 14

Battens disease and parenchymal administration

Answer 14

• 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

Question 15

Advantages of Parenchymal administration (4)

Answer 15

• 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

Question 16

Disadvantages of Parenchymal administration (3)

Answer 16

• Spread of virus is limited
• Not always feasible for whole brain pathology
• Surgically invasive.

Question 17

what is ICV delivery

Answer 17

intracerebroventricular - an intrathecal method.

Question 18

what is CM delivery
- intrathecal

Answer 18

cisterna magna delivery - one of three principal openings in the subarachnoid space. Problematic due to sensitive vasculature.

Question 19

what is IT delivery

Answer 19

intrathecal delivery.
- non-invasive and rapid delivery to the CSF.

Question 20

intrathecal delivery advantages (4)

Answer 20

1. direct delivery of vector to the CNS
2. administration of larger doses
3. widespread delivery throughout the CNS
4. clinically translatable

Question 21

intrathecal delivery Disadvantages (2)

Answer 21

1. invasive procedure - especially ICV/CM
2. penertation to deeper/distal areas remains a question

Question 22

systemic delivery

Answer 22

• 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.

Question 23

What is Capsid Engineering?

Answer 23

• novel synthetic capsids with improved neuronal tropisms express a hundred times more than standard AAV9
• Not always translatable to humans.

Question 24

Advantages of systemic administration (3)

Answer 24

• Minimally invasive.
• Can target the entire nervous system.
• Can also target other affected organs (global delivery).

Question 25

Disadvantages of systemic administration (3)

Answer 25

* High dose correlated with higher costs and risks * Immunogenicity and possible toxicity * Early intervention may be essential in certain diseases.

Question 26

what is mutagenesis

Answer 26

Mutagenesis is the process by which an organism's deoxyribonucleic acids (DNA) change, resulting in a gene mutation