Gene therapy

Question 1

In vivo gene therapy

Answer 1

Vector administered directly to the patient
Lungs, skin and muscles are accessible organs
Liver, retina and brain less accessible
Adenovirus, adeno-associated and some retroviral vectors
Used for single gene disorders and acquired diseases

Question 2

Ex vivo gene therapy

Answer 2

Cells removed from patient and vector added to cells in vitro before being returned to the patient

Haematopoietic, epidermal and neural stem cells

Retrovirus mostly used as gene must integrate into the genome

Question 3

Barriers to gene therapy

Answer 3

Vector may be destroyed by antibodies
Vector may be integrated into chromosome or exist as episome (replicated separately)
Gene may be inserted into heterochromatin (silenced)
Protein may be displayed by MHC, triggering immune response

Question 4

Adenovirus advantages

Answer 4

Large capacity (up to 30kb)
Easily purified
Infect broad range of cell types

Question 5

Adenovirus disadvantages

Answer 5

High incidence of neutralising antibodies (common cold virus)
Capsid protein highly immunogenic
Potentially fatal inflammatory response

Question 6

Adeno-associated Virus

Answer 6

Small 4.7kb ssDNA genome
Non-pathogenic, minimal immune response
rep/cap genes replaced with expression cassette
Maintained as episome (can be used in non-dividing cells)

Question 7

LCA gene therapy

Answer 7

Leber’s congenital amaurosis
Early onset blindness
Autosomal recessive
RPE65 encodes retinal pigment epithelium specific protein (photoreceptor function)

AAV2 serotype capsids injected. Virus taken up by retinal epithelium and RPE65 expressed from episome. Light sensitivity restored

Question 8

SCID gene therapy

Answer 8

No cell mediated or humoral immune response due to lack of IL-2R or ADA

Haematopoietic stem/progenitor cell isolated from bone marrow and transduced with y-retrovirus containing ADA/IL-2R
Long-term reconstitution of lymphoid lineages

Question 9

X-linked SCID gene therapy

Answer 9

Restored immune function in 20 patients but 5 developed leukaemia.
Retrovirus inserted into proto-oncogene LMO2 and activated, promoting cell proliferation

Question 10

Problems with y-retroviral vectors

Answer 10

Preference for insertion near promoters of active genes
Strong enhancer and promoter in LTRs (can activate nearby oncogenes)
Splice donor site downstream of 5’ LTR (can splice exons of oncogenes)

Question 11

Lentiviruses

Answer 11

LTRs lack strong enhancer

Self-inactivating vectors delete LTRs for addition safety

Question 12

DNA vectors

Answer 12

Simple plasmids/minicircles
No pre-existing immunity
High capacity
Delivery in vivo difficult (cannot determine where vector goes)

Question 13

Site-directed nucleases

Answer 13

Guide RNA attached to nucleosome matches faulty sequence
Cas9 nuclease cleaves the DNA
Can disrupt abnormal gene through non-homologous end joining
Can correct faulty gene through homology directed repair

Prevent immunological response to viruses and insertional mutagenesis

Question 14

Programmable nucleases

Answer 14

Modified Cas9 can swap bases by adding in base editing enzymes
Results in deamination of cytosine to uracil.

Question 15

ASP-RNAi

Answer 15

Allele specific gene silencing by RNA interference

Enables silencing of a gene on one allele without affecting the wild-type allele