GEN: Gene Therapy Flashcards
what is gene therapy?
use of recombinant genetic material under different forms or pharmaceutical preparations as a therapeutic agent
what are the two forms of gene therapy?
somatic gene therapy (legally permitted) and germline gene transfer (not permitted)
what are the two approaches of gene therapy delivery?
- in vivo
- ex vivo
what is in vivo?
systemic delivery or direct injection into affected organ
what is ex vivo?
isolation of cells from patient and place in tissue culture, genetically correct with vector (viral) and return cells to patient
what are 3 common components of gene therapy protocols?
- target tissue: accessible & manipulable
- efficient gene delivery: viral/non-viral vectors (genetic or chemical)
- transcriptional control: sustained & sufficiently high therapeutic effect
what relationship do these components of gene therapy protocols have?
mutual interdependency - requirements within one area puts restrains on others
what is the ideal cell target?
pluripotent, self-regenerating stem cells from patient being treated
Bone marrow*
Epidermis (skin)*
Skeletal muscle
Neural (numerous areas of CNS)
Adipose
*In clinical trials or approved
what are problems to consider when choosing target tissue?
- access
- non-dividing
- ex vivo procedure only
what are 5 examples of viral vectors?
adenoviral vector
adenovirus associated vector
herpes simplex virus
retroviral vector
lentviral vector
what are non-integrating viral vectors?
viral genetic material does not integrate into genome of targeted cells ⇒ restricted
adenovirus, AAV,
HSV
what are integrating viral vectors?
viral genetic material does integrate into genome of targeted cell ⇒ PERMANENT fixture ⇒ long-term expression
- retroviral, lentiviral
what is the major advantage of viral vectors?
highly efficient at infecting cells ⇒ high delivery efficiency
what are issues with viral vectors?
- retroviral infect/transduce dividing cells only
- lentiviral infect/transduce both dividing/non-dividing cells
- genetic size limitation - virus has a limited size
- mostly non-targetable, local delivery in vivo
- systemic delivery of some AAV vector serotypes; preferential tissue transduction
- ex vivo applications with retroviral/lentiviral vectors only
- some (adenoviruses) can be highly immunogenic ⇒ restrict repeat administration
- viral vectors difficult to produce/store ⇒ expensive manufacture
what are lentiviral vectors based on?
HIV
what type of virus are adenosine-associated virus (AAV) vectors?
non-pathogenic human virus
how can the AAV genome be described?
single stranded DNA with two genes: Rep, Cap
how is AAV converted into therapeutic vector?
removal of Cap and Rep and replace with therapeutic gene cassette by introducing to HEK293T cells: Cis, Trans and Helper virus
- the helper virus be?
adenovirus or herpes virus for manufacture
what was the targets for gene addition gene therapy via ex vivo procedures?
- haematopoietic stem cells
- epidermal stem cells in junctional epidermolysis bullosa
- t cells in car-t cell therapy for b-lymphoma
what are the clinical trials for gene addition gene therpay via in vivo procedures?
- haemophilia b
- parkinson’s
- leber’s congential amaurosis
- spinal muscular atrophy
- lipoprotein lipase deficiency
what vector targets haematopoietic cells?
retoriviral vectors
what is X-SCID?
x-linked severe combined immunodeficiency ⇒ boys have complete absence of NK + T-cells in peripheral blood due to a mutation of gamma chain blocking T cell development
how can X-SCID be treated
residence in sterile chamber, antibiotics, bone marrow transport is a cure
in the trial for X-SCID what gene therapy method and vector was used and why?
- ex vivo because direct delivery is not possible to hematopoietic stem cells
- gammaretroviral vector ⇒ permanently integrate genetic material into hematopoietic stem cells of patient ⇒ lifelong cure
- bone marrow CD34-positive stem cells isolated
what was seen in the severe adverse clinical trials for SCID-X1?
inappropriate activation of growth regulator oncogene in t-cell leukemia of patients - LMO2, CCND2
what is SCID-ADA?
deficiency of adenosine deaminase, a purine metabolic effect ⇒ accumulation of toxic metabolites adenosine/deoxy-adenosine ⇒ imparied lymphocyte development + function
what is the normal treatment for SCID-ADA?
enzyme replacement therapy and bone marrow HSC transplant
what is the gene therapy for SCID-ADA?
- accumulation of toxic metabolites and death of uncorrected cells
- selective advantage to cells producing sufficient vector-derived ADA = therapy
what vector is used for SCID-ADA?
ex-vivo, haemotopoietic gammaretroviral vector with BM CD34+ cells isolated
what has the patient undergone while waiting for vector for SCID-ADA?
partial BM destruction - non-myeloablative conditioning called busulfan chemotherapy to destroy diseased BM cells allowing gene corrected BM cells to become dominant component
what is X-CGD?
x-linked chronic granulomatous disease = primary immunodeficiency caused by defect in oxidative antimicrobial activity of phagocyte (neutrophils) ⇒ mutation of 4 genes encoding subunit of NADPH oxidase complex
what is the gene for X-CGD?
gp91phox
what was the result in the X-CGD trial?
both men in the trial were able to restore anti-microbial activity of gene corrected cells: with clearance of Staphylococcus aureus and aspergillus fumigatus infections but 27mo after gene therapy one man suffered colon perforation bacterial sepsis and died
SFFV promoter driving expression (not enhancer) was silenced ⇒ methylated
what is junctional epidermolysis bullosa?
mutation in gene encoding basement membrane component laminin 5, fatal skin adhesion disorder
what was the outcome OF junctional epidermolysis bullosa?
complete epidermal regeneration on both legs on day 8 with normal-looking epidermis maintained throughout 1yr follow up and full, normal, robust skin function was restored ⇒ LAMB3 restored
what is metachromatic leukodystrophy MLD?
autosomal recessive lysosomal storage disease resulting from mutations of ARSA gene (deficiency of arylsulfatase A enzyme)
what was the conclusion of metachromatic leukodystrophy MLD?
- stable engraftment: polyclonal, all lineages
- stable ARSA enzyme activity
what happens in beta-thalassaemia?
Hb has 2 alpha and 2 beta, but b-thalassaemia does not have enough beta chains
