future therapies and technologies Flashcards
week 12
What diseases have an excellent potential to be treated by gene therapy and why?
recessive
loss-of-function mutations so replacing defective gene eliminates symptoms
What Autosomal dominant diseases will gene therapy work for?
NFM and retinoblastoma
Why aren’t dominant diseases effective in gene therapy?
most involve gain-of-function
more = worsen disease
Somatic vs germ-line gene therapy
Somatic: inserting normal gene so that these cells will make missing gene product
Germline: corrections made or extra genes added to germ line
What is the issue with the mechanical/chemical methods of gene therapy?
lacks specificity
How do liposomes deliver gene therapy?
Liposomes: lipid membrane microspheres with DNA –> merge cell memebrane and deliver DNA
How do microprojectiles deliver gene therapy?
gold coated DNA –> mechanically ‘shot’ –> some randomly integrate
what is the suitibiility of:
a- ex vivo
b- in situ
c- in vivo
for CF?
a- not good
b-good but not great as CF is a multisystem disorder
c- best as a vector is introduced to body and homes in on damaged cells.
What was the first approved gebe therapy and:
what did it treat:
what vector did it use:
what treat: LPL deficiency
vector: adenovirus
what are the two chromosomal therapies?
XIST- inactivates chromosomes
HAG - Human Artificial Chromosomes
What are the two methods of tissue engineering and what is the ADVANTAGE of it?
Biopsy –> culture cells on scaffold = graft
Genetically identical
(no rejection issues)
what are the two methods to produce 3-person babies?
embryo repair work
egg repair work
Embryo repair work
2 embryos formed (parent and donor)
(the bad mitochondria of the mother stay but take the nucleus and put it in donor one with good mitochondria)
Parent pronuclei replace donor pronuclei in donor embryo
Healthy embryo implanted
Egg repair work
egg from mother and donor
Genetic material REMOVED from both eggs
Mother’s genetic material inserted into donor egg (can be fert)
How does CRISPR-Cas 9 work?
clustered regularly interspaced short palindromic repeats
edit the genome (and potentially epigenome) by gRNA guiding a Cas9 enzyme to target and cleave DNA
what are the problems with CRISPR?
Lack of specificity of gRNA
Timing (best time to cut DNA)
Off-target effects:
What are off-target effects in relation to CRISPR?
unintended changes in DNA seq from editing in regions outside the targeted area.
what is the treatment for:
a- recessive
b-trisomy
c-epigenetic
a- replace defective diseases
b- insert XIST gene on chromosomes
c- cut histone tails and CpG islands
what is a benefit and a disdvantage of 3-person babies do
prevents transmission of mitochondrial mutations (not mother to mother)
no longer trace maternal ancestry
Treatment for:
a- cystic fibrosis
b- Down/patau/ Edwards
c- Prader-wili/ Angelman
a- In vivo gene therapy
b- Gene therapy or CRISPR
c- HAG or CRISPR- Cas 9
Treatment for:
A- B-thalassemia
B- HIV
C- Retinoblastoma
A- Ex vivo blood cell therapy or CRISPR Cas-9 (hemoglobin switching)
B- CRISPR Cas-9
C- In-situ (eye) gene therapy
What is the best treatment for:
A- leigh syndrome
B- Huntington’s disease
A- 3 parent baby
B- RNA interference on mRNA of HTT
What are the different types of stem cells?
Embryonic stem cells
Adult (s0matic) stem cells
Induced pluripotent stem cells (iPSCs)
What are the essential properties of stem cells?
Self-renewal
Differentiate into different cell types
What are embryonic stem cells?
Can become any cell in body (toti or pluriopotent)
Characteristics and function of Adult stem cells (somatic)
Exist naturally in body, maintain and repair tissues
Multipotent
Induced pluripotent stem cells (iPSCs)
Made via reprograming of an individuals own differentiated cells
Add 4 genes –> All genes available
How us DNA Microarray achieved?
Denature DNA
Mix probe and sample together
Binding indicated by signal
What are the two types of DNA microarray?
Expression profiling
Mutation profiling
purpose of Expression profiling vs mutation profiling
EP = com[ares relative gene expression
MP= detects polymorphisms
what are the only two offences under Prohibition of Human Cloning for Reproduction Act 2002
Creating or developing a human embryo by fertilization that contains genetic material provided by more than 2 people
Heritable alterations to genome.
What are the ethical issues surroinding gene therapies
Sacrifice embryo for embryonic stem cell
Altering germline
Stem cell toursim
Briefly describe the main strategies used for gene therapy.
Ex vivo- target cells are removed from tge body, cultured with vector then re-inserted
In situ- target tissie is dirtectly exposed (useful when only one tissue affected)
In vivo - vector introducted to body non-specifically and targets site in body.
What does CRISPR stand for and what RNA is used in it?
Clustered Regularly Interspaced Short Palindromic Repeats.
RNA = gudie RNA or gRNA
What is:
T- totipotent
P- plurioptent ‘
M- multipotent
T= stem cell that can develop into a completely new oragnism, all cell types including extra-embrynic cells of placenta
P= stem cell that can give rise to all cell types but CANT form a new organism
M= stem cells that can deveop into more than 1 cell type but more limited than P.
What can microarray technology be used for?
1- detect presence of polymorphsism (SNPS)
2- assess relative expression of genes present in a articular sample via deerming if mRNAs are present or absent between two different samples
What is attached to an expression microarray slide or DNA chip?
many thousands of different DNA probes in a grid, each represents a single gene, each spot has mamny copies of the same DNA probe
What chromosomes is the XIST gene on and what is its role?
X chromosome
involved in X-inactivation and result in Barr Body formation
haploinsufficiency
one degenerative allele is enough to cause the disease
What was the name of the first EMA approved gene therapy?
Glybera
cDNA is copied from what?
mRNA
What is a major benefit of CRISPR-Cas9 over older gene therapy tools?
it delivers a gene to a specific location in the genome.
