Future treatments of genetic diseases Flashcards

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1
Q

under development “translation research”

A
pharmacologic chaperones
stop codon read-through
RNA interference
genome editing
stem cell therapy
gene therapy
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2
Q

chaperones help nascent proteins to

A

fold properly

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3
Q

pharmacologic charperones are

A

potent small molecule reversible competitive enzyme inhibitors. At inhibitory concentrations, these active site directed molecules bind to and induce/enhance conformational stability of the mutant enzyme at neutral pH in the endoplasmic reticulum.

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4
Q

function of pharmacologic chaperones

A

they enable/enhance proper transit from the Er for processing and trafficking to the site of function where the chaperone is displaced by high substrate concentration.

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5
Q

pharmacologic chaperone therapy

A

increased enzymatic activity predicts clinical benefit

orally administered

biodistribution to sites inaccessible to other therapies.

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6
Q

Kalydeco

A

a pharmalogic chaperone developed for cystic fibrosis. kalydeco is designed to improve the protein’s function once it has reached the cell surface.

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7
Q

chaperone Vx-809

A

a pharmacologic chaperone developed for cystic fibrosis. It moves the CF protein with deltaF508 mutation to the cell surface.

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8
Q

most common CF mutation

A

delta F508. It creates a defective protein that does not move to its proper place at the cell surface.

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9
Q

results from a phase 2 clinical trial of Kalydeco and VX-809 in multiple combinations showed significant improvements in

A

lung function in people with two copies of the deltaF508 mutation.

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10
Q

chaperone VX is designed to

A

move the defective delta F508 to the cell surface.

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11
Q

the triple combination of of the corrector VX-440 with tezacaftor (VX-661) and ivacaftor has the potential to

A

benefit people with CF who are homozygous and heterozygous for the F508 del mutation.

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12
Q

cavosonstat (for CF) works by

A

inhibiting an enzyme called S-nitrosoglutathione reductase, which is thought to modulate the unstable and defective CFTR protein.

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13
Q

when GSNOR is blocked,

A

GSNO levels are restored.

This modifies the chaperones, which are responsible for CFTR degradation, and stabilizes the CFTR protein.

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14
Q

In preclinical studies, cavosonstat was shown to

A

increase and prolong the function of the CFTR protein, leading to an increase in net chloride secretion.

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15
Q

aminoglycoside antibiotics such as gentamicin influence

A

the fidelity of reading of the stop-codon recognition process and enhance the exent of read-thorugh so that a stop codon could be misread as coding for an amino acid.

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16
Q

eteplirsen

A

it directs cellular mchinery to skip exon 51.

This allows enough dystrophin production to essentially transform Duchenne muscular dystrophy to Becker, a less severe form of the disease.

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17
Q

long double stranded RNAs can be used to

A

silence the expression of target genes in a variety of organisms and cell types.

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18
Q

upon introduction the long dsRNAs enter the

A

RNA interference RNAi pathway.

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19
Q

RNAi pathway

A

the dsRNAs get processed into 20-25 nt long siRNAs by an RNase III like enzyme called dicers.

The siRNAs assemble into RNA induced silencing complexes. The siRNA strands guide the RISCs to complementary RNA molecules, where they cleave and destroy the RNA.

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20
Q

gene therapy

A

a technique that uses inserted inserted wild type genes to treat or prevent disease.

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21
Q

gene therapy can involve

A

replacing a mutated gene that causes disease with a healthy copy of the gene.
inactivating, or “knocking out” a mutated gene that is functioning impropoerly
introducing a new gene into the body to help fight a disease.

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22
Q

ex vivo gene transfer

A

gene introduced via virus -> transduce in vitro -> return to patient -> explant cells

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23
Q

in vivo gene transfer

A
direct gene delvery via
adenovirus
liposomes
naked DNA
gene linked to ligands
24
Q

ideal viral vector

A
replication defective
high titer, infects broad cell range
stable, site specific integration or episomal
high level expression
unique promoters
non-toxic
25
Q

BMN 270 and hemophilia

A

patients with hemophilia A are not able to produce enough function factor VIII to prevent bleeding, and all high dose patients improved from severe to either moderate, mild or normal range in terms of factor levels.

26
Q

The correction of beta hemoglobinopathies by the efficient introduction of a

A

fully functional beta like globin transgene into HSCs.

27
Q

two major breakthroughs have enabled efficient HSC transduction and the terapeutic expression of beta like globin transgenes in HSc derived RBCs

A

the development of HIV-1-derived lentiviral vectors

the discovery of LCR HSs capable of boosting beta like blobin expression.

28
Q

the major problems with gene therapy

A

poor delivery of genes into cells
low or no gene expression (short term gene expression, vector/promoter shutdown, immune rejection)
chance of insertional mutagenesis causing cancer
high cost

29
Q

genomic editing

A

an approach in which the genome sequence is directly changed by adding, replacing or removing DNA bases using engineered nucleases or molecular scissors.

These nucleases created site specific double stranded breaks (DSBs) at desired locations in the genome which can be repaired in a manner to result in targeted mutations.

30
Q

for genome editing to be specific

A

there must be a way to direct a nuclease to the desired location where a DNA break is to be introduced.

31
Q

all nucleases consist of 2 components:

A

the nuclease itself that is responsible for DNA cleavage

a secondary component responsible for recognizing a specific DNA sequence.

32
Q

three classes of nucleases for genome editing

A

zinc finger nuclease technology
TALENS = transcription activator-like effector nucleases
CRISP-Cas9 system

33
Q

Zinc finger nucleases (ZFNs)

A

a nuclease component linked to a DNA binding component derived from an array of zinc finger proteins. Each finger protein can bind three nucleotides, so combinations of zinc fingers linked together an recognize specific genomic sequences.

34
Q

TALENs

A

are similar to ZFNs. They also have a nuclease domain linked to a DNA recognition domain. the difference is that TALENs have a DNA recognition domain with a series of amino acid repeats. TALENS can be designed to have different combinations of repeats to recognize specific genomic sequences.

35
Q

CRISPR/Cas system

A

It uses a nuclease called Cas9 to introduce a double stranded DNA break. Unlike ZFNs or TALENs, it does not use a protein based DNA recognition domain. Instead, an RNA sequence is designed to bind to a complementary DNA sequence, allowing fo rthe Cas9 nuclease to make a cut.

36
Q

Steps of CRISPR/Cas system

A
  1. An RNA guid molecule can be programmed to match any unique DNA sequence.
  2. An enzyme called Cas9 is attached to the RNA guide and finds the target sequence of DNA.
  3. The RNA aligns with the target DNA sequence and the Cas59 attaches and cuts both strands of the DNA double helix.
  4. The DNA cuts can be amended with an extra DNA insertion or a deletion of defective DNA.
37
Q

genome editing with Cas9 in adult mice corrects a disease mutation and phenotype

A

delivery of components of the Crispr-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type fah protein in 1/250 liver cells.

38
Q

the promise of stem cell research

A

bone marrow for leukemia and chemotherapy
nerve cells for parkinsons and alzheimer’s disease
heart muscle cells for heart disease
pancreatic islet cells for diabetes

39
Q

stem cell therapy: issues and challenges

A

major ethical issues (stem cells from embryos are destroyed)
Major technical challnges:
can adult stem cells be isolated?
can adult cells revert to embryonic like stem cells?
can methods be developed to generate different cell types?

40
Q

IPS cells

A

differentiated cells can form stem cells in tissue culture with the addition of transcription factor genes or gene productions

oct3/4 and Sox2: TF involved in maintenance of luripotency of ES cells.
c-Myc: enhances proliferation and cell transformation
Klf4: represses p53 and nanog.

41
Q

IPS cells have been successfully generated from

A

skin cells, stomach cells, liver, pancreatic, lymphocytes, testis germline and neural stem cells.

42
Q

future tasks for IPS cell researches with regard to modeling human diseases

A

development of an accurate assay system for disease associated phenotypes.
demostration of causative relationships between genotypes and phenotypes by genome editing: can IPS be used to proof a gene mutation cause a disease phenotype?
can IPS be used once causative genes are identified?
Can early treatment with IPS cells prevent symptoms of late onset diseases?

43
Q

one of the uses of IPS cells is to

A

correct genetic diseases by inserting the normal gene into the IPS cell and then putting the developed IPS cell into a patient.

44
Q

the major concern with the potential clinical application of iPSCs is

A

their propensity to form tumors.

iPSCs readily form teratoma when injected into immunodeficient mice. Teratoma formation is considered a major obstacle to stem cell based regenerative medicine and by the FDA.

45
Q

IPS cells and ethical concerns:

A

abnormal reprogramming occurs in the induction of human IPS cells and the stem cells generate tumors in the process of stem cell therapy.

46
Q

human IPCs should not be used to

A

clone human beings
produce human germ cells
make human embryos
informed consent should be obtained from patients in stem cell therapy.

47
Q

Up to _% of normal proteins misfold and are degraded by proteasomes.

A

30

48
Q

Patients take migalastat 2 hours prior to ERT

A

increased active enzyme in plasma, skin and leukocytes.

In Fabry mice,
prolonged T1/2 in circulation, stabilized enzyme
increased uptake in kidney, heart and skin
increased clearance of GL-3 in kidney, heart and skin

49
Q

Zinc finger nucleases (ZFNS) are specific “molecular scissors”

A

Zinc finger protein 1 identifies and binds to DNA sequence
Part of the ZFN makes a cute in DNA/gene (nuclease)
Zinc finger protein 2 identifies and binds to DNA sequence.

50
Q

Zinc finger nucleases are artificial restriction enzymes generated by fusing:

A

a zinc finger DNA binding domain

a DNA cleavage domain

51
Q

zinc finger domains can be engineered to target

A

desired DNA sequences.
ZFNs can target unique sequences within complex genomes.
Can precisely alter DNA sequences.

52
Q

TALENs are

A

restriction enzymes that can be engineered to cut specific sequences of DNA.

53
Q

TALENs are made by

A

fusing a TAL effector DNA binding domain to a DNA cleavage domain.
By modifying the amino acid repeats in the Tale, users can customize TALEN systems to specifically bind target DNA and induce cleavage by the nuclease between the two distinct TAL array binding sites.

54
Q

CRISPR/Cas9 system can intoduce

A

different types of mutations

55
Q

New methods of manipulating genes can

A

replace a mutation with a wild type base or insert a mutation into a wild-type gene.