Mitchell: Genomics in Clinical Flashcards
Tetraology of fallot and palatal abnormalities is associated with what 22q11.2 deletion?
DiGeorge syndrome
Velocardiofacial syndrome
Noonan syndrome has symptoms of low set ears, ptosis (droopy eyelids), short stature, congenital heart disease, and developmental delays all due to a gain of function because of a 1 bp change. Which technique would you use to detect this 1 bp change?
Sanger sequencing
based on selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication
Autism has a lot of copy number variants. What testing should first be performed to test for autism?
Comparative genomic hybridization (Microarray)
CNVs are imbalances that alter the diploid status of a locus so that copy numbers increase (duplications) or decrease (deletions).
For uncommon conditions with symptoms that cannot be directed to a particular disease, you would perform what test?
Whole exome sequencing
You would not do Sanger, CGH, or SNPS
Bioinformatics is what type of cloning?
Functional cloning
What is germline mosaicism?
Germline mosaicism, also called gonadal mosaicism, is a type of genetic mosaicism where more than one set of genetic information is found specifically within the gamete cells.
What is the first US approved gene therapy and what does it treat?
Kymriah
Non-Hogdkin’s lymphoma AKA B-cell acute lymphoblastic leukemia
What are the disadvantages of Kymriah?
dangerous drops in BP
Why do people we care about gene therapy?
we want to make the correct protein or alter it in away that it is therapeutic
What is the first US gene therapy to target a disease caused by a mutation with a specific gene?
Luxtuma
treatment for blindness
Immunotherapy and DNA editing are done on DNA. Antisense is done on pre-mRNA. Pharmacological therapies target what?
proteins
What makes protein therapy difficult?
really large molecules to manufacture (dystrophin)
How will we ensure that the correct amount of enzyme will be made from the newly introduced genes?
regulated expression
Traditional gene therapy consist of targets to what?
the DNA molecule is the target as opposed to the mutant protein product of this mutated gene
What are the advantages and disadvantages of cDNA therapy?
advantage: single versions of the gene delivered to replace the missing version of the protein
disadvantage: regulation of gene expression, ectopic expression may be a problem; delivery to specific cell type will be too difficult especially with large proteins; NOT used for dominant negative mutations (OI, Bcr-Abl in Chronic Myeloma leukemia)
What are methods used to making permanent corrections using gene therapy as opposed to delivery therapy every week for example?
you want it to be a permeant change
you can either:
-target stem cells that give rise to other cells
-target cells that are already differentiation
opposite ends of the spectrum
Adenovirus, lentivirus, and retroviruses are similar in that they can carry
8000 nucleotides
4500 nucleotides
Adenovirus, lentivirus, and retroviruses are similar in that they can carry what size of therapeutic gene? What about adeno-associated viruses?
8000 nucleotides
4500 nucleotides
gamm retroviral vector
- infects dividing cells
- integration near regulatory elements
- high risk of insertional mutagenesis
- packing capacity is 8,000 bases
adeno-associaited
- insert into dividing and non dividing
- stable in non dividing cells but not stable in dividing cells
- 5,000 bases (small packing capacity)
- no integration into host genome
- high production yield
- low immunogenicity
- long term expression
lentivirus
- works in dividing and non-dividing cells
- has integration into gene
- it is stable in dividing and non-dividing cells
Disadvantages
- packing capacity is 8,000 bases
- risk of insertional mutagenesis
Adeno-associated viruses are transient and good for long term correction due to what?
inability to integrate into genome
Which virus caused the first death in gene therapy and why?
Adenovirus
It very large and can cause immune system toxicity.
This incident led to a decrease in gene therapy for a good 10 years.
Early trials of gene therapy used this kind of retrovirus? Why is this significant?
gamma retroviruses mimic genes that suppress cancer
-need to ensure to make the vector correctly or you can cause
T-cell acute lymphoblastic leukemia
now we use Lentiviruses
What is first ex-vivo stem cell gene therapy to treat patients with ADA-SCID?
Strimvelis
What is the first gene therapy approved in Europe that counteracts lipoprotein lipase deficiency? It can also be used to treat what?
Glybera
Leber’s congenital amaurosis which is caused by a mutation in RPE65.
RPE65 gene is involved in recycling cis and trans retinal which then binds to rhodopsin. It is important for receiving photons and recognizing then and transmitting it to the brain. If this gene is mutated, it can cause severe blindness as you progress in age. Which type viral vector is used in gene therapy for this condition?
Adeno-associated virus which is small and can fit into the retina and is also protected from the immune system
CAR-T is considered what type of drug?
a living drug that is made by Novartis for acute B-cell lymphoblastic leukemia.
it trains your living T cells to recognize and kill the tumor cells
The engineered ________kills all B-cells expressing CD19 including memory B cells.
anti-CD19 T cells
T cells kill both normal and mutant cells
-you would become immunodeficiency (kills memory B cells)
How does CAR-T work?
- takes blood from patient
- isolate T cells
- transfect with a retrovirus that carries a modified gene that encodes a T cell receptor; this receptor has an antibody chain that recognizes an epitope that’s on B cells called CD19
What is the difficulty with CAR-T cell therapy getting approved by the FDA?
getting an experimental approach which is custom and made for each made
What are the main advantages of oncolytic viruses?
- selectively target tumor cells
- improve the spread of trans-gene products from cell to cell so increase efficiency of gene therapy
- In addition they exploit the defects in intracellular pathways which are commonly associated with tumor phenotype
What is the relationship of oncolytic viruses with p53?
they tend to associate with and propagate the effects of MUTANT p53 causing growth in tumors/cancer
Mutant genes (programs) can now be re-written (hacked) at the level of?
DNA (ROM) or RNA (RAM)
This essentially goes back to the idea of targeting the DNA instead of RNA or proteins in traditional gene therapy. We can fix what’s already there instead of introducing a lot of novel genes to the system.
So if you bring in an entirely new gene you have to bring the promotor and other regulatory elements with it.
What was the first treatment US FDA approved for the spinal muscular atrophy?
SPINRAZA (nusinersen)
What are methods of gene therapy at pre-mRNA and RNA level?
antisense
RNA interference
Advantages:
- you only need short sequences of genetic material
- one molecule can prevent or modify the expression of many mRNA
Disadvantages:
long term expression
off target effect a
saturation of cellular mechanisms (due to interference)
What is antisense?
- make complements to the bad RNA to form a double stranded RNA
- our cells don’t like double stranded RNA because they think it came from a virus (which it did)
- so cell brings RNAse to kill it
So you can make an antisense RNA that’s sitting on the splice site of a pre-mRNA and it blocks a splice site
▪ So now you get inclusion or exclusion of different exons
What is RNA interference?
RNAi is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules.
deliver an RNA with a particular structure and it gets cut up, and then gets put into a risk complex (which chops up the RNA and modifies the DNA)
What are examples of antisense therapy?
First is for a form of muscular dystrophy, it’s a potential therapeutic for about 15%
of the patients
The other is for spinal muscular atrophy, which is a disease in newborn children
FDA grants accelerated approval for which drug for DMD?
Exondys 51
exon 51 is skipped resulting in a nonsense stop codon that causes DMD and you end up not making dystrophin
What are examples of things that can go in and re-write the DNA?
Crisper-Cas9
Zinc Finger Nucleases, Talons, and
Meganucleases
What are the advantages and disadvantages of Crisper-Cas9?
Advantages
- can correct dominant negative mutant turning mutant to wild type
- size: replace portion of gene
- may be more immunogenic and thus does not require that you deliver some kind of enzyme that will cut the DNA
Disadvantages:
-off target effects because DNA is permanent
How does Crisper-Cas9 work?
- deliver the Cas9 protein and a guide RNA and DNA for correction
- cell division is required for gene correction and gene insertion
- targets proteins which is difficult because the targeted protein can be large
- can insert and turn the sequence of the therapy gene that is being inserted into whatever you want
How Zinc finger nucleases work?
- DNA binding protein
- need precise cutting by attaching the nucleases to another enzyme that is very close to the sequence you want to cut
What are two receptors required for HIV entry?
CCR5 on macrophages
CXCR4 on T cells
A mutation in this, which is found in CCR5, causing the loss of CCR5 expression. This protects you from HIV infection. What is the mutation in?
delta-32
Berlin HSC transplant from CCR5-delta 32 donor lead to HIV cure
-stems cells are harder to modify which is why
What occurs in trans-splicing?
all your genes get changed
we trick the machinery that’s doing to the cut and pasting/splicing into splicing between 2 different RNAs and that’s trans-splicing
-thus all you genes get changed
-advantage: it has all the advantages of DNA editing
-disadvantage:
RNA molecules are more unstable than DNA molecules
Splice sites are characteristically what?
a highly conserved
What is polypeptide base nanoparticles?
these cells are used to treat or target B cell leukemia through CD22 receptor
When the nanoparticle sits on the CD22 receptor, it transmits a signal that says stop growing or kill yourself
But, the patients who have the drug resistant leukemia have a mutation which causes skipping of exon 12, so they make a mutant CD22 that doesn’t translate or transmit the signal
Advantage:
- They developed a trans-splicing molecule that could repair any mutations in exons 10-14 (using the exons around 12 to fix the mutation of exon 12)
- doe not kill all the B cells (better than CAR-T)
What can trans-splicing also be used for?
used to repair factor 8 in knockout mice, repair mutations in the cystic fibrosis gene
When we put the trans-splicing gene in human cells, we got approximately 20% correction in both hemophilia A and cystic fibrosis, which is enough to change the disease
phenotype.
How can we use molecular imaging in HPV?
put the HPV inside the to make the rat glow in the areas that are causing problems
Is the vaccine for HPV effective is you’ve been infected with HPV already?
NOOOO
