Gene Therapy Flashcards
Define Gene Therapy
Delivery of genetic material into a patient’s cells as a drug
Describe the 4 ways gene therapy can work
1) Mutation in coding sequence - mutant protein
2) Mutation in promotor - more or less mRNA
3) Mutation in splice site - incorrectly spiced mRNA
4) Mutation in regulatory element in UTR - more or less protein
How do mutations in coding sequence work?
Example of a disease
Changes amino acid sequence affecting production or function of protein.
CF - F508 deletion, loss of phenylalanine causing miscoding and degradation
Premature termination - truncated protein
Frameshift - incorrect sequence downstream
How do mutations in promoters work?
Change expression level - remove site for transcription factor
How do mutations in splice sites work?
5’ or 3’ UTR leading to changes in regulation of expression by slicing or trans-acting factors e.g. - binding site prevents binding increasing production
Four challenges for gene therapy
1) Delivery to target cell
2) Maintenance in cell
3) High cost
4) Only suitable for diseases caused by reduced production of protein as can’t achieve precise level of expression.
Differences between in vivo and ex vivo
in - Directly into patient
ex - target cells out, introduce gene, return. Good for haematopoietic system. Precise targeting and increased efficiency
Pro’s and Con’s of Viral delivery
Pro’s - Efficient uptake, persist in cells modified to remove pathogenicity and tumorigenesis
Con’s - Immune response, pathogenicity, tumourigenesis, size of gene
Pro’s and Con’s of Non-Viral delivery
Pro’s - Low immunogenicity, Large scale production, larger DNA molecules
Con’s - Less efficient, difficult to target specific tissues, sustained expression
Viral delivery must…..
Attack and enter cell
Transport material to nucleus/maintenance as DNA
Sustained expression
Lack of toxicity/immunogenicity
Describe Retrovirus
Encodes reverse transcriptase (copies viral RNA to DNA) integrated into host genome.
Mutate to remove pathogenic element
Incorporation of gene into host genome and maintenance during devision
can’t control site of integration (can cause cancer)
Describe Lentiviruses
BETTER
Highly modified viruses
can integrate into non-dividing cells
Lower immunogenicity
Describe AAV
Non pathogenic/low immunogenicity Dividing and non-dividing cells Needs helper virus to replicate DNA retained in cell nucleus - specific site, rep and cap genes deleted, retained as emisomal concatemer serotypes = many cell types small - limited size of gene
What is CAR-T immunotherapy and what is it used for?
Adaptive T-Cell transfer
ALL - produce chimeric antigen receptors which recognise antigens on cancer cells
Retroviral
Cytokine release syndrome
How can we use gene therapy for haemophilia
Lack of clotting factors, Hepatocyte targeted AAV introduce factor IV to liver
Expression of enough - 7X more effective
Immunity - modifications of capsid
Liver cell damage - short term corticosteroids
Pro’s of mRNA therapy
Less stable expression, no danger of integration
Avoid immune response/degradation
What is CRISPR
Prokaryotic immune system, site-specific genome modification, most flexible and easy to use.
Off-target effects
Bacterial genome contains CRISPR - short repeated sequences with short sequences from viruses that affect bacterium inserted between.
Repeat transcribed to crRNA
Describe process of CRISPR
1) Double strand break at target site by cas9
2) Repaired with cellular machinery
3) NHEJ - error prone, small insertions/deletions disrupting target gene. Efficient but imprecise
4) Homology-directed repair - Template supplied matching cleavage site, desired mutation included. Precise but not efficient.
Describe TALENs
Transcription activator like effector nucleases
Amino acid sequence of DNA binding domain shows strong association with cleavage at specific nucleotides.
FOK1 nuclease to mediate site-specific dsDNA cleavage
Large, repetitive genes - difficult to deliver AAV or lentivirus
More specific, more complex and harder to engineer
How do antisense technologies work
1) Introduce oligonucleotide complementary to sequence on mRNA of interest
2) Prevents protein/RNA regulators by steric hindrance
3) Chemically modified bases/backbone
- Increased RNA binding
- Protect from nuclease degradation
- Improve cell entry
TWO ways antisense can be used
1) Splicing- mask sequence recognised by splicing factor
2) Translational inhibition - Bind across start codon
How can we use antisense for DMD
Exon51 frameshift
Exondys51 - causes exon51 skipping restoring reading frame
only partially functional - small deletion
Morpholine antisense oligonucleotide that targets splice enhancer preventing inclusion of exon
How can we use antisense for SMA
SMN2 almost identical except for splice site mutation(10%)
spirRAZA - modulate splicing of SMN2 making more production
How do siRNA’s work?
dsRNA processed by dicer, generate 21-23nt dsRNA duplexes with 2nt 3’ overhang.
One strand of duplex incorporated into RNAi-induced silencing complex. Binds to fully complementary RNA sequences and cleaves RNA
What can siRNA’s be used for
Reduce mRNA levels of over expressed gene
Reduce incorrectly spliced mRNA
Target viral RNA
Formulation of siRNA’s
antiviral mechanisms activated by dsDNA longer than 30 = siRNA’s too short.
Short hairpin RNA’s in vectors - processed by DICER allows viral delivery targeting specific organs
Issues with siRNA’s
1) Specificity
2) Efficiency (don’t completely knock out and viruses can envelope)
3) Delivery - maintenance difficult, some organs easier
Oligonucleotide delivery needs to achieve…
1) stability against serum nucleases
2) Entry into target cells
Oligonucleotide chemical modification
1) Phosphorothioate modification of backbone increased stability
2) GalNAc conjugates enhance uptake by hepatocytes
micro RNA’s background
Decrease in lin-14 protein necessary for normal development. Mediated by lin-4 = ss 22nt non-coding RNA
Binds to partially complementary sequence in lin-14 3’UTR repressing lin-14 expression
Describe how micro RNA’s work
1) Encoded in genome
2) Transcribed as part of longer RNA (pri-miRNA’s)
3) Nuclear processing to pre-miRNA hairpin by Drosha and exportin 5
4) In cytoplasm dsRNA hairpin processed by dicer to 21-23nt dsRNA duplex
5) one strand retained as mature miRNA
How are miRNA’s involved in cancer?
CLL = loss of heterozygosity at 13q14.
Tumour suppressor genes = miR-15a & 16-1 which target BCL2 which is anti-apoptotic
How are miRNA’s involved in HepC?
Highly specific liver miR-22 interacts directly with 5’UTR required for replication
