gene therapy and gene editing

human genetics lecture 14

gene therapy

a process in which one or more copies of a gene are introduced into a patient’s body with the help of a vector to repair the effect of a malfunctioning gene which is causing a genetically-based disease or condition

episome

similar to a plasmid, attaches to chromosomes, replicate normally alongside chromosomes

approaches of gene therapy

• a normal gene inserted to compensate for a non-functional gene

• a normal gene replacing an abnormal gene via homologous recombination

• an abnormal gene repaired through selective reverse mutation

first gene therapy case

Ashanti DeSilva, 1990

• severe combined immunodeficiency (SCID)

• ex vivo → doctors removed her white blood cells, inserted the missing gene into the white blood cells, then put them back in her blood stream

steps of gene therapy

1. a therapeutic gene is inserted/cloned into a vector (usually an altered viral agent)

2. the viral vector infects the cell and delivers the therapeutic gene into a patient’s target cells

3. the genetic material is integrated into the target cell’s genome

4. functional proteins are created from the therapeutic gene causing the cell to return to a normal state

tropism

the targeting of specific cells (by recognizing target antigens) by a virus

integrase

enzyme that reverse transcribes and inserts a retrovirus’ genes into the host genome

adenovirus in cancer gene therapy

an adenovirus encoding the gene for an enzyme that converts an inactive pro-drug to a potent cytotoxic drug can be used to infect tumour and normal cells

linkage of inserted gene to a tumour-specific promoter

ensures a higher level of enzyme transcription in tumour cells compared to normal cells

adenoviruses

target cells that are undergoing replication, can be modified so that it is only copied in cells that have dysregulated cell cycle activity

oncolytic viruses

natural or genetically modified viruses that specifically target cancer cells, useful for immunotherapy

problems with gene therapy

• immune response to viral vectors

  • patient may have toxic, immune, inflammatory response

• may induce cancer if integrated into a tumour suppressor gene

  • insertional mutagenesis

• gene therapy doesn’t last long, multiple rounds of therapy are needed

• not as useful for multi-gene disorders

  • need to introduce more than one gene

genome editing

targets disease-causing genes to correct or remove a defect permanently, corrections will persist throughout the life span of the transformed cells

non-homologous end joining (NHEJ) gene editing

indels introduced through NHEJ can be useful for knocking out gain-of-function mutations

homology-dependent repair (HDR) gene editing

useful for introducing a new sequence into the genome to correct loss-of-function mutations

non-homologous end joining (NHEJ)

error-prone repair, leading to the introduction of variable length insertion or deletion (indel) mutations

homology-directed repair (HDR)

crossing over with the donor template, can lead to the introduction of precise nucleotide substitutions or insertions by supplying a double-stranded DNA donor template

methods of inducing site-specific double-stranded breaks

• zinc-finger nucleases (ZFNs)

• transcription activator-like effector nucleases (TALENs)

• mega-nucleases

• CRISPR/Cas system

zinc-finger nucleases (ZFNs)

artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain (Fok1)

CCR5 ∆32

polymorphism giving immunity to HIV

Fok1

restriction enzyme, non-specific cleavage site

transcription activator-like effector (TALE) proteins

transcription activators that bind to plant genes that, when over-expressed, facilitate infection

transcription activator-like effector nucleases (TALENs)

nuclease (Fok1) associated with a transcription activator-like effector (TALE)

main difference between ZFNs and TALENs

TALENs consist of natural DNA-binding modules

benefits of TALENs over ZFNs

easier to design and more versatile

benefits of ZFNs over TALENs

smaller and easier to get into a cell

mega-nucleases

endonucleases with a long recognition sequence, highly specific to a targeted locus, not easy to edit, limited to using ones that naturally occur

CRISPR/Cas9 genes

essential in adaptive immunity in certain bacteria and archaea, enable them to respond to, and eliminate, invading genetic material

acquisition and adaptation in CRISPR/Cas

a bacterium remembers previous invaders by incorporating identifying pieces of the phage genome into their own into CRISPR arrays, expression of the piece, linked with the Cas nuclease, will lead to cleavage of the viral genome if it tries to re-infect

crRNA

CRISPR RNA, incorporated viral RNA in CRISPR array

gRNA

guide RNA, required for binding to Cas9, the gRNA is ligated to the target sequence

PAM

sequence of nucleotides required by Cas9 to bind at the 3’ end of the target sequence, 5’-NGG'-3’ for Cas9

SCR7

inhibitor of DNA ligase IV

upregulation of SCR7

inhibits non-homologous end joining

CRISPR stabilizing RAD51

upregulates homology-directed repair pathway

waiting to activate Cas until the G₂ or S stage of cell replication

increases likelihood of homology-directed repair

risk of upregulating homology-directed repair pathway

may increase oncogenic expression, decrease immune function

regenerative medicine

regrow or replace damaged or diseased cells, tissues, or organs

phase 1 clinical trial

dose ranging, small sample size

phase 2 clinical trial

~100 volunteers, testing efficacy and side effects

phase 3 clinical trial

upscale, 1000+ individuals, test efficacy and safety across demographics