Lecture 22: Gene therapy Flashcards
(34 cards)
define gene therapy
the delivery of therapeutic genes into the human body to correct conditions created by a faulty gene or genes
gene therapy - Cystic Fibrosis
- conventional treatment includes back clapping to drain lung mucus, drugs to thin mucus, antibiotic treatments
- the CFTR gene was cloned in 1989 and by 1993 scientists had begun gene therapy trials. the challenge, however, was the right amount of CFTR.
0 in early 1990s, medical researchers placed the normal allele of the gene into the respiratory tissue of mice with the disease. these mice produced a functional CFTR protein
gene therapy - sickle cell anaemia
- most widespread inherited blood disorder in North America
- homozygosity for the HbBs allele
- current treatment consists of a prescription drug with toxic side effects or bone marrow transplantation
- in 2001, a Harvard research team successfully treated a sickle-cell mouse model with gene therapy
two primary strategies for gene therapy
ex vivo gene therapy
in vivo gene therapy
ex vivo gene therapy
cells are removed from the patient -> gene introduced into the cells -> cells reintroduced into the body
eg bone marrow blood precursor cells
in vivo gene therapy
introducing genes directly into tissues and organs in the body (injected into retinal cells, inhaled into the lungs)
what is the main challenge with in vivo gene therapy?
delivering genes only to intended tissues
use the liver as an example to compare in vivo gene therapy with ex vivo gene therapy
patient with liver cell genetic defect, lacks gene for blood-clotting protein.
ex vivo:
- remove small portion of liver to isolate cells
- grow cells in culture
- introduce normal genes for clotting protein
- transplant liver cells back into patient. genetically altered proteins provide clotting protein
in vivo:
- normal gene for blood-clotting protein is placed into viruses
- these viruses act as vectors for gene delivery
- directly introduce normal gene for clotting protein into patient’s liver cells
SCID
SCID = severe combined immunodeficiency
- defect in gene called adenosine deaminase (ADA)
- produces an enzyme involved in the metabolism of nucleotide dATP
- accumulation of dATP is toxic to T cells
- without T cells, B cells cannot recognise antigen and make antibodies
ex vivo gene therapy - SCID
first human gene therapy (1990)
1. remove ADA-deficient T cells from the SCID patient
2. culture cells in the laboratory
3. infect the cells with a retrovirus that contains the normal ADA gene
4. rein fuse the ADA gene containing T cells back into the SCID patient; genetically altered T cells produce ADA
in vivo gene therapy - Leber’s congenital amaurosis
- UPenn and Children’s hospital of Philadelphia
- LCA is a degenerative disease of the retina that affects 1/50000-100,000 infants each year and causes severe blindness
- young adult patients with defects in the RPE65 gene were given injections of the normal gene
- complete vision was not restored but 4 children gained enough vision to play sports and several months after a single treatments patients can see more light/navigate an obstacle course
in what way do we rely on viruses as vectors for gene delivery?
we can use the viral genome to carry a therapeutic gene or genes -> infect human cells, introducing the gene
list the various types of viruses we can use for gene delivery
- adenovirus (common cold): immunogenicity
- Adeno associated virus (AAV): does not integrate into genome
- retrovirus
- HIV based (lentiviral vectors): integrates into the genome
- herpes virus (cold sores, some cause STDs): target CNS
when we use viruses as vectors, what must we make sure to do?
we must make sure the virus has been genetically engineered so that it can neither produce disease nor spread throughout the body
mode of infection of viruses
- attachment: selective binding to cells
- injection: release genetic material into nucleus or cytoplasm
- human cell acts as a host to reproduce the viral genome and to produce viral RNA and proteins
why viruses make good vectors
- they are efficient at infecting many types of human cells
- retroviruses or lentiviruses (HIV) permanently inserted their DNA into host cell genome (integration)
- some viruses infect only certain types of cells - good for targeted gene therapy
gene therapy with a retroviral vector
- attachment
- injection
- RNA -> dsDNA -> insert viral DNA into host genome
gene therapy with an AAV vector
- attachment
- injection
- ssDNA -> dsDNA -> viral DNA not incorporated into host, usually extrachromosomal
how to package a gene into retroviral particles
- make the recombinant retroviral RNA genome:
- LTRs (Long Terminal Repeats): required for integration and transcription
- Ψ (psi) packaging signal: ensures RNA is packaged into the viral capsid
- gene (downstream of promoter) - package recombinant genome into viral particle:
check video
SCID-X1
X-linked severe combined immune deficiency
- LOF mutation in IL2-RG (necessary to promote the growth of several immune system cells)
- in 2000, several SCID-X1 patients were cured substantially by gene therapy
- out of the 9 children treated, 8 are still alive
- however, 4 eventually developed leukaemia due to insertional mutagenesis, 1 succumbed to the cancer
potential risks of viruses as vectors
- death of 18 year old Jesse Gelsinger in 1999 due to complications related to adenovirus vector
- death of two children in France in 2002
- temporary cessation of a large number of gene therapy trials and FDA stopped most retroviral studies
- trials eventually resumed with greater patient monitoring
alternative gene delivery options (non-viral)
liposomes -> lipid nanoparticles with an active pharmaceutical/genetic ingredient
how can we tackle dominant negative or toxic alleles?
using RNA interference (RNAi) technology
how does RNAi work?
uses complementary RNA to silence gene expression
