BIOC2306 Gene therapy vectors

define gene therapy

deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic or diagnostic purposes

aims of gene therapy

correct a gene defect e.g. Cystic fibrosis

eradicate tumour cells (leukaemia)

stim immune system (cancer)

control autoimmune disease (rheumatoid arthritis)

gene editing

vaccination

what diseases are mainly targeted by gene therapy studies

cancer

monogenic diseases

infectious disease

what are non viral vectors (chemical)

liposomes

aqueous cavity in centre of spherical PL bilayer that contains DNA

can be anionic and cationic (typically use cationic as they attract the DNA (dont repel)

pros and cons of chemical vectors

easy to prod and formulate with nucleic acids

problem with specificity and targeting

but still deliver DNA or mRNA

viral vector types

non-integrating, transient expression (adenovirus)

non-int. sustained expression (herpes virus)

integrating, sustained expression (retroviruses, adeno-associated virus)

pros of viral vectors

efficient entry into cells

many cell surface receptors

often tissue specific entry (e.g. adenovirus and respiratory tract)

can intergrate or persist (1 administration req e.g. herpes, retroviruses, adenoassoc)

can create disabled viruses that are replication deficient

cons of viral vectors

inflammation due to viral proteins

pre-existing immunity against virus

reapeat dosing req if not sustained exp (so raise immune response)

integrating virus may insert into critical host genes

expensive to produce

alternative approach to giving viral vector

give many plasmids, each encoding an individual viral gene e.g. pol, env, gag. and a plasmid with the therapeutic gene into a human cells in culture.

The therapeutic retrovirus is then secreted into the cell culture medium, harvest and give

adeno-assoc virus

ssDNA virus, non pathogenic. 4.6 kb

enters human cells and establishes a latent infection

needs coinfection with adenovirus (or herpes virus) for growth

integrates into specific region of ch 19

multiple types (AAV2 currently used for gene therapy)

AAV vector vs adenovirus

no replicase or capsid protein, has promoter and cDNA instead with 3’ polyA tail encoding section too

is why need adenovirus to rep (for rep and cap gene)

want to grow it when in a lab, but not really after give to patient

benefits of different AAV types

give tissue specificity

can coinfect cells with mulitple AAVs to produce larger genes taht can encode larger proteins

herpes virus

large DNA virus 150 kb

has many non-essential genes that can be replaced

can infect non-dividing cells (e.g. cardiac muscle, neurones, mature RBCs)

non-integrating, latent infection

what is an episome

genetic element in cell that replicates independently of chromosomes e.g. recombinant herpes virus (non-integrating)

adenoviruses

have linear dsDNA (not ssDNA like AAV)

36 kb

infect epithelial cells that like resp tract, conjuctiva (eyes), bladder and kidneys

infect non-dividing cells

30-40 genes (some nonessential)

adenovirus infection pathway

taken up into endosome by binding to CAR receptor and integrin

acidification of endosome, release from endosome and breakdown of capsid in cyto

DNA enter thru NPC into nucleus

pros of adenoviruses for gene therapy

realtively safe (can revert)

nonpathogenic (except in immunocomp or v young)

can have large inserts

infect a wide range of cells (dep on serotype too)

cons of adenovirus gene therapy

wide distribution of CAR receptor so hard to target

lose CAR receptor in cancer tissues

immune response to adenovirus proteins

inflamm response

therefore re-administration difficult

what are transgenes

gene that is transferred from one organism to another (typically thru genetic eng)

what is SCID

severe combined immuno deficiency - 19 types, severity varies

v vulnerable (no immune defense) against pathogens , autosomal recessive

norm v short life (bubble boy)

e.g. IL-2 receptor mutations

can have no T cell reponse, B cells never mature, only NK cells

gene therapy for SCID

take bone marrow from patient, treat with retrovirus so it now expresses a functional e.g. cytokine receptor (IL-2 receptor) (ex vivo treatment)

but req give immunosuppressants as will get immune response to vector (retrovirus)

but problem with leukaemia reported (insert into LMO2 gene) (~random integrate)

cause prevmature stop codon to form in LMO2 gene

large side effect of adenovirus vectors

liver toxicity

benefit of retrovirus for treating cystic fibrosis

could inhale the adenovirus containing CFTR

resp tract where want to restore function so very targeted

but only showed transient improvement of Cl- transport, immune memory too

general probems with gene therapies

large cost - specialised manufacture process and administration process (IV), limited patient populations (rare diseases often target)

ethics: cost, accessibility and patient needs

T-VEC

first live virus approved by FDA to treat melanoma

herpesvirus

infects healthy and cancer cells, but only replicates in tumour cells, killing them

CRISPR-Cas9 overview

Cas9 enzyme guided by RNA to cut at specific locations

allow edit, delete insert within a gene

e.g. ras oncogene, cut, hope repair is inefficient and a stop codon is introduced

thereby making nonfunc truncated protein (silencing the gene)

how is CRISPR used to treat sickle cell

edit haemopoitic stem cells to induce production of foetal haemoglobin, to compensate for defective haemoglobin