GENE-BASED THERAPIES Flashcards
FUNDAMENTAL CONCEPTS – GENE THERAPY
*Gene therapy
* The introduction, removal, or change in the content of a person’s genetic code with
the goal of treatment or curing a disease.
* The transferred genetic material changes how a single protein or group of proteins
is produced by the cell.
*Gene therapy can be used to reduce levels of a disease-causing version of a
protein, increase production of disease-fighting proteins, or to produce
new/modified proteins
FUNDAMENTAL CONCEPTS – CELL THERAPY
*Cell therapy
* The transfer of intact, live cells into a patient to help lessen or cure a disease.
* The cells may originate from the patient (autologous) or a donor (allogenic).
* The cells can be classified by their potential to transform into different cell types.
* Pluripotent cells can transform into any cell type
*Multipotent cells can transform into a few cell types
*Differentiated or primary cells are fixed type
GENE THERAPY– CLINICAL PROBLEM
Human genome contains > 25,000 genes encoding a variety of proteins necessary for
maintaining life
Genetic alteration is a part of human evolution, but sometimes can lead to dysfunctional
protein → disorder, disease
~ 80% of rare diseases are caused by a genetic dysfunction
~ 300 million people in the world are affected by a genetic disorder
Conventional small molecule (i.e. chemical) drugs only offer temporary mitigation and
sometimes only symptomatic management
Protein drugs (e.g. monoclonal antibodies) are costly/difficult to produce, exhibit less desirable
pharmacokinetic properties, and can cause significant life-threatening toxicities (discussed in
lectures 2 & 3)
Gene therapy(ideal therapy, in theory?): “one-time treatment”, “long-lasting production of
therapeutic protein”, “localized to the target tissue”, “natural therapy – minimal toxicity”
GENE THERAPY – GENERAL PRINCIPLES
overall approaches
Germ line: gene therapy of the reproductive cells such as sperm and
eggs. Changes are inheritable. Not conducted in humans secondary to
ethical problems.
Somatic line: all cells, except sperm and egg, can be treatment targets.
Changes are NOT inheritable.
Overall approaches:
1) maintain long-term expression of proteins associated with transferred
(wild-type) gene (AKA “augmentation gene therapy”)
2) suppress expression of a mutated, harmful gene using RNA
interference
3) genome editing to correct a mutated sequence
GENE- THERAPY PHARMACOLOGY
a) gene augmentation: Provides a functional copy without
deleting the dysfunctional gene
Examples: spinal muscular atrophy, X-linked
severe combined immunodeficiency,
inherited retinal diseases
A is most common and easiest because you insert a gene into genome
b) gene suppression: Reduces the accumulation of toxic
/dysfunctional proteins by RNA interferenceExamples: Huntington’s disease
c) genome editing:Generates DNA break, then rejoining
via homologous template or non-homologous
nucleotide editing
Examples: Strictly experimental; no clinical
application in 2022 yet
GENE-THERAPY PHARMACOLOGY
General methods for the delivery of therapeutic genetic materials:
1) In-Vivo technique: gene delivered directly into the patient (e.g. targeting
postmitotic cells not in active division; to achieve long term expression in the
cell; usually achieved with adeno-associated viral vectors)
E.g. voretigene neparvovec-rzyl for treatment of RPE65 inherited retinal disease
2)FYI: Ex-Vivo technique: genetic-modification of cells taken from the subject, and
subsequent re-introduction into the patient (e.g. genetically-engineered tumortargeting lymphocytes)
Ideal properties of viral vectors for delivering genetic material:
1) Challenges:
Difficult for DNA to pass through cellular membranes (large molecular weight and highly charged)
Presence of cellular endonuclease can break down un-protected DNA
2) Viral vectors:
Able to accommodate therapeutic genes of various sizes (next slide)
Increased chances of transduction success
Increased chances of stable protein expression
Ability to target specific cells (both stem and post-mitotic cells)
Minimal immunogenicity and pathogenicity (next slide)
“Relatively” easily manufactured
IN VIVO EXAMPLE – VORETIGENE NEPARVOVEC
RPE65-mediated inherited retinal diseases (Leber’s congenital amaurosis)
Condition: Autosomal recessive retinal pigment epithelial 65 kDA protein (RPE65)
genetic mutation
Loss of production of retinoid isomerohydrolase, responsible for the visual cycle
Prevalence of 1 in 50,000-100,000 (rare!)
Symptoms: progressive retinal degeneration → severe visual impairment
Nyctalopia, nystagmus, visual field constriction, and loss of visual acuity (legal
blindness) as early as infancy
Treatment: Voretigene Neparvovec (VN) is the 1st approved gene therapy for a
hereditary genetic disease in the USA and Canada
VN: genetically-altered, non-replicating adeno-associated virus (AAV) carrying the
functional human RPE65 gene
“Gene-augmentation” therapy
Voretigene Neparvovec
pharmacology
Utilizes adeno-associated virus (AAV) vector to deliver the functional RPE65 gene to the retinal pigment epithelial cells
AAV viral vectors are triple-transfected using a HEK293 primary cell line (i.e. inserting the RPE65 gene into the virion)
AAV: small (~25 nm in diameter), single-stranded viruses (benefits: nonpathogenic, non-replicating, non-integrating, ability to transduce non-dividing
cells, low-immunogenicity)
Pharmacology
1) AAV vector carrying RPE65 binds to cell surface receptor
2) AAV vector gets endocytosed via endosomes
3) either proteosome-mediated degradation (i.e. cellular protection)
OR
4) gains entry into the nucleus
5) uncoating in nucleus → releasing single-stranded AAV genome (as a
stabilized extragenic episome; lack of integration into the host genome)
6) conversion to double-stranded DNA
7) transcription into mRNA
8/9) translation and production of RPE65 protein (constitutively
