Biotechnology of drugs – Gene therapy
Doc. RNDr. Jan Hošek, Ph.D.
hosekj@pharm.muni.cz
Department of Molecular Pharmacy
FaF MU
What is gene therapy?
A treatment or procedure for
ameliorating the manifestations of a
genetic disorder using a patient's
genetically modified cells with a
therapeutic benefit to the patient
Symptomatic treatment of genetically determined
diseases does not treat the essence, although the
cause of the disease is known
Gene therapy treats the cause of the disease
Gene therapy × symptomatic treatment
➢ Supplying the missing enzyme in enzymopathies
➢ Delivery of other missing substances (substrates,
proteins...)
➢ Avoidance of substrate that cannot be metabolized
properly (special diets)
➢ Surgical interventions
➢ Pharmacological influence of disturbed physiological
processes
➢ Pharmacological improvement of quality of life
➢ Other influencing of physiological processes or quality
of life (devices, aids)
➢ Transplantation of an organ damaged by disease
Symptomatic treatment
➢ It includes any procedure intended to
treat a disease by genetically modifying
the patient's cells
➢ The following are transferred into the
cells: genes, their parts or
oligonucleotides
Gene therapy
https://www.science.org/doi/10.1126/science.aan4672
They date back to 1990 - the first clinical studies were conducted in the treatment of adenosine deaminase
deficiency (ADA). In this study, peripheral blood lymphocytes from ADA-deficient patients were transformed with a
retroviral vector that expressed functional adenosine deaminase.
Even 10 years after this intervention, one patient's lymphocytes expressed a functional enzyme, which means that
gene therapy can have a long-term effect.
Another patient developed an immune reaction to the transporter system and therefore did not express the
therapeutic gene, which already highlighted the problems associated with gene therapy.
The beginnings of gene therapy
N Engl J Med 2019;381:455-64.
➢ Gene therapy in vitro (ex
vivo): collection of cells,
execution of gene
modification outside the
organism, return of cells
➢ Gene therapy in vivo:
intravenous transformation
directly by gene construct
➢ Gene therapy in situ:
(modification of in vivo) a
gene construct is injected into
or near the affected tissue
Gene therapy techniques
N Engl J Med 2019;381:455-64.
➢ Congenital disorders (genetic deficiency of
the gene product, inappropriate gene
expression)
➢ Tumor diseases (disorders in the biological
function of proto-oncogenes, tumor
suppressor, apoptotic and repair genes)
➢ Diseases of the immune system (allergies,
inflammation and autoimmune diseases)
➢ Infectious diseases (viral or bacterial
pathogen)
What can be treated with gene therapy?
➢ We need to know the exact cause of the genetic
disease - i.e. the gene, its location, the nature of
the product and, above all, the mechanism of
the pathological effect
➢ A properly designed gene therapy strategy
➢ The strategy also includes the choice of a
suitable vector and the selection of target cells
for gene therapy
➢ Considering the certain controversy of this
therapy, gene therapy should only be performed
if it is successfully tested and approved for use
When can gene therapy be used?
➢ Is the goal of gene therapy to correct an inherited genetic
disorder or to introduce a new function into recipient cells? If it
concerns, for example, the treatment of cystic fibrosis, then it is
a matter of correcting a defective condition. Conversely, in the
case of AIDS treatment, we can try to transform cells with a gene
with a new function; e.g. a gene whose product will interfere
with the replication of the HIV virus.
➢ Should the therapeutic gene work for a long or short time?
Mostly, it will be a requirement that the gene works for a long
time, but in the case of the treatment of tumor growth or the
requirement to introduce a DNA vaccine, the effect may be
relatively short-lived.
Additional questions for use of GT - 1
➢ For most applications, continuous
expression of the therapeutic gene is
required. In some cases, it is necessary to
regulate the expression, for example in the
treatment of diabetes mellitus.
Additional questions for use of GT - 2
➢ Selection of target cells
According to the nature of the disability; for example, in the case
of familial hypercholesterolemia, it is necessary to introduce a
gene encoding a receptor for LDL (low density lipoprotein) into the
hepatocytes.
When low levels of clotting factors saturate (leading to
hemophilia), a specific cell population needs to be targeted; here it
is necessary to introduce the gene producing said clotting factors
into such cells that will be able to bring them to the site of action,
i.e. into the bloodstream. In the case of factor IX, myoblasts that
secreted this clotting factor into the blood were successfully
transformed.
The choice of target cells is also dependent on the available cell
manipulation techniques.
Additional questions for use of GT - 3
1) Creation of genetic information (recombinant DNA
methods) that is intended for transport into cells
2) Selection of cells into which modified genetic
information will be introduced
3) Selecting the appropriate vector
4) After carrying out gene therapy, the patient's
condition must be carefully monitored, noting
both the improvement of the health condition and
the onset of possible complications
Gene therapy implementation includes
CLASSIC
➢ Optimal expression of the
inserted gene
➢ Creating the missing
product
➢ Direct elimination of
diseased cells
➢ Activation of the immune
system
NON-CLASSICAL
➢ Inhibition of pathogenic
gene expression
➢ Restoration normal
expression
➢ siRNA
Gene therapy strategies
Its goal is to deliver genes to suitable target cells
in such a way as to achieve optimal expression
of the introduced genes and
➢ Ensure the production of the substance that
is missing
➢ Activate immune system cells to help
eliminate diseased cells
Gene therapy "classic"
➢ Inhibition of the expression of genes
associated with pathogenesis
➢ Correction of the genetic defect and
restoration of normal gene expression
"Non-classical" gene therapy
Gene therapy by cell type
Germ cells
➢ A copy of the correct version of the
relevant gene is delivered to the
fertilized egg and the egg is
implanted back into the mother's
body
➢ The gene is usually present in all
cells of a new individual
➢ It is performed by microinjection of
DNA into the egg
➢ Theoretically applicable to cure any
hereditary disease
Somatic cells
➢ Handling cells that can be taken
from the body, transfected and put
back into the body
➢ Promising for the treatment of
hereditary diseases of blood cells
➢ Genes are introduced into bone
marrow stem cells
➢ Viruses serve as vectors retroviruses,
adenoviruses
➢ Problem with dominant characters
stem cell
new gene
transfection implantation
diferentiation
All mature cells contain the new gene
T-lymphocyte
B-lymphocyte
bazophil
eozinophil
neutrophil
macrophage
Gene therapy of stem cells
➢ Current gene therapy is limited to somatic
mutation therapy
➢ Ethical issues with potential germline
mutation therapy
Somatic or germ cells?
➢ Creation of genetic information (recombinant
DNA methods) that is intended for transport into
cells
➢ Selection of cells into which modified genetic
information will be introduced (in vivo, in vitro)
➢ Selecting the appropriate vector
➢ After carrying out gene therapy, the patient's
condition must be carefully monitored, noting
both the improvement of the health condition
and the onset of possible complications
Gene therapy implementation includes
Ex vivo (in vitro)
➢ Transfer of cloned genes into cells in culture
(transplantation of autologous genetically
modified cells)
In vivo (in vivo, in situ)
➢ The transfer takes place directly into the
patient's tissue using liposomes or viral
vectors
Genetic transfer
The cDNA with the complete DNA coding sequence is
modified to ensure a high level of expression, e.g. using
a potent viral vector
The subsequent insertion of the gene takes
place
➢ to the chromosome
➢ extrachromosomally
Principles of genetic transfer
➢ the gene will spread to other cells
➢ high level of expression (stem cells)
➢ random insertion - different localization
➢ different level of expression
➢ death of individual cells
➢ tumor degeneration (oncogene activation,
suppressor or apoptotic gene deactivation)
(advantage of ex vivo transfer)
Chromosome insertion
➢ Uncertain long-term effect
➢ The gene is expressed throughout the life of the
cell
Extrachromosomal insertion
1) Increase in gene expression – gene dose
effect
2) Killing of "sick" cells
3) Killing cells with the assistance of the
immune system
4) Targeted inhibition of gene expression
5) Targeted mutation repair
Implications of gene therapy
Principle of ex vivo gene therapy
N Engl J Med 2019;381:455-64.
The principle of in situ / in vivo gene therapy
NEnglJMed2019;381:455-64.
Example I. – insertion of a functional gene
deficient cells
gene T for toxin
T
T
T
Cells killed by expressed toxin
gene P for prodrug Cells killed by the drug
P
P
P
Example II. – targeted cell killing
Example II.
deficient cells
gene for antigen
gene for cytokine cell elimination
antigen exposure
cells of the immune system
cytokine production
Example III. - Killing of cells with the
participation of the immune system
Example III.
deficient cells
antimeditor gene
Inhibition of gene expression
Blocking the production of
the "pathological" protein
A
A
A
Example IV. - Targeted inhibition of gene expression
Example IV.
X
-
X
-
X
cell
with X- deficienci
gene X+
Gene conversion
Normal phenotype
X
+
X
+
X
+
gene X+
gene XExample
V. - Targeted mutation correction
Example V.
replicating virus
non-replicating virus
Cell lysis
Synthesis of virions
Synthesis of viral and
recombinant proteins,
incomplete virions
retrovirus
Integration into the cell
genome, synthesis of
recombinant proteins
Viral transfer
➢ Retroviral vectors
➢ Lentiviral vectors
➢ Adeno-associated virus vectors
➢ Adenoviral vectors
➢ Herpes simplex virus vectors
Mammalian viral vectors
5‘LTR 3‘LTRGag Pol Env
Protein of core
Replication/integration
Envelope proteins
Packing
signal
Retrovirus
5‘LTR 3‘LTRMarker Promoter Gene of interest
Identification/separation
Exprese genu
Reparing gene
Packing
signal
Retroviral gene therapy procedure
DOI: 10.3390/biomedicines4020009
• Integration into the genome
Gene therapy with an adenoviral vector
• Does not integrate into the genome = episomal DNA
• It infects various types of dividing and non-dividing cells
• Strongly immunogenic → used in the production of vaccines and anti-tumor drugs
• Replication-defective viruses → gene therapy and vaccines
• Replicating (oncolytic) viruses → antitumor drugs
https://doi.org/10.1016/j.gendis.2017.04.001
Gene therapy with adeno-associated virus
• Does not integrate into the genome = episomal DNA
• Use in in vivo gene therapy
• Recombinant AAV is created from non-pathogenic nonenveloped
parvovirus
• The efficiency of virus packaging is greatly reduced if the
transgene sequence is longer than 5 kbp
• In a mouse neonatal model, the formation of
hepatocellular carcinoma was demonstrated when a
high dose of AAV was used, in humans it was not
observed
Gene therapy for spinal muscular atrophy
N Engl J Med 2019;381:455-64.
Actual news
• Gene therapy of romatic L‐amino
acid decarboxylase (AADC)
deficiency
– Eladocagenum exuparvovecum is a gene
therapy drug that expresses a human
enzyme aromatic L-amino acid
decarboxylase (hAADC). It is a nonreplicating
vector of recombinant
adeno-associated virus serotype 2
(AAV2) containing cDNA of the human
dopadecarboxylase (DDC) gene under
the control of the cytomegalovirus
immediate early promoter.
– Eladocagenum exuparvovecum is
produced in human embryonic kidney
cells by recombinant DNA technology.
– Prize – 3,600,000 €
EMBO Mol Med. 2021 Sep 7; 13(9): e14712.
Approved
gene
therapies
N Engl J Med 2019;381:455-64.
According to Decree 84/2008 Coll. (Decree on good pharmacy practice,
closer conditions for handling medicines in pharmacies, medical facilities
and other operators and facilities dispensing medicinal products) it is
possible to prepare gene therapy products in a pharmacy (§3(8d)), in
vacuum safety boxes with vertical laminar flow air cleanliness class A and
with extraction outside the space, which are located in the space of air
cleanliness class B and are reserved for this purpose (§5(f)).
What if I wanted to start using gene therapy in the
Czech Republic?
➢ Receptor-mediated endocytosis
➢ Transfer via liposomes
➢ Direct DNA transfer
➢ Particle bombardment
Non-viral transfer of genes into cells
In vivo transfer via liposomes
Gene therapy of SMA
N Engl J Med 2019;381:455-64.
CAR-T therapy
• T-cells carrying chimeric antigen receptor (CAR) (stable transfection)
• CAR → antigen binding domain behind Ig or TCR + intracellular domain for T-cell
activation
• Mainly targeted treatment of tumors
• Marked systemic toxicity and unclear "tumor-off" effect.
https://www.cancer.gov/about-
cancer/treatment/types/immunotherapy/t-cell-transfer-
therapy
https://doi.org/10.3390/cancers12092360
Examples of gene therapy of cancer
Ethical aspects of gene therapies
➢ Handling safety
➢ Validation in mammalian models
➢ Hierarchical protocol approval
➢ Danger of side effects
➢ Gene therapy of germ cell
➢ Very high financial demands
➢ Technical and technological complexity
➢ Low success rate of therapy if there are problems
with the "attachment" of the introduced genetic
information
➢ Random insertion of genetic information =
disruption of proto-oncogene, tumor-suppressor
gene = malignant transformation of the cell
➢ Gene therapy is ethically problematic
Disadvantages of gene therapy
Risks of gene therapy
• Insertional mutagenesis – integration of a vector into the genome can
disrupt the function of the original DNA
• With an in vivo approach, an immune response to the viral vector
used may arise
NEnglJMed2019;381:455-64.
• Saving the lives of patients with severe genetic diseases or cancer
• Making life more pleasant for many other people whose illness is not so serious,
but who are still dependent on supportive therapy
• Necessary demarcation of the line between what is still ethical to use gene
therapy and what is not.
• Will there be "custom babies" in the future? Will we be able to determine the
color of our children's eyes, hair or height?
• Will we see an era of superhumans?
• Will gene therapy become such a business as plastic surgery is today?
The future of gene therapy
Genome editing (e.g. CRISPR/Cas9 technique) – gene knock-in,
knock-out, targeted mutagenesis
CRISPR/Cas9 technique
• CRISPR = Clustered
Regularly-Interspaced Short
Palindromic Repeats
• Cas9 = endonucleases
• A prokaryotic immune
system that provides
resistance to foreign genetic
elements such as phages
and thus represents a form
of acquired immunity.
Use of CRISPR/Cas9 in GT
https://www.sigmaaldrich.com/CZ/en/technical-documents/technical-
article/genomics/advanced-gene-editing/human-ipsc-crispr-protocol
https://doi.org/10.
3390/horticulturae
7070193
https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-
genome-editing-a-new-era-in-molecular-biology
Xenotransplation
• Xenotransplantation = transplantation of animal organs to human
• Modification of animal tissues/organs to become transplantable into human
• Posible obstacles:
–Strong immune incompatibility
–Posibility of transfer of animal viruses and bacteria to human
• March 2024 – first succesful transplantation of porcine kidney to human
(https://doi.org/10.1111/ajt.16930 - transplantation to man with brain dead)
–10-GE pig = 10 genetic modifications: targeted insertion of two
human complement inhibitor genes (hDAF, hCD46), two human anticoagulant genes
(hTBM, hEPCR), and two immunomodulatory genes (hCD47, hHO1), as well as deletion
(knockout) of 3 pig carbohydrate antigens and the pig growth hormone receptor gene