Gene therapy (GT)?

Treatment the disease by transferring

genetic material to the human cells
Term used the first by Friedmann and Roblin (1972)

Gene therapy (GT)

may theoretically concern
germ line cells
foetal cells
somatic cells

Gene therapy (GT)

may theoretically concern
germ line cells
foetal cells
somatic cells

Types of GT
Supplementary
Suppressory
Conferring new phenotypic
features to cells

Strategies of GT

In vivo
Ex vivo

In vivo or Ex vivo

In vivo or Ex vivo

Basic techniques of GT

Molecular cloning

DNA recombination
Cell transfection

vector
insert
transgene

Some terms

Vector
Insert
Transgene
Vectortransfer
Not-vectortransfer
Sense
Antisense

Somatic cell GT attempts

Typical diseases:

Hereditarygeneticdefects(monogenediseases)
Recessive
Dominant

Tumors
Infectiousdiseases
Otheracquireddisorders(usuallymultigenedetermineddiseases=complexdiseases)

Examples of genetic diseases as

targets of GT:
GROUP

EXAMPLE

Coagulopathies

HemofiliaA,B

Hemoglobinopathies

Sicklecelldisease

Immune deficiences

ADA(adenosinedeaminaze)deficiency

Lipidoses

Gaucherdisease

Disorders of urea cycle

Ornithinetranscarbamoylasedeficiency

Disorders of uric acid

metabolism

Lesch-Nyhansyndrome

Hyperlipidemias

Familialhypercholesterolemia

What disorders can be treated

with GT?
Life-threatening conditions
No effective conventional therapy
In hereditary disorders only monogenic disease with
known cloned gene sequence should be cosidered
The regulation of gene expression: it cant be precise
Solving technical problems concerning
gene delivery to the target cell
gene expression in the cell

First trials
DNAtransferto
tumorcell

Suplementation
(substitution)ofthe
sickgene

Treatmentof
melanoma

ADAdeficiency
treatment

1989

1990!

Two patients
Ashanti da Silva / Jessi Gelsinger

Techniques of gene transfer to

the target cell
-DNAprecipitationwithcalciumions
- Electroporation(currentshock)
- InjectionofnakedDNAtothecellnucleus
(micropipets)
- TransferofnakedDNAdirectlytotheskeletal
muscles
- DNAtransferwithliposomes
- DNAtransferwithcationiclipidsmacromolecules
- TransferofproteincoatedDNA
- Viral vectors
- Plasmids

Central dogmat of
molecular biology

The most popular viral vectors

Adenovirus
Retrovirus
Lentivirus (subtype of Retrovirus)
AAV(Adeno-Associated virus)
Herpes virus
Vaccinia virus

Adenovirus
Linaerdouble-strand
DNA as a genetic
material
Theydonotintegrate
withthehostgenome
Theytransfectwide
rangeofhumancellular
tarhets,includingnotproliferatedcells

Adenovirus vectors

Hightransductionefficiency(in vivo, ex vivo)

Largespectrumoftransfectedcells
Highleveloftransgeneexpression
Highconcentrationtransfectionagents

Cytotoxicity
Short-termtransgeneexpression(nointegrationwith
genome)
Intensiveimmuneresponse(repeatedtransfection?)

Adenovirus

E1

late genes

MLP

E3
ITR

ITR
10

20

IVa2

30

40

50

60

70

80

(E2F, pRB , TBP)

E2

90

100

E4
3.6 kb

1st generation Ad vectors

Late genes (L1-L5)

E1

ITR

10

ITR

? E3

Transgene
20

30

40

50

60

70

80

90

100

E2

E4
3.6 kb

293 cells

E1
ITR

Retrovirus vectors
Genetic material: single-strand RNA
Small genome with 3 characteristic genes
gag encoding core and structural proteins
pol encoding reverse transcriptase and
integrase
env encoding capsule proteins
These genes expression in controled by flank
sequences known as LTR (5'-long terminal repeat)
Basic genome element is signal sequence which
drives viral RNA packing to the protein capsule

Gag-Pol-Env

TRANSGEN
TRANSGEN

TRANSGEN
TRANSGEN

TRANSGEN

TRANSGEN
TRANSGEN

TRANSGEN

TRANSGEN
TRANSGEN

TRANSGEN

TRANSGEN
TRANSGEN

TRANSGEN

TRANSGEN
TRANSGEN

TRANSGEN

TRANSGEN
TRANSGEN

Retrovirus as GT tool

Quickintegrationwithcell
genome
Lowdegradationrate
(contrarytootherDNAand
RNAvectors)
Hightransfectionefficacy
Singletransgenecopyin
specificgenomesite

Limitedsizeoftrasgene
Carcinogenesisrisk
Contaminationwith
geneticmaterialcoming
fromforeigncells

Lentivirus as GT tool
Retrovirus subgroup, HIV-1 as the best known
Transfection of not dividing cells
Long-term transgene expression
Inter alia, good transduction of bone marrow
stem cells
SHORTCOMINGS:

Difficult in construction and control Insertion

mutations possible

Most frequently viral vectors in GT

Summary
Vector/ virus

Genetic
material

Genome Cytopathogenic
activity
integration

Maximal
transgene
size

Retrovirus

RNA

Low

9000bp

Adenovirus

DNA

Low

8000bp

AAV

DNA

-/+

Low

5000bp

HSV

DNA

High

25000bp

Vaccinia

DNA

High

GLYBERA
Transgene:

lipoprotein lipase gene, LPL (S447X)

Vector: AAV (adenovirus-associated virus)

Tiparvovec (AAV1-LPL S447X)
Action: phenotype correction in hiperchylomicronemia
patients (hiperlipidemia I i V type)

including remerkable decrease of trigliceridemia,

frequency reduction of acute pancreatitis episodes

Approval:
Administration:

FDA and European Comission in July 2012

I.M.

Gene transfer into the cell by AAV

vector

GENDICINE (rAD/p53)

Transgene:
p53
Vector:
AdV
GTsupplemntarytechnique
Target: head and neck squamous cell cancer
(HNSCC).

Approval Schenzhen SiBiono GeneTech in China; up to date

have not been approved in US and EU

Target:cancercells
Strategy: transfection of p53 gene copy affects in
cellcyclesuppressionandapoptosisintumorcells

Target cells in GT 1
Hematopoietic stem cells
Source: bone marrow
umbilical blood
Application:
Genetic deffects of hematopoieic and immune
system
Cell pluripotency and extremaly high
proliferation potential

Target cells in GT 2
Fibroblasts &
keratinocytes
Easy to obtain and grow in culture
They can secrete many compounds characterized by
high biologic activity (e.g. Factor IX)
Early side effect: graft rejection (use of aloogenic
models limited)

Target cells in GT 3
Hepatocytes
Easy to collect
Retrovirus transfected cells are to be
transferred back to the liver by the spleen
Specific hepatocyte receptors to be used
Gene expression for months (>6)

Target cells in GT 4
Skeletal muscle cells
Oportunity to inject plasmid directly to the
muscle cell
Gene expression for months
Application of foreign DNA in patient
vaccination (DNA vaccines)

Target cells in GT 5
Lymphocytes
Easy to collect (from peripheral blood, PBL)
Long cell live, including laboratory conditions
Key role in immune response
Key role in anti-tumor combat

Specifity of GT?
1
Tissue

Promoter

myelin proteins
Albumin
CK
Tyrosinase, TRP-1, -2
NSE

Glial cells
Hepatocytes
Miocytes
Melanocytes
Neurons

Specifity of GT?
2
Tumor
Adenocarcinomas
Breast cancer

Liver cancer
Inradiated tumors
Chemoresistant tumors

Promoter
CEA
erbB2
ALA
BLG
CEA
egr-1
mdr-1