7/25/2019 Gene Therapy 3-4-2016

1/43

Gene Therapy

7/25/2019 Gene Therapy 3-4-2016

2/43

What is Gene Therapy?

Gene therapy is a technique for

introducing the genetic material of a gene

in a patient that lacks that gene becauseof a mutation. (= Gene transfer)

It represents the App l icat ion of nucleic

acids for treatment of diseases

7/25/2019 Gene Therapy 3-4-2016

3/43

Gene therapy targets In principle, any disease can be targeted for gene therapy. In

practice, we need to know the following

(1) Does the disease arise from a mutation in one (or a few)

genes?

(2) Has the mutant gene been identified ? (3) Has the corrected gene been cloned?

(4) Where is the gene expressed (tissues)?

(5)Will a normal gene solve the problem (dominant negative

mutations)?

(6) Can we deliver a normal gene to the affected tissue?

7/25/2019 Gene Therapy 3-4-2016

4/43

Gene Delivery:

Gene delivery is the Key to successful gene therapySuccessful gene delivery involves:

(1) Targeting: repaired gene must specifically enter the

correct cells

(2) Activation: repaired gene needs to enter nucleus and

be successfully transcribed in response to regulatory cues

(3) Integration: for long term protection, the repaired genemay need to integrate into the genome and be replicated

(4) No side effects: the introduction of any foreign

biological material runs the risk of being toxic, damaging

the cells or stimulating an immune response

7/25/2019 Gene Therapy 3-4-2016

5/43

Barriers for introducing Nucleic acid into cells/tissues

1- Crossing plasma membrane:

A- Cell membrane has a negative charge & would

block attachment of DNA/RNA to cell membrane.

B- Also DNA/RNA are hydrophilic & would block

direct fusion with lipid plasma membrane.2- Release from phagosomes (Phagocytosis)

3- Dissociation of nucleic acid from the chemical

vehicle/carrier

4- Transportation to the nucleus (can be improved by

adding ORI sequence from SV40 virus to the nucleic

acid or adding to the chemical vehicle a protein with

nuclear localization signal,NLS,e.g.SV40

7/25/2019 Gene Therapy 3-4-2016

6/43

7/25/2019 Gene Therapy 3-4-2016

7/43

Vectors

The way you insert the normal gene in the

patients cell is by vectors.

Vector: A carr ier fo r therapeut ic nucleic acid

for i ts del ivery to target t issues/ cel ls

Vehicle: A chemical substance, which

improves delivery of nucleic acid to the cells

The most common vectors that are used in

gene therapy are virus vectors

7/25/2019 Gene Therapy 3-4-2016

8/43

Transformation: is I ntr oduction of foreign genes into prokaryotic cells

such as bacter ia

Transfection: Introduction of foreign genes to eukaryotic cells by

means of plasmid or vectors

Transduction: I ntroduction of genes by means of viral vectors

7/25/2019 Gene Therapy 3-4-2016

9/43

7/25/2019 Gene Therapy 3-4-2016

10/43

7/25/2019 Gene Therapy 3-4-2016

11/43

7/25/2019 Gene Therapy 3-4-2016

12/43

Qualities of a suitable vector for gene therapy

1) An optimal vector should be one that can be made available ina highly intense form using an ideal and reproducible production

plan.

2) The vector must protect the genetic material from degradation

by host.3) Those vectors that can accomplish site-specific integration are

more preferred and desirable in case of insertional mutagenesis.

4) A perfect vector should be harmless and should not cause any

noxious effect on human health.

7/25/2019 Gene Therapy 3-4-2016

13/43

Why Viruses?

Viruses through the time of evolution have

evolved to infect the cells with great specificity

Viruses tend to be very efficient at transfectingtheir own DNA into the host cell genome.

This allows them to produce new viral particles

at the period of synthesis of the cell

7/25/2019 Gene Therapy 3-4-2016

14/43

Types Of Viruses

Retrovirus

Adenovirus

Adeno associated virus

Lentiviruses

Poxviruses and Herpes Viruses

7/25/2019 Gene Therapy 3-4-2016

15/43

Which Virus to Use?

Depends how well they transfer the genes to cells

Mainly on which cells they can recognize and infect

and whether they alter the cells DNA permanently(integration) or temporarily (Transient)

Whether cells are dividing or not?

Viral vectors have natural host cell populations that they infect most

efficiently. Retroviruses have limited natural host cell ranges. Adenovirus

and adeno-associated virus are able to infect a relatively broader range

of cells efficiently. And are the most popular vectors for use in gene

therapy trials.

http://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.html

7/25/2019 Gene Therapy 3-4-2016

16/43

Advantage Disadvantage

Retrovirus -Stable integration into chromosome

- Moderate transduction efficiency- (ss RNA virus with Reverse transcriptase &

integrase enzyme)

- Only infect actively dividing cells;

especially useful for tumor therapy- Limited size of introduced gene

about 8 kb

- Insertional mutagenesis possibility

Adenovirus

(ds DNA

virus)

-Higher transduction efficiency

- Broad range of target cells

- Does not require dividing cells- Low risk for insertional mutagenesis

- Most successful vector with 30 kb capacity

-Transient expression

- Immuno genecity Respiratory tract

infection)

Adeno-

associated

virus (AAV)

(ss RNA)

-Does not require cell division

- Site specific integration

-Not immunogenic

-Infect both dividing and non-dividing cells

- limited size of introduced gene

about 4.5 kb

Herpes

simplex

(ds DNA)

- Large size of introduced gene about 30 kb

-Can infect a wide range of cells, including

neurons

- Cytotoxic

Non viral -No limitation for size

- Easy to produce

- Not immunogenic

- Low efficiency

7/25/2019 Gene Therapy 3-4-2016

17/43

Non viral vectors are biocompatible moieties which can directly deliver genetic material

(DNA/RNA) into a specific cell with minimal toxicity. DNA is encapsulated with liposomes

(cationic lipids mixed with nucleic acids) or nanoparticles and then injected into a

specific cell. They can be produced in large scale, easy to manipulate and are easy forcell or tissue targeting. The only disadvantage of non-viral vector is its reduced

transfection efficiency in cells or tissues. Non-viral delivery systems utilize different

genetic materials such as antisense oligonucleotide ( AON ), plasmid DNA, small

interfering RNA ( siRNA ), short hairpin RNA ( shRNA ), and micro RNA (miRNA) that

works on electrostatic interaction. On the other hand cationic vectors such as lipid and

polymers help in the formation of lipoplexes and polyplexes.

1- Liposomes in gene therapy: (hollow sphere surrounded by a lipid bilayer)

lipos meaning fat and soma meaning body. A liposome is a tiny bubble (vehicle)

made out of the same material as a cell membrane. Liposomes can be filled with DNA

and can be used to deliver for cancer and other diseases. Membranes are usually made

of phospholipids which are molecules that have a head group and a tail group. Head is

hydrophillic and tail is long hydrocarbon chain and is hydrophobic. Usually phospholipids

are bilayer stable membranes. During packaging of the polynucleotide material in

liposome, a number of structures are formed. Each structure formed is the most

energetically favorable conformation based upon characteristics of specific lipids used.

7/25/2019 Gene Therapy 3-4-2016

18/43

2- Lipoplexes and polyplexes in gene therapy:

To improve the delivery of DNA into the cell, it must be protected from damage and its

entry into the cell must be facilitated. Plasmid DNAs are covered by lipids in an

organized structure like a micelle or liposome. When the organized structure is

complexed with DNA, it is called lipoplex. There are three types of lipids: cationic,anionic and neutral. Cationic lipids are most preferred for gene delivery because these

are positively charged and thus complex well with negatively charged DNA and interacts

with the cell membrane, endocytosed by the cell and finally release the DNA.

Can be used to transfect cells in lung in cystic fibrosis treatments

Poor targeting and Inactivated in the blood

3- Naked DNA in gene therapy:

The simplest non-viral gene delivery system uses naked DNA as a vector. Direct

injection of free DNA into certain tissues specially muscles produce high levels of gene

expression. It is particularly applied to cancer tissues where the DNA can be injected

either directly into the tumor or can be injected into muscle in order to express tumorantigens that might function as a cancer vaccine. But the expression is usually non

uniform and fails to correct the underlying histological and functional abnormalities of the

disease. Though it leads to gene expression but level of expression is much lower than

with either viral or liposomal vectors. It is unsuitable for systemic administration due to

the presence of serum nuclease. Therefore its application is limited

to muscle cells and skin.

7/25/2019 Gene Therapy 3-4-2016

19/43

4- Immunotherapy comprises of majority of the gene therapy trials for cancer. It has

been well established that cancer cells are weakly immunogenic because they are self

and by virtue of many ways to down regulate host immune response. Most of the studies

related to cancer gene therapy involve manipulation of tumor cells ex vivo to enhancethe production of interleukins, interferon gamma and tumor necrosis factor

Currently, antigen presenting cells such as dendritic cells isolated from a patient are

modulated to enhance the immune response against cancer.

5- Virosome is a drug or vaccine delivery system. It is a virus-like particle that acts as a

vaccine carrier and adjuvant thereby acting as an immune enhancing system. Vaccinesthat are manufactured using virosome technology show high efficacy with high purity

and hence it is a safe and an effective way to vaccinate infants and adults. Virosomes

are reconstituted viral envelopes including membrane lipids and viral spike glycoproteins

but devoid of viral genome. They are highly effective as vaccine antigens and adjuvants

because they stimulate humoral immune response because of the presence of viral

glycoproteins. The main advantage of virosomes over other drug delivery system likeliposomal and proteoliposomal carrier system is that the virosomes protect

pharmaceutically active substances from proteolytic degradation and low pH within

endosomes. This helps the contents to be in intact form till it reaches cytoplasm. . It is

target specific and can stimulate both cellular and antibody immune response to

maximize protection against the targeted disease. In addition to this, they are completely

biodegradable.

7/25/2019 Gene Therapy 3-4-2016

20/43

Cells removed from body

Transgene delivered

Cells cultured

Cells returned to the body

Ex Vivo In Vivo

Transgene delivered

directly into host

Strategies for Transgene Delivery

7/25/2019 Gene Therapy 3-4-2016

21/43

7/25/2019 Gene Therapy 3-4-2016

22/43

7/25/2019 Gene Therapy 3-4-2016

23/43

Target site for somatic gene therapy

There are different target sites for the gene therapy

Endothelium: The advantage of using endothelium as a target site for gene therapy

includes formation of new blood vessels and ease to get the gene of interest directly intothe blood stream. Eg- Hemophilia

Muscle: It is easily accessible to blood stream and muscle specific promoter is well

studied. Eg- Duchenne Muscular Dystrophy.

Liver: It has a capacity to regenerate and involve in a variety of functions. Thyroid

binding promoter is well studied for liver specific gene transfer. Eg- Familial

hypercholesterolemia.Skin: Grafting of skin is possible and small piece can be grown to a large.

Brain: Important for nervous system related illnesses.

Lung:Airway epithelium is easily accessible organ. Eg- gene therapy for cystic fibrosis.

7/25/2019 Gene Therapy 3-4-2016

24/43

Naked DNATarget

Cell

TherapeuticProtein

AAV

Retrovirus/Lentivirus

Adenovirus

Nucleus

Gene Therapy Principles

7/25/2019 Gene Therapy 3-4-2016

25/43Adenovirus Cell Entry

7/25/2019 Gene Therapy 3-4-2016

26/43

Lesch-Nyhan Syndrome (LNS); is a rare, inherited disorder caused by a

deficiency of the enzyme hypoxanthine-guanine phosphoribosyl

transferase(HPRT). LNS is an X-linked recessive disease

The lack of HPRT causes a build-up of uric acid crystals in all body fluids, in

the joints, kidneys, central nervous system, leading to gout-like swelling in the

joints and severe kidney problems with poor muscle control With lip and finger

biting

Neurological symptoms include facial grimacing, involuntary writhing, and

repetitive movements of the arms.

Treatment for LNS is symptomatic. Gout can be treated with allopurinol tocontrol excessive amounts of uric acid. Kidney stones may be treated by using

shock waves or laser beams. There is no standard treatment for the

neurological symptoms of LNS.

7/25/2019 Gene Therapy 3-4-2016

27/43

HPRT deficiency- results in Lesch Nyhan syndrome

ADA deficiencyresults in severe immunodeficiency syndrome

7/25/2019 Gene Therapy 3-4-2016

28/43

The first gene therapy experiment was conducted on a 4-years-old American girl in

1990. She was suffering from a genetic disease having deficiency of adenosine

deaminase enzyme required for the proper metabolism of purine. Any malfunctioning of

adenosine deaminase leads to severe combined immunodeficiency in affected patients.

The white blood cells of the girl was taken out and corrected with a human gene usingretrovirus as a vector.

7/25/2019 Gene Therapy 3-4-2016

29/43

First partial success (~1990)

Young child with Severe Combined

Immuno Deficiency (SCID)

- point mutation in a single gene of white

blood cells

- withdrew blood; introduced normal gene;transfused blood

Improved quality of life (not a cure) but

required the procedure be repeatedevery 3-4 months

(Note: blood cells have life span of ~120

days)

7/25/2019 Gene Therapy 3-4-2016

30/43

The molecular basis of SCID (Severe combined immunodeficiency

disease)

ADA-SCIDAdenosine Deaminase defect. Adenosine metabolites build up in the

blood and kill T cells. Therefore, no T cell immunity.

First genetic disease targeted with gene therapy 1990; 2 girls. T cells

taken out, infected with ADA virus, re-infused Problem: Too low alevel of ADA expression Correction: LTR contained element that

suppressed transcription in stem cells--eliminated!

X-SCID

Kids lack the IL2 receptor gamma common (C) chain,One of the components of the IL2, IL4, IL7, IL15 receptors.

T cells can not expand after stimulation (IL2, IL4).

B cell do not undergo class-switching (IL7, IL15). Have no natural

killer T cells. In 1998, the French study of 11 individuals was initiated

to replace gamma C in blood stem cells

7/25/2019 Gene Therapy 3-4-2016

31/43

7/25/2019 Gene Therapy 3-4-2016

32/43

7/25/2019 Gene Therapy 3-4-2016

33/43

7/25/2019 Gene Therapy 3-4-2016

34/43

7/25/2019 Gene Therapy 3-4-2016

35/43

Cancer gene therapy

1. Direct attack on tumor cells

a) transfer of tumor suppressor gene (p53)

b) Inhibition of oncogenes

-anti-sense therapy

-ribozymes (Degrading enzymes)

c) Suicide genes

d) Oncolytic viruses

2. Harnessing immuneresponse to tumor antigens-overexpression of cytokines genes

-tumor vaccines

3. Anti-angiogenic therapy

7/25/2019 Gene Therapy 3-4-2016

36/43

Oncolytic viruses

7/25/2019 Gene Therapy 3-4-2016

37/43

Strategy of genetically modified tumor vaccines;

1.Isolate tumor cells from a patient=autologic cells

2. Alternativeculture other tumor cells-eg, cell line of

The same typei.e. allogeneic cell line (Dead cancer cell)

3. Transduce such cells with vectoreg. Retroviral vector

Harboring cytokine gene

4. Inject such modified cells into patients

5. Allogeneic tumor cells stimulate immune system, and also

cytokines enhance this response to kill cancerous tissues.

7/25/2019 Gene Therapy 3-4-2016

38/43

Disorder Affected Gene

SCID adenosine deaminase (ADA)

Lesch-Nyhan

syndrome

hypoxanthine-guanine

phosphoribosyltransferase (HGPRT)

Familial

hypercholesterolemia

(FH)

LDL receptor

Phenylketonuria

(PKU)phenylalanine hydroxylase

-Thalassemia -Globin

Hemophilia B Clotting Factor IX

Cystic fibrosis Cystic fibrosis transmembrane receptor

Melanoma Tumour necrosis factor (TNF)

http://themedicalbiochemistrypage.org/scid.htmlhttp://themedicalbiochemistrypage.org/lesch-nyhan.htmlhttp://themedicalbiochemistrypage.org/lesch-nyhan.htmlhttp://themedicalbiochemistrypage.org/fh.htmlhttp://themedicalbiochemistrypage.org/fh.htmlhttp://themedicalbiochemistrypage.org/fh.htmlhttp://themedicalbiochemistrypage.org/pku.htmlhttp://themedicalbiochemistrypage.org/pku.htmlhttp://themedicalbiochemistrypage.org/betathalassemias.htmlhttp://themedicalbiochemistrypage.org/betathalassemias.htmlhttp://themedicalbiochemistrypage.org/hemophilia-b.htmlhttp://themedicalbiochemistrypage.org/hemophilia-b.htmlhttp://themedicalbiochemistrypage.org/betathalassemias.htmlhttp://themedicalbiochemistrypage.org/pku.htmlhttp://themedicalbiochemistrypage.org/fh.htmlhttp://themedicalbiochemistrypage.org/lesch-nyhan.htmlhttp://themedicalbiochemistrypage.org/scid.html

7/25/2019 Gene Therapy 3-4-2016

39/43

7/25/2019 Gene Therapy 3-4-2016

40/43

7/25/2019 Gene Therapy 3-4-2016

41/43

Gene therapy using germ cells result in permanent changes that are

subsequently passed on to next generation. Gene therapy using somatic

cells are not passed on to the next generation. Somatic cell gene therapy is

safer than the germ line gene therapy. Somatic cell gene therapy can bedone exterior (where cells are modulated in vitro and then transplanted

back) or interior (where genes are changed in vivo ).

ex vivo, which means exterior (where cells are modified outside the body

and then transplanted back in again). cells from the patients blood or bonemarrow are removed and grown in the laboratory. The cells are exposed to

the virus that is carrying the desired gene. The virus enters the cells and

inserts the desired gene into the cells DNA. The cells grow in the laboratory

and are then returned to the patient by injection into a vein. This type of

gene therapy is called ex vivo because the cells are treated outside thebody.

in vivo, which means interior (where genes are changed in cells still in the

body). This form of gene therapy is called in vivo, because the gene is

transferred to cells inside the patients body.

http://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.htmlhttp://www.genetherapynet.com/glossary-of-gene-therapy-terms.html

7/25/2019 Gene Therapy 3-4-2016

42/43

Today's Science Fiction

Gene therapy was viewed as Science

Fict ion 40 years ago when the genes

were shown to be basic units of

heredity

Gene therapy is now a reality and there

are many successful treatments in thatfield (still years to go ...)

So what can we look forward to (be

afraid of? in another 40 ears ....

7/25/2019 Gene Therapy 3-4-2016

43/43

Most diseases can be(potentially) treated with

gene therapy, because everydisease has a genetic

background!