©1999 timothy g. standish pharmaceutical biotechnology phg 424 mounir m. salem, ph.d....

©1999 Timothy G. Standish

Pharmaceutical BiotechnologyPharmaceutical BiotechnologyPHG 424PHG 424

Mounir M. Salem, [email protected]

King Saud UniversityCollege of PharmacyDepartments of Pharmaceutics/ Pharmacognosy


Gene Therapy


Gene TherapyGene Therapy

Genes, which are carried on chromosomes, are the basic physical and functional units of heredity.

Genes are specific sequences of bases that encode instructions on how to make proteins. it’s the proteins that perform most life functions and even make up the majority of cellular structures.

IntroductionIntroduction



When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result.

Each of us carries about half a dozen defective genes. We remain blissfully unaware of this fact unless we, or one of our close relatives, are amongst the many millions who suffer from a genetic disease.

Introduction….Introduction….


What is Gene TherapyWhat is Gene Therapy

It is a technique for correcting defective genes that are responsible for disease development

There are four approaches:

1. A normal gene inserted to compensate for a nonfunctional gene.

2. An abnormal gene traded for a normal gene

3. An abnormal gene repaired through selective reverse mutation

4. Change the regulation of gene pairs



Gene Therapy is ExperimentalGene Therapy is Experimental

Advances in understanding and manipulating genes have set the stage for scientists to alter a person's genetic material to fight or prevent disease.

Gene therapy is an experimental treatment that involves introducing genetic material (DNA or RNA) into a person's cells to fight disease.



The Beginning…The Beginning…

In the 1980s, Scientists began to look into gene therapy.

– They would insert human genes into a bacteria cell.

– Then the bacteria cell would transcribe and translate the information into a protein.

– Then they would introduce the protein into human cells



The First CaseThe First Case

The first gene therapy was performed on September 14th, 1990

– Ashanti DeSilva was treated for SCID

Sever combined immunodeficiency

– Doctors removed her white blood cells, inserted the missing gene into the WBC, and then put them back into her blood stream.

– This strengthened her immune system

– Only worked for a few months



How It WorksHow It Works

A vector delivers the therapeutic gene into a patient’s target cell

The target cells become infected with the viral vector

The vector’s genetic material is inserted into the target cell

Functional proteins are created from the therapeutic gene causing the cell to return to a normal state



Principles of Gene therapyPrinciples of Gene therapy

A normal gene may be inserted into a non-specific location within the genome to replace a non-functional gene. This approach is most common.

An abnormal gene could be swapped for a normal gene through homologous recombination.

The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.

The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.



Gene Therapy Depends on Delivery of Corrective Gene Therapy Depends on Delivery of Corrective GenesGenes

Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells.

This process can be performed inside a living organism (in vivo) or in cell culture (in vitro).

Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect.



Viruses are used as Delivery TollsViruses are used as Delivery Tolls

Viruses are used as vectors to introduce the genetic material inside the bodies.

These viruses are inactivated, they are not able to reproduce

Adenoviruses most common

Herpes viruses DNA tumor viruses

Retroviruses RNA tumor viruses most common



Why Viruses?Why Viruses?

Viruses through the time of evolution have evolved to infect the cells with great specificity

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

This allows them to produce new viral particles at the period of synthesis of the cell


Majority are TrailsMajority are Trails

Gene therapy is being studied in clinical trials (research studies with people) for many different types of cancer and for other diseases.

It is not currently available outside a clinical trials.



What Gene therapy can AchieveWhat Gene therapy can Achieve

Replacing a mutated gene that causes disease with a healthy copy of the gene.

Inactivating, or “knocking out,” a mutated gene that is functioning improperly.

Introducing a new gene into the body to help fight a disease.



Uses of gene therapy Uses of gene therapy

Replace missing or defective genes;

Deliver genes that speed the destruction of cancer cells;

Supply genes that cause cancer cells to revert back to normal cells ??

Deliver bacterial or viral genes as a form of vaccination;

Provide genes that promote or impede the growth of new tissue; and;

Deliver genes that stimulate the healing of damaged tissue.



Delivering desired GenesDelivering desired Genes


ES = Embryonic stem cells. HLA= human leukocyte antigen. SCNT = somatic-cell nuclear transfer


Gene Therapy CorrectsGene Therapy Corrects

Gene therapy is a technique for correcting defective genes responsible for disease development.

Researchers may use one of several approaches for correcting faulty genes:



Steps in Gene TherapySteps in Gene Therapy


AAV = adeno-associated virus


Manipulation corrects the Defective GenesManipulation corrects the Defective Genes



Gene Therapy delivers ProteinsGene Therapy delivers Proteins

Today, gene therapy is the

ultimate method of protein

delivery, in which the delivered

gene enters the body's cells and

turns them into small "factories"

that produce a therapeutic

protein for a specific disease

over a prolonged period.



Antisense therapyAntisense therapy

Antisense therapy is a form of treatment for genetic disorders or infections.

When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off".



Antisense TherapyAntisense Therapy

Antisense therapy is not strictly a form of gene therapy, but is a genetically-mediated therapy and is often considered together with other methods



Making the new Genetic Material FunctionalMaking the new Genetic Material Functional

Gene that is inserted directly into a cell usually does not function.

Instead, a carrier called a vector is used to introduce the therapeutic gene into the patient's target cells.

The most common vector that is used is a virus that has been genetically altered to carry normal human DNA.



Somatic and Germ Line Gene Therapy Somatic and Germ Line Gene Therapy

Gene therapy can target somatic (body) or germ (egg and

sperm) cells.

In somatic gene therapy the recipient's genome is changed,

but the change is not passed on to the next generation;

whereas with germ line gene therapy the newly introduced

gene is passed on to the offspring.



Safety Safety

Safety: Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them.



Making safe ProtocolsMaking safe Protocols

Low toxicity:

The viral vector should have a minimal effect on the physiology of the cell it infects.

Stability:

Some viruses are genetically unstable and can rapidly rearrange their genomes.



Cell type specificityCell type specificity

Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible.

However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell.



LentivirusLentivirus

Lentivirus (lenti-, Latin for "slow") is a genus of slow

viruses of the Retroviridae family, characterized by a

long incubation period.

Lentiviruses can deliver a significant amount of

genetic information into the DNA of the host cell, so

they are one of the most efficient methods of a gene

delivery vector. HIV, SIV, and FIV are all examples

of lentiviruses.



RetrovirusesRetroviruses

Retroviruses can infect only dividing cells.

The viral genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme



Vectors deliver the Genetic MaterialsVectors deliver the Genetic Materials

The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides.



AdenovirusesAdenoviruses

As opposed to lenti viruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division.

Adenoviral vectors are occasionally used in in vitro experiments.



Choosing non infective AdenovirusChoosing non infective Adenovirus

Their primary applications are in gene therapy and vaccination. Since humans commonly come in contact with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences

To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity.



Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species.

AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.

AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.

These features make AAV a very attractive candidate for creating viral vectors for gene therapy


Adeno-associated virusesAdeno-associated viruses


Limitation of Direct Gene InductionLimitation of Direct Gene Induction

The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA.



Nonviral approachNonviral approach

Nonviral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane



Nonviral Vectors: Liposome's less ImmunogenicNonviral Vectors: Liposome's less Immunogenic

DNA/lipid complexes are easy to prepare and there is no limit

to the size of genes that can be delivered. Because carrier

systems lack proteins, they may evoke much less

immunogenic responses.

More importantly, the cationic lipid systems have much less

risk of generating the infectious form or inducing tumorigenic

mutations because genes delivered have low integration

frequency and cannot replicate or recombine.



Nanoengineered substancesNanoengineered substances

Nonviral substances such

as Ormosil have been used

as DNA vectors and can

deliver DNA loads to

specifically targeted cells in

living animals. (Ormosil

stands for organically

modified silica or silicate)



Transfection and NanoengineeringTransfection and Nanoengineering

Transfection is the process of introducing nucleic acids into cells by non-viral methods.

The term "transformation" is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells;

"transduction" is often used to describe virus-mediated DNA transfer.



Problems of Large Gene

It would be a large vector capable of carrying substantial

amounts of genetic code, and scientists anticipate that,

because of its construction and autonomy, the body's immune

systems would not attack it.

A problem with this potential method is the difficulty in

delivering such a large molecule to the nucleus of a target

cells.



Gene Therapy Uses AIDS Virus to Fight AIDS

In the study, immune cells were removed from the patients' bodies, modified with a disabled AIDS virus known as a lentivirus, and then intravenously returned.

The genetically altered cells disseminated anti-HIV material and prevented HIV from reproducing

( 07 November, 2006)



Technical Difficulties in Gene Therapy

Gene delivery: Successful gene delivery is not easy or predictable, even in single-gene disorders. For example, although the genetic basis of cystic fibrosis is well known, the presence of mucus in the lungs makes it physically difficult to deliver genes to the target lung cells.

Delivery of genes for cancer therapy may also be complicated by the disease being present at several sites.

Gene-therapy trials for X-linked severe combined immunodeficiency (X-SCID), however, have been more successful



Problems with Gene Therapy Short Lived

Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long time

Would have to have multiple rounds of therapy Immune Response

new things introduced leads to immune response increased response when a repeat offender enters

Viral Vectors patient could have toxic, immune, inflammatory response also may cause disease once inside

Multigene Disorders Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are

hard to treat because you need to introduce more than one gene May induce a tumor if integrated in a tumor suppressor gene because

insertional mutagenesis



What are the ethical issues surrounding gene therapy?

How can “good” and “bad” uses of gene therapy be distinguished?

Who decides which traits are normal and which constitute a disability or disorder?

Will the high costs of gene therapy make it available only to the wealthy?

Could the widespread use of gene therapy make society less accepting of people who are different?

Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?


The Future of Gene Therapy

Current uses of gene therapy focus on treating or curing existing conditions.

In the future, the focus could shift to prevention.

As more of the human genome is understood, medicine will know more about which genes contribute to or cause disease. With that knowledge in hand, gene therapy could be used to head off problems before they occur.


Creating 47th Chromosome

Researchers are also experimenting with introducing a 47th artificial chromosome to the body.

It would exist autonomously along side of the other 46, not affecting their workings or causing any mutations.

It would be a large vector capable of carrying substantial amounts of genetic information and the body’s immune system would not attack it.


Several Diseases have Genetic basis

Gene mutations probably play a role in many of today's most common diseases, such as heart disease, diabetes, immune system disorders, and birth defects.

These diseases are believed to result from complex interactions between genes and environmental factors.

When genes for diseases have been identified, scientists can study how specific environmental factors, such as food, drugs, or pollutants interact with those genes.


Gene therapy of pain: emergingstrategies and future directions

Gene therapy to alleviate pain could appear surprising and perhaps not appropriate when opioids and other active molecules are available. However, the possibility of introducing a therapeutic protein into some targeted structures, where it would be continuously synthesised and exert its biological effect in the near vicinity of, or inside the cells, might avoid some drawbacks of "classical" drugs.


Pain – Cancer a major research area

Numerous other molecules involved in pain processing or associated with chronic pain have been identified and the gene-based techniques might be particularly adapted for the evaluation of the possible therapeutic interest of these new potential targets


Cancer Gene Therapy

…Using Tumor Suppressor Genes.


Overview

Gene Therapy

p53

Using Gene Therapy to Treat Lung Cancer

Problems


Types of Viruses…

Retrovirus Adinovirus Lentiviruses Poxviruses and Herpes Viruses


Adenovirus

36 kb Double Stranded DNA Genome

Entry through CAR receptor and integrin co-receptor


E1A E3 E1B

E2A E4E2B

L1 L2 L4L3 L5

Latest Generation Adenoviral Vector

“Gutless”; Helper-dependent; Minimal Ad

Therapeutic Transgene

Stuffer DNAStuffer DNA ITRITR


Which Virus to Use?

Depends how well they transfer the genes to cells which cells they can recognize and infect and whether they alter the cell’s DNA permanently

or temporarily


Cells removed from body

Transgene deliveredCells cultured

Cells returned to the body

Ex Vivo In Vivo

Transgene delivereddirectly into host

Strategies for Transgene Delivery


Which cells are the target cellsWhich cells are the target cells

Both Healthy and Cancerous cells can be

a target Ex of targeting Healthy cells

– One way is by replacing a missing or altered gene with a “normal” one


Cont: Which cells are the target Cont: Which cells are the target cellscells

Ex of targeting Cancer Cells Scientists can target cancer cells with genes that can

be used to destroy the cells. In this technique, cancer cells are introduced to what is called “suicide genes”


Naked DNANaked DNATarget Target

CellCell

Therapeutic Therapeutic ProteinProtein

AAVAAV

Retrovirus/LentivirusRetrovirus/Lentivirus

AdenovirusAdenovirus

NucleusNucleus

Gene Therapy Principles


Adenovirus Cell Entry


p53 Pathwayp53 Pathway


Using Gene Therapy to Treat Using Gene Therapy to Treat Lung CancerLung Cancer

In this clinical trial the scientist used gene therapy in combination with radiation therapy so they can treat lung cancer in 19 different patients


Treatment: Gene therapy and Treatment: Gene therapy and Radiation.Radiation.

Intratumoral needle injections of Ad-p53 on days 1, 18 and 32 of the treatment.

tumors ≥ 4 cm where injected with 10 ml tumors ‹ 4 cm were injected with 3 ml

Radiation therapy


ResultsResults

17/19 patients made it through the entire therapy

complete response in 2 patients (11%) partial response in 4 patients (21%) stable disease in 1 patient (5%) progressive disease in 11 patients (57%)


Results Not That GoodResults Not That Good

57% of the patients showed that the cancer progressed to worse stages

Why?


Major Problems that Scientists Major Problems that Scientists Must OvercomeMust Overcome

Identify more efficient ways to deliver the genes to the patients’ genetic material

Develop vectors that can specifically focus on the targeted cells

Ensure that vectors will successfully insert the desired genes into each of these target cells


ContCont. . Major Problems that Major Problems that Scientists Must OvercomeScientists Must Overcome

Deliver genes to a precise location in the patient’s DNA

Ensure that transplanted genes are precisely controlled by the body’s normal physiologic signals

©1999 timothy g. standish pharmaceutical biotechnology phg 424 mounir m. salem, ph.d....

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