use of gene therapy in the treatment of disease by ming

8/3/2019 Use of Gene Therapy in the Treatment of Disease by MING

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Whats gene therapy?

Imagine that youaccidentally broke oneof your neighbor'swindows.

I. Stay silent: no one will ever findout that you are guilty, but thewindow doesn't get fixed.

II. Repair it with some tape: not thebest long-term solution.

III. Put in a new window: not only doyou solve the problem, but alsoyou do the honorable thing.

Many medical conditions result from flaws, or mutations,in one or more of a person'sgenes.So, if aflawed gene caused our "brokenwindow," can you "fix" it? What areyour options?

I. Stay silent: ignore the geneticdisorder and nothing gets fixed.

II. Try to treat the disorder with drugsor other approaches: dependingon the disorder, treatment may ormay not be a good long-termsolution.

III. Put in a normal, functioning copyof the gene: if you can do this, itmay solve the problem!


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How to fix it

A

B C a beneficial geneA

virus modified virus

A virus is found which replicates byinserting its genes into the host cell's genome.This virus has three genes -A, BandC.

GeneA encodes a protein which allows this virus to insert itself into the host'sgenome.

GenesBandCactually cause the disease this virus is associated with. ReplaceBandCwitha beneficial gene. Thus, the modified virus could introduce your

'good gene' into the host cell's genome without causing any disease. So we use the modified virus to fix the broken window


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Gene therapy using an Adenovirus vector.A new gene is inserted into an adenovirus vector, which is used to introducethe modified DNA into a human cell. If the treatment is successful, the newgene will make a functional protein.


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Background

In the 1980s, advances in molecular biology had already enabled humangenes to be sequenced and cloned. Scientists looking for a method of easilyproducing proteins, such as the protein deficient in diabetics insulin,investigated introducing human genes to bacterial DNA. The modifiedbacteria then produce the corresponding protein, which can be harvestedand injected in people who cannot produce it naturally.

Scientists took the logical step of trying to introduce genes straight intohuman cells, focusing on diseases caused by single-gene defects, such ascystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia, opticnerve disease1, wound repair and regeneration2, and cardiovasculardisease3.

However, this has been much harder than modifying simple bacteria,primarily because of the problems involved in carrying large sections of

DNA and delivering it to the right site on the genome.


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Cystic Fibrosis

Background Cystic fibrosis was first described as a disease in the late 1930s by

Dorothy Hansine Andersen. In 1988, the first mutation for CF,F508, was discovered by Francis Collins, Lap-Chee Tsui and John

R. Riordan on the 7th chromosome of the human genome.Research has subsequently found over 1000 different mutations thatmay cause CF, however F508 accounts for approximately 70% of

CF patients in Europe (this percentage varies regionally).

CF is an autosomal recessive disease and is the most common lethal

genetic disease among whites. There are 30,000 cases in the United States,3,000 cases in Canada, and 27,000 cases in Europe4.

The mutation for CF on the 7th chromosome
http://gslc.genetics.utah.edu/units/genetherapyhttp://gslc.genetics.utah.edu/units/genetherapyhttp://gslc.genetics.utah.edu/units/genetherapyhttp://gslc.genetics.utah.edu/units/genetherapy


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Gene Therapy for Cystic Fibrosis

Cystic fibrosis should be an ideal candidate forgene therapy, for four main reasons:

(1) it is a single gene defect; (2) it is a recessive condition, with heterozygotes

being phenotypically normal (suggesting genedosage effects are not critical);

(3) the main pathology is in the lung, which isaccessible for treatment;

(4) it is a progressive disease with a virtuallynormal phenotype at birth, offering a therapeuticwindow.

Cystic Fibrosis


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Choices of Vectors

The ideal vector system would have the following characteristics: (1) an adequate carrying capacity; (2) to be undetectable by the immune system; (3) to be non-inflammatory;

(4) to be safe to the patients with pre-existing lung inflammation; (5) to have an efficiency sufficient to correct the cystic fibrosis phenotype; (6) to have long duration of expression and/or the ability to be safely re-

administered.

Cystic Fibrosis

Viral vectors:

Vetrovirus

Adenovirus

Adeno-associated virus

Herpes Simplex Virus

Non-viral vectors:LiposomeDNApolymer conjugatesNaked DNA


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1993 vector used: Adenovirus

The first cystic fibrosis gene therapy clinical trialsused an adenovirus vector to deliver the full-length CFTR (cystic fibrosis transmembrane

regulator) gene to cells. Adequate doses of adenovirus vector will

probably cause an immune response. If theadenovirus is to be useful, researchers need to

find ways to both improve the viruss ability toenter cells and reduce the chances of immuneresponse.

Cystic Fibrosis


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1995 liposome

Trials using liposome-mediated CFTR gene transferbegan in 1995.

Non-viral vectors have the potential to avoid some of thecritical problems observed with viral vectors, such as the

immune response, limited packaging capacity, andrandom integration5 .

Liposomes may be mildly effective, but their activity doesnot last. For this approach to work, researchers need tofigure out how to improve delivery, make the effects

more permanent and reduce the adverse side effects. To date, only cationic liposome-based systems have

been tested in clinical trials in cystic fibrosis subjects6.

Cystic Fibrosis


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1998 adeno-associated virus

Trials using adeno-associated virus todeliver the CFTR gene began in 1998.

Because it is safe, the adeno-associated

virus as we predicted earlier holdspromise for being a good way to deliverthe CFTR gene to patients airway cells.But researchers need to learn more abouthow the virus infects cells in order to makeit an effective delivery method.

Cystic Fibrosis


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Mode of delivery

The majority of experience in terms of vector deliveryto the lungs has involved the instillation of largevolumes of vector-containing fluid into the lung via thenose.

However,I. this mode of delivery poses safety problems because

of the potential for aspiration.II. In addition, the instillation of large volumes of fluid

leads to enhanced alveolar exposure, as a result ofbulk flow into the lung parenchyma. This exposure isundesirable because it may induce adverse reactions.

III. At the same time, it is likely that airway epithelial cells,rather than alveolar epithelial cells, are the appropriatetarget for CFTR gene transfer.

Cystic Fibrosis


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Another mode of lung delivery for vector-containing fluidis by oral inhalation of aerosolized vectors.

However, aerosolization of a fluid is typically achieved bymeans of a nebulizer, and most nebulizers have been

designed to generate small particles. This is becausemost nebulizers have been developed to deliver drugs totreat patients with asthma, and in asthma the targetregion of the lungs is often the peripheral airways. Smallparticles enhance delivery to the peripheral airways and

the alveolar region of the lung, but this is againundesirable for gene vector delivery because of thepossibility of inducing adverse effects.


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One way to avoid alveolar deposition is to generate anaerosol that is composed primarily of large droplets.

Delivery of the vector by means of a spray device that isinserted into a bronchoscope may have anotheradvantage over nebulization.

Research suggests that spray delivery of the vectorcould provide a means of targeting the larger, centralairways , avoiding deposition in the smaller airwaysand alveolar region , which is more likely withnebulizers that generate small aerosol particles.

The findings from studies using spray technologyindicate that efficient and targeted delivery of aerosolizedgene vectors to the lungs may be possible in the future.


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Current treatment

Modern treatment now includes

the intake of digestion enzymes,nutritional supplements,

percussion and postural drainageof the lungs, improved antibiotics

inhalation of aerosols containingmedication.

The most visible gene therapydrug under development is inhaledcomplementary DNA to treat CF.

Cystic Fibrosis

A typical breathing treatment forCystic Fibrosis, using a nebulizerand the ThAIRapy Vest


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challenges

The goal of developing an effective genetic therapy for CF lungdisease has led to the attainment of several milestones in the largerfield of gene therapy. These include:

the first published in vivogene transfers with adenovirus (Ad)7, andwith recombinant adeno-associated virus (rAAV), and

the first phase I clinical trials using each of these vector systems.8 Choice of vector, mode of delivery to the airways, translocation of

genetic information, and expression of normalized CFTR in sufficientamounts to correct the CF phenotype in the lungs of CF patientscontinue to be hurdles in the development of gene therapy for CF9.

A few attempts at gene therapy were initially successful, but failed toproduce acceptable long-term results.

Cystic Fibrosis


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References: 1. Eye 2004, 18, 1049-1055

2. Wound Rep. Reg. 2000, 8, 443-451 3. Circulation Journal 2002, 66, 1077-1086 4. Respiratory Care 2005, 50, 1161-1174 5. Am. J. Med. 2003, 115, 560-569 6. Biochem. J. 2005, 837, 1-15

7. Science 1991, 252, 431-434 8. Hum. Gene Ther. 1996, 7, 1145-1159 9. Chest 2001, 120, 124S-131S

Additional resources:

http://gslc.genetics.utah.edu/units/genetherapy/

http://www.asgt.org/

http://www.congrex.se/esgt/

http://web.archive.org/web/20030219034830/http://www.gtherapy.co.uk/

http://www.cheng.cam.ac.uk/research/groups/biosci/index.html

http://www.gene-watch.org/
http://gslc.genetics.utah.edu/units/genetherapy/http://www.asgt.org/http://www.congrex.se/esgt/http://web.archive.org/web/20030219034830/http://www.gtherapy.co.uk/http://www.cheng.cam.ac.uk/research/groups/biosci/index.htmlhttp://www.gene-watch.org/http://www.gene-watch.org/http://www.gene-watch.org/http://www.gene-watch.org/http://www.cheng.cam.ac.uk/research/groups/biosci/index.htmlhttp://web.archive.org/web/20030219034830/http://www.gtherapy.co.uk/http://www.congrex.se/esgt/http://www.asgt.org/http://gslc.genetics.utah.edu/units/genetherapy/


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The End

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use of gene therapy in the treatment of disease by ming

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