Gene therapy by bdollar

CURRENT TRENDS OF GENE THERAPYMphil Biochemistry 1

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MPhil Biochemistry 2

INTRODUCTION• Genetics and genomics both play roles in health and disease.

• Genetics helps individuals and families learn about how conditions are inherited in families and what treatments are available.

• Genomics is helping researchers discover why some people get sick from certain infections, environmental factors, and behaviors, while others do not.

• All human beings are 99.9 percent identical in their genetic makeup.

• Differences in the remaining 0.1 percent hold important clues about the causes of diseases.


• Understanding more about diseases caused by a single gene (using genetics) and complex diseases caused by multiple genes and environmental factors (using genomics) can lead to earlier diagnoses, interventions, and targeted treatments.

• The family history can serve as the cornerstone • for learning about genetic and genomic conditions in a

family

• and for developing individualized approaches to disease prevention, intervention, and treatment.


What’s gene therapy?

• Imagine that you accidentally broke one of your neighbor's windows.

I. Stay silent: no one will ever find out that you are guilty, but the window doesn't get fixed.

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

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

So, if a flawed gene caused our "broken window," can you "fix" it? What are your options?

I. Stay silent: ignore the genetic disorder and nothing gets fixed.

II. Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution.

III. Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem!


What 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


What makes a condition a good candidate for gene therapy?• Could the condition be corrected by adding one or a few

functional genes?

• Do you know which genes are involved?

• Do you understand the biology of the disorder?

• Will adding a normal copy of the gene fix the problem in the affected tissue? Or could getting rid of the defective gene fix it?

• Can you deliver the gene to cells of the affected tissue?


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 Case• The first gene therapy was performed on September 14th,

1990

• Ashanti DeSilva was treated for SCID• Severe 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 Works• A vector delivers the therapeutic gene into a patient’s

target cell

• The target cells become infected with the 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


http://encarta.msn.com/media_461561269/Gene_Therapy.html

http://encarta.msn.com/media_461561269/Gene_Therapy.html


• A gene can't easily be inserted directly into your cells.

• Rather, it usually has to be delivered using a carrier, called a vector.

• The most common gene therapy vectors are viruses

• Researchers remove the original disease-causing genes from the viruses, replacing them with the genes needed to stop disease.


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 long duration of expression and/or the ability to

be safely re-administered.

• Viral vectors:• Vetrovirus• Adenovirus• Adeno-associated virus• Herpes Simplex Virus

Non-viral vectors:LiposomeDNA–polymer conjugatesNaked DNA


• The most common gene therapy vectors are viruses because they can recognize certain cells and carry genetic material into the cells' genes.

• This technique presents the following risks:

Unwanted immune system reaction.

Targeting the wrong cells

Infection caused by the virus

Possibility of causing a tumor



Use of Liposome• Trials using liposome-mediated gene transfer began in 1995.

• Non-viral vectors have the potential to avoid some of the critical problems observed with viral vectors, such as the immune response, limited packaging capacity, and random integration .

• Liposomes may be mildly effective, but their activity does not last.

• For this approach to work, researchers need to figure 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 subjects.


Is gene therapy available to treat any disorder?• Gene therapy is currently available only in a research

setting.

• The U.S. Food and Drug Administration (FDA) has not yet approved any gene therapy products for sale in the United States.

• Hundreds of research studies (clinical trials) are under way to test gene therapy as a treatment for genetic conditions, cancer and HIV/AIDS.


EYE DEFECTS AND GENE THERAPY• The eye is a sense organ that react to light

• Cells of the rod and cones in the retina allow conscious light perception and vision including color differentiation

• There are so many defect of the eye and include • Leber’s Congenital Amaurosis• Retinitis Pigmentosa • Achromatopsia • Choroideremia• Usher Syndrome


Leber’s Congenital Amaurosis (LCA)• LCA is a heterogeneous group of AR diseases which is

inherited from parent.

• This is a Gene defects affecting retinal pigment epithelium–specifically the protein (RPE65)

• This defect or mutation result in the narrowing of the retinal blood vessels which intern affect the cell that detect light in the retina i.e. the rods and cones.


• This may cause severe sight impairment in childhood and progressive retinal degeneration

• There is so far No treatment for this form of eye defect but scientist through gene therapy has made positive efforts to getting the cure to it.


Gene Therapy for Leber’s Congenital Amaurosis• Gene therapy trial was carried out on dogs and 12 humans.

• A recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 cDNA at doses of 1x1011 or 1x1012 vector genomes were administed to the 12 subject and studied for three years.

• Improvements in retinal sensitivity were evident in 6 human subjects and lasted for up to 3 years

• In a parallel study they administered the same vector to RPE65-deficient dogs

• In dogs, administration of the same vector at lower doses resulted in improved visual behavior,


GENE THERAPY FOR HIV INFECTION• Hematopoietic Stem Cell (HSC)-based gene therapy against HIV

infection has emerged as a promising approach for the treatment

of HIV/AIDS

• HIV-1 attachment to susceptible cells involves binding of gp120

to CD4 receptor and subsequently to a chemokine co-receptor,

either CCR5 or CXCR4

• Studies have shown that a 32-bp deletion in CCR5 (CCR5∆32)

generates a nonfunctional gene product (Lai, 2012)


• A single proof of concept was provided by the so-called “Berlin patient,” who

remained free of detectable HIV (for 20 months) after receiving a bone

marrow transplant from a CCR5-∆32 homozygous donor (Berkhout and

Herrera-Carrillo, 2015)

• Homozygosity for CCR5∆32 confers profound resistance against HIV

infection, and heterozygous mutation confers delayed progression to

acquired immune deficiency syndrome (AIDS) (Lai, 2012)

• The use of HSC-based gene therapy against HIV infection is based on the

concept that, after transplantation, genetically modified HSCs carrying anti-

HIV transgenes would engraft, divide and differentiate into a population of

cells that express antiviral genes


• CCR5 gene knockdown inhibits functional expression of CCR5 in

HSCs which produce daughter cells resistant to HIV

• A study developed an anti-CCR5 ribozyme which were transduced

using a retroviral vector into human CD34+ cells purified from fetal

liver

• These hematopoietic cells underwent normal differentiation in vitro

into mature macrophages that expressed the ribozyme and

showed marked resistance to the infection of CCR5-tropic virus

HIV-1 (Lai, 2012)


Gene therapy for blood diseases• Factor VII bleeding disorder affects both sexes

• Patients lack a specific clotting factor

• Mostly treated with regular transfusions of clotting factor

• An adeno associated virus (AAV) which doesn’t cause disease is used as a vector to deliver the DNA into the cell where it can express enough factor to make blood clot normally.


• Hemophilia B clotting factor IX is lacking

• Used the lentivirus vector- a large retrovirus

• Hemophilia A lack clotting factor VIII

• Platelet precursor cells put in a gene therapy vector that expresses factor VIII

• Been used successfully in dogs that have the same condition.

• Yet to be used in humans


• Beta – thalassemia, defect in beta globin gene, patients do not have enough red blood cells to carry oxygens to all tissues.

• Blood stem cells taken from patients bone marrow and treated with a retrovirus to transfer a working copy of the beta globin gene.

• Modified stem cells were transfered back into patient and it gave rise to healthy red blood cells.


Fat Metabolism Disorder• In 2012, Glybera became the first viral gene-therapy

treatment to be approved in Europe.

• The treatment uses an adeno-associated virus to deliver a working copy of the LPL (lipoprotein lipase) gene to muscle cells.

• The LPL gene codes for a protein that helps break down fats in the blood, preventing fat concentrations from rising to toxic levels.


Cancer• Several promising gene-therapy treatments are under

development for cancer.


• It replicates (makes more of itself) inside the cancer cells until they burst, releasing more viruses that can infect additional cancer cells.

• A completely different approach was used in a trial to treat 59 patients with leukemia, a type of blood cancer.

• The patients' own immune cells were removed and treated with a virus that genetically altered them to recognize a protein that sits on the surface of the cancer cells.

• After the immune cells were returned to the patients, 26 experienced complete remission.

CARDIOVASCULAR DISEASES• Hypertension

• Atherosclerosis

• Heart Attack Stroke

• Congestive Heart Failure

• Angina Pectoris

• Arrhythmia

GENE THERAPY FOR HEART FAILUREChronic heart failure is associated with increased

sympathetic outflow.

Chronic beta-adrenergic receptor (βAR) stimulation contributes to worsening of the pathophysiology.

A number of alterations in the βAR signaling cascade have been described. and these include

βAR downregulation upregulation of βAR kinase (βARK) increased inhibitory G-protein alpha-subunit (GαI) function


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?


• Current gene therapy research has focused on treating individuals by targeting the therapy to body cells such as bone marrow or blood cells.

• This type of gene therapy cannot be passed on to a person’s children.

• Gene therapy could be targeted to egg and sperm cells (germ cells), however, which would allow the inserted gene to be passed on to future generations.

• This approach is known as germline gene therapy.

• The idea of germline gene therapy is controversial.


• While it could spare future generations in a family from having a particular genetic disorder, it might affect the development of a fetus in unexpected ways or have long-term side effects that are not yet known.

• Because people who would be affected by germline gene therapy are not yet born, they can’t choose whether to have the treatment.

• Because of these ethical concerns, the U.S. Government does not allow federal funds to be used for research on germline gene therapy in people.


In summary• Gene therapy is the transfer of a therapeutic or working

copy of a gene into specific cells of an individual

• The technique may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favourably modify the symptoms of a condition

• Gene therapy is still an experimental technique and much research remains to be done before this approach to the treatment of conditions will realise its full potential


References• Burdette, Walter J. The Basis for Gene Therapy. Springfield: Charles C

Thomas, 2001.• Crayton, Stephanie. “First Clinical Trial Of Gene Therapy For Muscular

Dystrophy Now Under Way.” Medical News Today. 1 April 2006. University of North Carolina at Chapel Hill. 11 November 2006 <www.medicalnewstoday.com>.

• Gene Therapy. Human Genome Project Information. 18 November 2005. U.S. Department of Energy Office of Science, Office of Biological and Environmental Research, Human Genome Program. 12 September 2006 <http://www.ornl.gov/hgmis>.

• McCormack, Matthew P. “Activation of the T-Cell Oncogene LMO2 after Gene Therapy for X-Linked Severe Combined Immunodeficiency.” The New England Journal of Medicine. http://content.nejm.org. 346: 1185-1193, Apr 18, 2002.

• Peel, David. “Virus Vectors & Gene Therapy: Problems, Promises & Prospects.” Virus Vectors & Gene Therapy. 1998. Department of Microbiology & Immunology, University of Leicester. 11

November 2006 <http://www.tulane.edu/~dmsander/WWW/335/peel/peel2.html>.


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Gene therapy by bdollar